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This article discusses a wide range of developmental early onsets of puberty, specifically in that of women and their early production of hormones that cause the growth of underarm hair and breast development at increasingly younger ages. In this paper, it is also discussed that there was a study conducted to measure the effects of EDCs over a several generations. The results observed a wide variety of developmental changes with the most drastic effects being shown in third generations. Some of these effects include delayed vaginal opening and a less frequent cycle.

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Neonatal Intensive Care Unit, Department of Medical and Surgical Sciences of the Mothers, Children and Adults, University of Modena and Reggio Emilia, via del Pozzo 71, 41124 Modena, Italy

Post Graduate School of Paediatrics, Department of Medical and Surgical Sciences of the Mothers, Children and Adults, University of Modena and Reggio Emilia, via del Pozzo 71, 41124 Modena, Italy

Pediatric Unit, Department of Medical and Surgical Sciences of the Mothers, Children and Adults, University of Modena and Reggio Emilia, via del Pozzo 71, 41124 Modena, Italy

Author to whom correspondence should be addressed.

Received: 3 March 2020 / Revised: 12 March 2020 / Accepted: 14 March 2020 / Published: 18 March 2020

Puberty is the process of physical changes between childhood and adulthood during which adolescents reach sexual maturity and become capable of reproduction. It is considered one of the main temporal windows of susceptibility for the influence of the endocrine-disrupting chemicals (EDCs). EDCs may act as single chemical agents or as chemical mixtures; they can be pubertal influencers, accelerating and anticipating the processing of maturation of secondary sexual characteristics. Moreover, recent studies have started to point out how exposure to EDCs during puberty may predispose to breast cancer later in life. In fact, the estrogen-mimicking endocrine disruptors (EEDs) may influence breast tissue development during puberty in two main ways: the first is the action on the proliferation of the breast stromal cells, the second concerns epigenetic mechanisms. The aim of this mini-review was to better highlight what is new and what is not completely known regarding the role of EDCs during puberty. View Full-Text

Lucaccioni, L.; Trevisani, V.; Marrozzini, L.; Bertoncelli, N.; Predieri, B.; Lugli, L.; Berardi, A.; Iughetti, L. Endocrine-Disrupting Chemicals and Their Effects during Female Puberty: A Review of Current Evidence. Int. J. Mol. Sci. 2020, 21, 2078. https://doi.org/10.3390/ijms21062078

AMA Style

Lucaccioni L, Trevisani V, Marrozzini L, Bertoncelli N, Predieri B, Lugli L, Berardi A, Iughetti L. Endocrine-Disrupting Chemicals and Their Effects during Female Puberty: A Review of Current Evidence. International Journal of Molecular Sciences. 2020; 21(6):2078. https://doi.org/10.3390/ijms21062078

Chicago/Turabian Style

Lucaccioni, Laura, Viola Trevisani, Lucia Marrozzini, Natascia Bertoncelli, Barbara Predieri, Licia Lugli, Alberto Berardi, and Lorenzo Iughetti. 2020. "Endocrine-Disrupting Chemicals and Their Effects during Female Puberty: A Review of Current Evidence" International Journal of Molecular Sciences 21, no. 6: 2078. https://doi.org/10.3390/ijms21062078

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# data[].attributes.attachment_link.title
Endocrine-Disrupting Chemicals and Their Effects during Female Puberty: A Review of Current Evidence

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Puberty is the process of physical changes between childhood and adulthood during which adolescents reach sexual maturity and become capable of reproduction. It is considered one of the main temporal windows of susceptibility for the influence of the endocrine-disrupting chemicals (EDCs).

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https://onlinelibrary.wiley.com/share/JKCHRHAIR5C3EXNXV7VM?target=10.1002/tox.22013
This study looks at the exposure of EDCs on mice and how it can lead to sytnhesis of fatty acids and oxidation in the liver of mice, which on a large scale basis in a human body can influence puberty.

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Oral exposure of pubertal male mice to endocrine‐disrupting chemicals alters fat metabolism in adult livers

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Alana Williams: "The evaluation of possible role of endocrine disruptors in central and peripheral precocious puberty" BPA

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Alana Williams: "The evaluation of possible role of endocrine disruptors in central and peripheral precocious puberty" BPA

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https://www.tandfonline.com/doi/abs/10.3109/15376516.2016.1158894

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Abstract
Exposure to environmental chemicals can affect genetic and epigenetic molecular pathways and may cause altered growth and development. Among those exposures, endocrine-disrupting chemicals (EDCs) are of particular concern as humans are abundantly exposed to these chemicals by various means in every period of life. Several well-known environmental chemicals, including phthalates and bisphenol A (BPA), are classified as EDCs. These EDCs are suggested to play roles in early onset of puberty in girls. The aim of this study is to determine plasma phthalate (di(2-ethylhexyl)phthalate [DEHP] and its main metabolite mono(2-ethylhexyl)phthalate [MEHP]) and urinary BPA levels in girls with idiopathic central precocious puberty (CPP) and peripheral precocious puberty (PPP). This study was performed on newly diagnosed idiopathic central precocious puberty (CPP) patients ( n = 42) and peripheral precocious puberty (PPP) ( n = 42) patients, who were admitted to Keçiören Training and Research Hospital, Clinic of Pediatric Endocrinology between August 2012 and -July 2013. Nonobese healthy girls ( n = 50) were used as the control group. Urinary BPA levels were not statistically different in control, PPP and CPP groups (medians 10.91, 10.63 and 10.15 μg/g creatinine, respectively; p > 0.05). Plasma DEHP levels were significantly higher in PPP group when compared to control. Plasma MEHP levels were not significantly different in control and PPP groups ( p  > 0.05). However, in CPP group, both plasma DEHP and MEHP levels were significantly higher than control and PPP groups. This study showed that phthalates might play a role in the occurence of CPP in girls.

Declaration of interest

The authors declare no conflicts of interest.

# data[].attributes.attachment_link.title
The evaluation of possible role of endocrine disruptors in central and peripheral precocious puberty

# data[].attributes.attachment_link.description
Abstract Exposure to environmental chemicals can affect genetic and epigenetic molecular pathways and may cause altered growth and development. Among those exposures, endocrine-disrupting chemicals (EDCs) are of particular concern as humans are abundantly exposed to these chemicals by various means in every period of life.

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Lindsey Duckett: Endocrine disrupters and human puberty. Study found that earlier age of menarche was found in girls who were exposed to PCBs, PBBs, and DDT

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Lindsey Duckett: Endocrine disrupters and human puberty. Study found that earlier age of menarche was found in girls who were exposed to PCBs, PBBs, and DDT

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Michelle Consolver: Being exposed to BPA (found in plastics and chemical additives) has an estrogenic  and obesogenic similarities. It has been suggested to cause early great bud development as early as 4 years old. Though studies have been inconclusive, it may be discovered upon further research the effect BPA has on early onset puberty. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5615581/   

# data[].attributes.subject
Michelle Consolver: Being exposed to BPA (found in plastics and chemical additives) has an estrogenic  and obesogenic similarities. It has been suggested to cause early great bud development as early as 4 years old. Though studies have been inconclusive, it may be discovered upon further research the effect BPA has on early onset puberty. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5615581/   

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Abstract

Many scientific studies have revealed a trend towards an earlier onset of puberty and have disclosed an increasing number of children that display precocious puberty. As an explanation, some authors have considered the global socio-economic improvement across different populations, and other authors have considered the action of endocrine disrupting chemicals (EDCs). Among these, bisphenol A (BPA), an aromatic compound largely used worldwide as a precursor of some plastics and chemical additives, is well known for its molecular oestrogen-like and obesogenic actions. We reviewed the medical literature of the previous 20 years that examined associations between BPA exposure and the age of puberty in humans, considering only those referring to clinical or epidemiological data. Of 19 studies, only 7 showed a correlation between BPA and puberty. In particular, the possible disruptive role of BPA on puberty may be seen in those with central precocious puberty or isolated premature breast development aged 2 months to 4 years old, even if the mechanism is undefined. Some studies also found a close relationship between urinary BPA, body weight, and early puberty, which can be explained by the obesogenic effect of BPA itself. The currently available data do not allow establishment of a clear role for BPA in pubertal development because of the conflicting results among all clinical and epidemiological studies examined. Further research is needed to fully understand the potential role of exposure to EDCs and their adverse endocrine health outcomes.

Keywords: bisphenol A, menarche, puberty, pubertal development, premature thelarche

1. Introduction

Endocrine-disrupting chemicals (EDCs) are a group of hormonally active agents to which we are directly or indirectly exposed on a daily basis and that have been widely used in industry for almost a century [ 1]. They interfere with natural hormones, stimulating or inhibiting their action by binding their receptors or by affecting their synthesis, transport, metabolism, and elimination [ 2]. As a result, they may cause different kinds of diseases. Concerning EDCs' toxic effects on health, many studies have confirmed that foetuses, infants, and children are more vulnerable than adults, primarily because of the important role of hormonal balance in both growth and development [ 3, 4]. Moreover, they receive more exposure to these substances than adults, as demonstrated by the higher quantities of EDCs found in urine and blood [ 5, 6].

Bisphenol A (BPA), one of the most studied EDCs, is an aromatic compound largely used worldwide as the precursor of plastics and chemical additives [ 7]. It is widely used in the production of polycarbonate plastics (very common for transparency, heat resistance, and mechanical properties) and epoxy resins for coating of cans for food and beverages. Humans are exposed to these products mainly through their diet [ 8] but also dermal absorption and inhalation [ 9]. Among these, we find EDCs in food and drink packaging, baby feeding bottles, recycling containers, polyvinyl chloride, toys, and dental sealants [ 10]. EDCs can also be transferred to the baby via the placenta or breast milk [ 11, 12]. Although many countries including Europe (EU Directive 2011/8/EU) have recently drawn up laws restricting the use of BPA in the production of basic necessities [ 13], this substance is still used on a large scale [ 14], with more than 3.5 million tons being produced every year [ 7]. The United States of America is the world's largest producer, with more than a 1 million tons of BPA produced per year (almost 25% of the global production) [ 15].

Despite the seriousness of prenatal exposure to EDCs' toxic effects, the pathological role of BPA becomes more evident during the pubertal period, when neuroendocrine mechanisms, during the development of reproductive organs and the appearance of secondary sexual characteristics, become more vulnerable to environmental stimulation [ 16, 17]. The aim of our review was to find all studies examining the association between BPA exposure and age of puberty in humans. A PubMed search via MEDLINE was undertaken to identify studies matching the following keywords: "bisphenol A" and puberty", "precocious puberty", "female puberty", "male puberty", "premature thelarche", "menarche", and "pubertal development". The same research was conducted with these keywords replacing the word "bisphenol A" with the most widely used acronym "BPA". We reviewed all papers published in the last 20 years, and the date of our last search was March 2017. We retrieved 111 studies, excluding animal studies, in vitro studies, BPA chemical analysis studies, and BPA pharmacology studies, and considering only articles that referred to clinical and/or epidemiological data. From the initial collection, we selected 17 scientific papers based on the above-listed criteria; we later added 2 other papers not found in the PubMed database but that were tracked through the bibliographical references. In the end, we split all works into three subgroups: BPA and female puberty, BPA and isolated premature thelarche, and BPA and male puberty.

2. Puberty Onset

Puberty is a period of transition between adolescence and adulthood. During this period, full reproductive maturity is reached through the maturation of gonads, permanent genital development, and appearance of secondary sexual characteristics [ 18]. The harmonic evolution of the phenomena characterizing puberty is closely linked to the activation of the hypothalamic-pituitary gonadotropic cells-gonadal axis [ 19]. Clinical and experimental evidence has indicated that the hypothalamic-pituitary-gonadal system is in a condition of repression during childhood.

Mechanisms responsible for the reactivation of this axis are currently unknown. Recently, a neuropeptide known as kisspeptin, encoded by the gene KISS-1, and its receptor GPR54 have been revealed as major regulators of the beginning of the reproductive pubertal axis, acting as potent stimulators of pulsatile secretion of the hypothalamic gonadotropin-releasing hormone (GnRH) [ 20, 21]. GnRH neurons have sex steroid receptors through which oestrogen and progesterone can modulate the activity of kisspeptin [ 22]. Leptin, a hormone produced by adipocytes and an important regulator of both fat mass and gonadal function, is among the factors leading to increased kisspeptin expression during puberty [ 23]. Other factors involved in controlling the levels of the GnRH network are the transcriptional regulator enhanced at puberty 1 (EAP1) and Ying Yang 1 [ 24], which operate as inhibitory regulators, and thyroid transcription factor 1 (TTF1) [ 25], which operates as a stimulator within the GnRH neuronal network.

Other possible puberty regulators may be RFamide-related peptide-3 (RFRP3) neurons, which seem to play an inhibitory action on the GnRH pathway [ 26, 27]: they were first identified in birds and have recently also been found in humans [ 28, 29]. Some authors have suggested that kisspeptin behaves as an accelerator of GnRH, while RFRP3 acts as a brake; through their modulation, the timing of pubertal development is scanned [ 30].

We know that, at the level of the tonic hypothalamic centre, sensitivity to the inhibition of gonadal hormones is greater in childhood and decreases at the onset of puberty. In fact, in childhood, the very small amount of hormones produced by gonads is able to inhibit the release of GnRH and the secretion of gonadotropins [ 31]. When puberty approaches, there is a progressive reduction of sensitivity of the tonic centre receptor to the sex steroids' actions. Then, a critical time coinciding with the onset of puberty arrives, during which gonadal hormones are no longer able to inhibit the hypothalamus neurosecretory activity. There is a massively elevated release of GnRH and LH-FSH gonadotropins, and the consequent activation of the gonads leads to an increased circulating level of androgens and oestrogens [ 32, 33].

Oestrogen plays a very important role in sexual differentiation, which occurs at the pubertal stage and is closely linked to several changes in the child's phenotype: breast growth [ 34], effect on pubertal growth spurt for both sexes (stimulation of linear growth at low concentrations and closure of the epiphyseal plates and cessation of linear growth at higher concentrations) [ 35], maturation of sexual characteristics [ 36], beginning of menses [ 37], and mutual influence on the hypothalamic-pituitary axis [ 38]. The last data explain why, in girls with early pubertal signs, elevated levels of oestradiol can be found in blood, even if the mechanism remains unclear [ 39]. Three different receptors through which oestrogen carries out its functions have been identified: two are nuclear receptors (αER and βER ⁰) [ 40, 41], while another one, the most recently discovered, works as a membrane receptor or a cytoplasmic receptor [ 42].

Androgens play a crucial role in male sexual development, and they have two origins: adrenal or gonadal. Adrenal androgens (DHEA, DHEAS, and androstenedione) increase in their secretion at 6-8 years. This moment corresponds with adrenarche, a process that precedes gonadarche by approximately 2 years, which instead represents the true onset of puberty in males. Gonadarche is regulated by increased production of testosterone by the testicular Leydig cells, which in turn are stimulated by activation of the pituitary gland via GnRH pulsatile secretion. Adrenal androgens are responsible for the initial stages of linear growth and pubic hair, changes of tone, appearance of acne, and appearance of hair in the androgen-sensitive areas. However, the most important role in puberty is played by testosterone in collaboration with pituitary hormones, which are implicated in linear growth, increase in testicular volume, appearance of male secondary sex characteristics, and increase in lean body mass [ 43].

During pre-pubertal age, the levels of endogenous sex steroids are very low, although receptors for oestrogens and androgens are expressed since early childhood [ 44]. This explains how a small change in levels, even from the outside, may cause a major change in balance of child hormones, such as several pubertal changes [ 45]. Diseases related to puberty include early and delayed puberty. Precocious puberty is defined as the onset of puberty characteristics in females under the age of 8 years and in males under the age of 9 years [ 46, 47]. The main clinical manifestations of suspicion for precocious puberty in girls are breast development and acceleration of growth rate; other signs include pubic or underarm hair development, acne, and puberty sweating. In boys, an increase in testicular volume (>4 mL) may occur. Late signs may include modifications of penis and scrotum in appearance and size, acne, pubic hair, underarm or facial hair development, and acceleration of linear growth [ 48]. Pubertal status is conventionally evaluated using Tanner's staging; for both boys and girls, early puberty is conventionally summed up in that a Tanner stage 2 or higher may occur earlier than expected [ 49, 50].

Precocious puberty is more prevalent in girls (almost 0.2%) than boys (almost 0.05%) [ 51]. This condition can be caused by early activation of the pituitary gonadostat (central precocious puberty, CPP) and ectopic production of gonadotropins or sex steroids (peripheral precocious puberty, PPP); it may have different origins, such as idiopathic, sporadic, familial, or neurogenic, which is secondary to central nervous system abnormalities [ 52]. Idiopathic central precocious puberty (ICPP) is the most common cause in females, being characterized by anticipated activation of the hypothalamic-pituitary-ovarian axis [ 53]. PPP is a rare condition and is usually characterized by the overproduction of sex hormones (oestrogen and testosterone) [ 54].

A careful remote and family medical history allows for suspicion of forms of early secondary and familiar precocious puberty, respectively. The clinical data listed above are important in association with other information dealing with signs of pubertal development at the pelvic ultrasonography in females (increase in uterine volume, longitudinal diameter >35 mm, uterine body/cervix ratio >1, thickening of endometrial rhyme, or increase in the diameter of the ovaries, longitudinal diameter >3 cm or increase in size of follicles, diameter >9 mm); with detection of testosterone in males (>50 ng/dL) and oestradiol in females (>20 pg/mL, even if for females low estradiol concentrations do not rule out the diagnosis of precocious puberty); or assessment of bone age at the left hand X-ray film (resulting higher than at least 1 year in comparison with the chronological age).

A definite diagnosis is based on the GnRH test: CPP is present when there is an LH blood peak >5 mUI/mL and LH/FSH ratio >0.66 after stimulation with the hypothalamic hormone analogue [ 55, 56, 57, 58, 59]. Magnetic resonance imaging allows discrimination between forms caused by neurologic diseases and those not (including idiopathic forms) [ 60]. On the other hand, PPP can be diagnosed by the same criteria except the detection of high oestradiol and low gonadotropin blood levels, LH peak <5 mUI/mL, absence of LH/FSH inversion after GnRH stimulation test, and negative cerebral magnetic resonance imaging [ 61, 62]. Pathological effects related to early puberty include insulin resistance, metabolic syndrome, psychosocial disorders, cardiovascular diseases, adult-onset asthma, and breast or reproductive system tumours [ 63, 64, 65, 66, 67].

3. Age of Puberty

Many studies have reported a current counter-trend of age for puberty onset in girls compared to the past. The mean onset of thelarche, pubarche, and menarche has decreased over the last decades [ 49, 68]. This trend has been observed both in America [ 69] and Europe [ 70]. A study by Biro et al. showed that 16% of girls (with a peak of 23.4% in black non-Hispanic girls) began pubertal development at the age of 7 years, while 30% (with a peak of 42.9% in black non-Hispanic girls) began at the age of 8, well below the previous average [ 71]. Askglaede highlighted the different average age for onset of secondary sexual characteristics between the beginning of the 1990s and the early years of this decade (in a time interval of approximately 15 years) in Denmark. If the average was 10.8 years old during the 1990s, it dropped 1.02 years between 2006 and 2008 (to 9.86 years old). Girls get their first periods on average 0.29 years earlier (13.13 years) [ 71]. Genetics, with the A2 polymorphism of the CYP17 gene [ 72], Oct-2 gene [ 73], and GPR54/KiSS-1 at the neuroendocrine level [ 74], and the environment play a role in the definition of puberty onset; in this respect, the general improvement of socio-economic and nutritional conditions of the population only partially justifies this anticipation [ 75]. Weight-gain, insulin resistance, and metabolic syndrome are well-known factors linked to this condition. Glucose and leptin have been shown to be involved in the control of GnRH secretion, but their role in the timing of puberty remains controversial [ 75]. What is certain is that obese girls are more likely to have early menarche [ 76]. Studies have shown that further decreases in the age of onset of pubertal activation signs (breast development in girls and increased testicular volume in boys) have occurred in recent years, despite the maintenance of substantially unchanged standards of life [ 70]. For this reason, industrialization and daily exposure to environmental pollutants are considered among the causes of this trend because of their ability to interfere with normal bodily function [ 77, 78]. It also explains why the number of studies dealing with EDC and their effects on pubertal development is increasing [ 37, 79]. One of those studies was conducted by Massart et al. in Italy, where in a limited area of northern Tuscany, they have reported a higher proportion of early puberty: the study has raised the suspicion that this growing incidence might be correlated with environmental factors [ 80].

Sun et al., comparing previous studies, found an anticipation of puberty also in males, but only for white and Hispanic males (not for non-Hispanic black males) [ 81], although other studies investigating the male pubertal timing trend have presented less reliable data [ 82].

4. Bisphenol A (BPA) and Its Oestrogen-Like Role

As an EDC, BPA is able to alter reproductive, nervous, cardiovascular, and immune systems and interferes with thyroid and pancreatic functions [ 82]. According to the pharmacological studies, the maximum BPA value to which we can daily expose ourselves as recommended by the European Food Safety Authority is almost 4 mcg/kg/day [ 83]. However, some studies recommend lower thresholds for exposure without toxicological relevance [ 84].

Although most of the molecular mechanisms of its effects on the human body are still unknown, BPA is widely studied for its oestrogen-like action. Because of its structural similarity with 17β-oestradiol, many studies have shown that BPA may act as a leading actor in the pathogenesis of several genital and reproductive disorders, such as male and female reproductive tract alterations, female and male infertility, precocious puberty, externalizing behaviour in girls, breast and prostate cancer, menstrual irregularities, and polycystic ovary syndrome (PCOS) [ 85, 86, 87, 88, 89, 90]. It is a xenooestrogen and competes with natural oestradiol for oestrogen receptors (αER, βER, and the membrane receptor) [ 91, 92, 93]. Furthermore, it may act antagonistically in the presence of oestrogens, such as 17β-oestradiol, and can interfere with oestrogen nuclear receptors [ 82, 94]. The possibility of simultaneously causing early puberty and having a negative role on fertility seems to confirm its dual aspect. This means that it behaves as an agonist or antagonist through the classical or alternative endocrine receptor-dependent signalling pathways [ 95]. Moreover, its interference with steroid hormones seems to be expressed by antagonizing the action of androgens and thyroid hormones [ 96, 97].

The pathologic mechanism of precocious puberty caused by BPA exposure is related to its oestrogen-like activity, which triggers a positive feedback process for the activity of the GnRH pulse generator, with increased LH and FSH central secretion despite the weak effect as a xenooestrogen [ 98]. Animal studies have shown the effects of BPA on the hypothalamic-pituitary-gonadal axis through alterations in brain sexual differentiation, higher GnRH secretion, and alterations in the oestradiol-induced LH surge [ 86]. In a recent study, Mueller et al. showed that BPA is able to induce precocious puberty by stimulating factors implicated in pubertal activation, such as KiSS1, and repressing factors inhibiting the onset of puberty, such as EAP1 and YY1. Epigenetic changes may also enable this mechanism. The same study demonstrated the stimulatory effect of BPA on Dnmt3b, a DNA methylase, with a possible effect of altered methylation of the genes belonging to the neuronal network of GnRH [ 99].

Another relevant issue is the correlation between BPA and obesity. This correlation was first studied in rats; when exposed prenatally to BPA, they develop dyslipidaemia, hyperleptinaemia, hyperglycaemia, and insulin resistance [ 100]. In a second step, the obesogen action of BPA in human beings was evaluated and confirmed [ 101, 102]. An interesting study by Trasande et al. showed high levels of urinary BPA in obese children and adolescents [ 103]. A more recent study by Li et al. found that, in Shangai, girls aged between 9 and 12 years with high levels of urinary BPA were linked to a more than 2-fold risk of obesity [ 104]. As obesity has an important role in puberty induction, it is not difficult to imagine a possible indirect action of BPA mediated by its interference with glucose and lipid metabolism [ 105]. Although several hypotheses have been raised, many uncertainties still remain.

5. Bisphenol A (BPA) and Female Puberty

One of the largest and longest clinical studies on pubertal development in girls and exposure to environmental phenols was conducted by Wolff et al. [ 106]. In 2008, they investigated the exposure to EDCs and pubertal status in a multiethnic group of 192 healthy 9-year-old girls living in New York City. In addition to phytooestrogens, 1,1′-dichloro-2,2′-bis(4-chlorophenyl)ethylene (DDE) and polychlorinated biphenyls (PCBs), they also measured the urinary BPA in this group of subjects. The choice of selecting 9-year-old patients stemmed from the opportunity of having a relatively equal distribution between subjects with puberty signs (53% had breast development predominantly at Tanner stage 2, and 31% had pubic hair development) or subjects with no signs of sexual development. Despite taking into account BMI as a possible modifier of hormonal exposure, they revealed no correlation between the values of urinary BPA and pubertal development [ 106].

Later, in 2010, Wolff and other colleagues expanded the study by taking into account 1151 American girls among those enrolled in the Breast Cancer and Environment Research Centers (BCERC) [ 107]. The last group was established by the National Institute of Environmental Health Sciences and the National Cancer Institute that studied environmental chemicals' influences on the timing of pubertal development in girls. Between 2004 and 2007, they recruited girls aged between 6 and 8 years and followed them throughout puberty. As in the previous study, the authors compared the pubertal stage of these girls and exposure to 3 classes of chemical contaminants: phenols (including BPA), phthalates, and phytooestrogens. The proportion of girls with breast or pubic hair development (30% and 22%, respectively) was lower 1 year after the recruitment compared to the previous study, given the lower age considered. A sample of urine taken at enrolment and another taken 1 year later was also analysed, but no association was found between the level of urinary BPA and pubertal stage [ 107].

In 2015, the same group evaluated 1239 girls taken from BCERC, all of whom were 6-8 years old at enrolment, in order to detect age at first puberty appearance in a 7-year follow-up and correlated this moment with urinary values of 10 different phenols, including BPA [ 108]. This study estimated a relative risk of early or late puberty linked to phenol exposure. In approximately 85% of girls, signs of pubertal activation (growth of breasts and pubic hair) appeared in the extended follow-up time, but no statistical correlation was found between levels of BPA and early pubertal development. According to this study, there was no correlation between BMI and levels of urinary BPA [ 108].

A similar study with a smaller group was conducted in Europe by Frederiksen et al., who investigated pubertal status in relationship with urinary levels of 8 hormonally active phenols in 129 Danish children and adolescents aged between 6 and 21, showing the absence of correlation between urinary BPA and pubertal development [ 109].

Other works have considered the correlation between BPA exposure and age at menarche. McGuinn et al. evaluated this association in girls through the study of the National Health and Nutrition Examination Survey (NHANES) in the period 2003-2010 [ 110]. All girls analysed were aged 12-19 years, and it was therefore possible to consider the effect of BPA exposure close to the age of menarche (indeed, 72.2% had reached menarche). Age, ethnicity, country of birth, annual household income, level of guardian graduation, and body max index were also considered as potential confounders. In this case, the index of exposure to BPA was represented by urinary BPA. Among the 987 girls that were studied, those with moderate levels of urinary BPA had delayed menarche compared to those with the lowest values. The girls layering for potential confounders did not reach statistical significance, although a common result with other studies was the finding of elevated values of urinary BPA in overweight girls (BM ≥ 85th percentile) who were more likely to have early onset menarche [ 111].

Another important American study was conducted by Buttke, Sircar, and Martin in 2012 [ 112]. They considered the link between exposure to certain EDCs and age of menarche in a group of 461 girls aged between 12 and 16 years who had been recruited from Centers for Disease Control and Prevention's National Health and Nutrition Examination Survey (NHANES)) between 2003 and 2008. In that survey, the study group was given a questionnaire on demographics and related health behaviours, and each subject underwent clinical and laboratory evaluations. Among the 461 girls, it was possible to analyse the levels of urinary BPA in 440 subjects. To obtain the most reliable results, the interaction between urinary phenols and BMI and ethnicity were also considered, as these factors affect the age of menarche. However, regardless of the adjustments made, urinary BPA levels were not significantly associated with age at menarche [ 112].

While these studies did not show a significant correlation between BPA and puberty, different results have been obtained from studies that took into account a population of girls with early puberty. Most of these works were carried out in Asian populations. Durmaz et al. tested the difference in urinary BPA between 28 non-obese Turkish girls affected by idiopathic central precocious puberty and 25 healthy age- and ethnicity-matched control girls [ 113]. Both were aged 4-8 years. In the control group, the girls considered had no signs of pubertal activation at the time of the study. The level of urinary BPA in the affected group of 26 girls was significantly ( p = 0.001) higher than that in the control group. The study also took into account the absence of correlation between urinary BPA levels and serum LH, FSH, and oestradiol levels. They did not observe any significant differences between early puberty and use of feeding bottles or nipples.

Another more recent study by Supornsilchai et al. considered the values of urinary BPA and pubertal stage in 88 Thai girls [ 114]. The girls were divided into two groups: 41 patients affected by precocious puberty and 47 healthy age-matched controls. Those with signs of early puberty had higher levels of BPA in the urine compared to the control group. In addition, obese or overweight girls with signs of pubertal activation had higher values of urinary BPA compared to normal weight girls and normal pubertal overweight or obese girls. As in other studies, BPA levels were not associated with levels of FSH, LH, or oestradiol [ 114].

To determine if there was an association between the onset of precocious puberty and levels of EDCs in the plasma, Kwon performed a study in 2011 that confirmed the association between BPA and early puberty [ 115]. In that case, the determination of exposure to BPA was obtained by dosing the concentration of BPA in serum associated with that of kisspeptin. Both the levels of serum kisspeptin and BPA were significantly higher in the study group, consisting of 31 girls who were diagnosed with central precocious puberty, compared to the control group of 30 healthy age-matched girls. Instead, no correlation was found between serum kisspeptin, BPA, LH, and FSH peak values [ 115].

In 2010, Qiao et al. compared the plasma levels of 3 different phenols (bisphenol A, octylphenol, and 4-nonylphenol) in 110 girls affected by precocious puberty and 100 normal girls [ 116]. Those values were compared with the volume of the uterus and ovaries and the value of oestradiol. BPA was identified in the blood of 40.9% of girls with precocious puberty compared to 2% of healthy controls. This confirmed that higher values of BPA are found in subjects with early onset of puberty. In such subjects, exposure to this contaminant positively influenced the volume of ovaries and the uterus.

Yum et al. did not agree with the results of previous trials conducted on populations of children with early puberty. In fact, they recruited 150 female patients affected by precocious puberty and 90 control subjects in the Seoul area, measuring plasma values of 10 different EDCs, including BPA [ 117]. In that study, the levels of BPA of precocious pubertal girls were lower than in healthy children, unlike other compounds, such as monobutyl phthalate (MBP), that was 1.3 times higher in the affected group compared to controls.

Similar results may be extrapolated from previous works conducted on the Korean population by Lee et al. in 2009 and Han et al. in 2008. In Lee's study, 30 patients (29 girls and 1 boy) with idiopathic CPP (diagnosed on the basis of clinical and hormonal tests) were included [ 118]. Levels of serum BPA in this group were not significantly different than those obtained from 30 normal control children. Han et al. came to the same conclusion one year earlier in his study evaluating serum BPA in 100 subjects (50 with precocious puberty and 50 without) [ 119].

In 2016, Buluş et al. were the first to evaluate the BPA and peripheral precocious puberty ratio [ 120]. Among the Turkish population, they selected 42 patients with idiopathic central precocious puberty (ICPP), 42 patients with peripheral precocious puberty (PPP), and 50 healthy non-obese age-matched girls as controls. The authors did not identify any statistically significant dissimilarity between the mean values of urinary BPA in the three groups. Moreover, they obtained additional data through a questionnaire investigating the possible source of BPA exposure in daily life; the duration of breastfeeding, playing time with plastic toys, and use of creams or cosmetics did not influence the development of early puberty. As a retrospective questionnaire, the answers might be biased. In contrast, both the groups with ICPP and PPP had a higher presence of polyvinyl chloride windows in their houses compared to the control group. In addition, LH, FSH, and oestradiol serum levels were detected in all three groups, but the results were affected by the underlying disease [ 120].

A more recent study that correlated peripheral precocious puberty and urinary levels of BPA was carried out by Lee et al. [ 121]. They selected 42 patients suffering from central precocious puberty and 40 patients with peripheral precocious puberty, comparing them with a group of 37 age- and sex-matched healthy subjects through the dosage of 84 different sex steroid hormones. The study was born from the idea that the biological changes involved in early puberty may cause changes in the metabolism of human steroids. Furthermore, there may be exposure to EDCs, in particular to bisphenol A. They found that the urinary levels of BPA were slightly higher but not statistically significant in both PPP and CPP groups than in the control group. They also showed that testosterone, androstenedione, androstenediol, 16-alpha-hydroxy-dehydroepiandrosterone, and 5-alpha-androstenedione were higher in both groups with precocious puberty compared to controls. Moreover, the levels of 17-beta-estradiol were higher in subjects with CPP than in the other two groups, and many other oestrogens were slightly increased in both precocious puberty groups. Furthermore, the levels of testosterone, 17-beta-estradiol, and pregnenolone were significantly higher in patients with elevated levels of urinary BPA [ 121].

describes the main epidemiological studies investigating the relationship between BPA and precocious puberty in girls.

Table 1

Reference

Study Area

Type of Study

Study Group

Subjects' Age

Exposition Measure

Correlation with PP

Other Remarks

BPA exposition and CPP

31 girls with CPP + 30 healthy girls

6-8 years old

Serum BPA

YES

-neither correlation between levels of serum kisspeptin and BPA; nor between BPA and LH or FSH

6. Bisphenol A (BPA) and Isolated Premature Thelarche

As isolated premature thelarche may anticipate a framework of central precocious puberty and result from exposure to xenooestrogens, it is appropriate to consider studies referring to this phenomenon [ 122, 123]. It is well known that physiological breast development accompanies an upsurge in steroid hormones, chiefly oestrogens [ 124]. Limited clinical or epidemiological studies have evaluated the relationship between the exposure to EDCs and premature thelarche [ 125]. One of the latest studies investigating BPA was conducted in 2014 on 251 girls aged 4 months to 2 years [ 126]. Chen et al. selected only patients with at least stage 2 thelarche per the criteria by Tanner and without other signs of pubertal development. The results obtained showed a serum concentration of BPA significantly higher in premature thelarche girls compared to 33 healthy age- and sex-matched controls. Moreover, within the study group, there was a different distribution of BPA values based on the age of girls: the youngest girls had much higher values than the 4-year-old girls. This highlights the possible harmful effect of higher exposure to BPA in the early stages of life [ 126].

A more recent study contrasts with this result. In 2015, Ozgen et al. measured the levels of urinary BPA in 28 girls with central precocious puberty, 28 girls with premature thelarche, and 22 pre-pubertal children as controls, without identifying any significant differences between the three groups [ 127]. In their study, they also measured the serum levels of kisspeptin and found higher concentrations in the CPP group but no correlation between the values of BPA and oestradiol. The contrast with Chen's study may derive from the different ages of patients selected (girls with isolated premature thelarche aged between 5.41 and 8.12 years were analysed); in fact, the previous study found higher values of urinary BPA in the younger girls.

summarizes the main epidemiological studies investigating the relationship between BPA and isolated premature thelarche.

Table 2

7. Bisphenol A (BPA) and Male Puberty

Only 2 studies were identified belonging to this specific category. The first work was carried out by Ferguson et al. in 2014, who studied the correlation between prenatal exposure to phthalates and bisphenol A by evaluating maternal urine collected in the third month of pregnancy [ 128]. They also considered postnatal exposure by sampling the urine of children born to those mothers, with levels of sex hormones and development of secondary sexual characteristics evaluated in 113 boys aged between 8 and 14 years. Prenatal exposure to BPA did not show a clear association with any of the analysed hormones, in contrast to prenatal exposure to phthalates, which seemed to have an inverse relationship with levels of DHEAS and inhibin B and a direct relationship with SHBG (while there was no correlation with the levels of oestradiol, free testosterone, or total testosterone). With regard to exposure during childhood, BPA seemed to be positively associated with levels of SHBG and inversely correlated with total values of testosterone and free testosterone. The most interesting result, probably correlated with the anti-androgenic nature of BPA, was that prenatal exposure to BPA seemed to be associated with a reduced age of onset of adrenarche [ 128].

The second study, conducted by Wang et al. in 2016, evaluated urinary BPA in 671 Chinese boys aged between 9 and 18 years and their pubertal development [ 129]. The group consisted of young people from three stages of puberty: early puberty, late mid-puberty, and puberty (100% of 13-year-old patients enrolled in the study had begun pubertal development at the time of recruitment and had testicular volume >3 mL, Tanner stage G2). The results showed that moderate levels of urinary BPA seemed to correlate with the early appearance of pubic hair and testicular development, although this latter association was not statistically significant. On the other hand, there was a direct correlation between values of urinary BPA and the delayed appearance of genital development's final stages. According to this study, BPA exposure in boys anticipated both pubarche and adrenarche [ 129]. However, it slowed the progression of puberty, especially with regard to the maturation of genitals. Additional information offered by this study was the apparent importance of BMI as not interfering in the relationship between BPA and pubertal development, emphasizing the uncertain role of fat in male puberty in contrast to in females [ 130, 131].

shows the main epidemiological studies investigating the relationship between BPA and male precocious puberty.

Table 3

8. Critical Analysis of the Relationship between Bisphenol A (BPA) and Puberty

The first data on BPA and puberty was obtained from studies conducted on animals. A growing number of animal experiments, especially on rats, has highlighted that early puberty onset may be caused by exposure to BPA in the female population [ 132, 133, 134, 135, 136]. Other studies have published results that are inconsistent with these data, in particular those referring to BPA exposure in utero and during breastfeeding [ 137]. However, other studies on rats have shown no clear correlation with puberty early onset, even among works evaluating postnatal exposure to BPA [ 138, 139].

Among the limited number of human studies, similar equivocal results have been found. Only 7 of the 19 scientific articles considered in this review showed a correlation between BPA and pubertal development. Surveys that evaluated female patients with central precocious puberty and isolated premature breast development aged 2 months to 4 years have confirmed that early exposure to EDCs may lead to risk of developing signs of early puberty. However, a direct relationship between BPA levels and age of menarche or pubertal stage has not been found. The reason for the elevated serum or urine values of BPA in children affected by central precocious puberty has not yet been explained.

The underlying molecular mechanism is not clear: only 2 studies evaluated serum levels of kisspeptin, and both found no association with BPA levels. This might suggest that BPA action on precocious puberty plays a different role towards the activation of kisspeptin's network. In fact, even if an indirect action of BPA on puberty through interference with the GnRH axis is possible, some studies have ruled out any correlation between blood levels of BPA, FSH, LH, and oestradiol. However, the possibility of identifying elevated levels of BPA in obese or overweight girls is accepted as is the possible obesogenic effect of exposure to BPA and the close relationship between urinary BPA, body weight, and early puberty [ 101]. Another consideration can be made about Wang's study in Chinese boys, as a delay in sexual development was found. This result seems to confirm the idea that children with premature pubertal onset undergo a compensatory slowdown in the late-pubertal stage [ 140, 141].

The data obtained from surveys are conflicting, perhaps because of the lack of standardization of studies. A further explanation might be the different geographical areas where the epidemiological investigations have been carried out. The most studied populations were in America and Asia, in 5 and 13 papers, respectively. None of the American studies found an effect of BPA on puberty, whereas 7 of the 13 Asian studies found a correlation. The different exposures based on geographic location can probably explain this difference. Furthermore, different pathological expressions resulting from exposure to EDC can be suspected in various human ethnic groups [ 142]. The association between BPA and early puberty could reflect changes in personal habits, such as familiar and socio-economic conditions or variable pharmacokinetics and metabolism that are difficult to quantify. Zawatski and Lee have advanced another hypothesis for the conflicting results: EDCs show non-monotonic dose-response relationships instead of the fluctuation of hormone levels during pubertal transition; thus, measurements cannot be considered representative of the hormonal condition [ 43].

In the end, the different results of epidemiological studies may be attributed to the various methods used for the determination of BPA exposure. Regarding BPA metabolism, it has a short half-life (5-6 h) in our bodies and is almost completely eliminated after 24 h [ 143]. Regarding urinary BPA determinations, several studies have demonstrated that single spot samples of urine give an average value of prolonged exposure to such EDCs [ 144, 145]. Teitelbaum stated that single measurements of urinary phenols are fairly representative of 6-12 months of exposure in children [ 146]. Another advantage of BPA urinary determination is the easy detection (more than 90% of the general population, according to some studies) [ 147].

9. Conclusions

BPA plays a role only in female patients with precocious puberty or premature thelarche and shows no correlation with pubertal development in healthy subjects. Instead, studies on its role in male puberty are still limited and did not provide definitive data. The molecular mechanism underlying its activity as a puberty disruptor is not clear, but a potential interference with metabolism has been hypothesized. However, this is only speculation because the currently available data do not allow establishment of a clear role for EDCs, BPA in particular, on pubertal development. Moreover, the considered populations are often limited, and many final considerations are derived from cross-sectional studies. All findings related to BPA and puberty should therefore be interpreted with caution with respect to these limitations, highlighting the need for more research on the potential role of any EDCs in adverse endocrine outcomes.

Acknowledgments

This study was supported by a grant from the World Association of Infectious Diseases and Immunological Disorders (WAidid).

Author Contributions

Alberto Leonardi wrote the first draft of the manuscript; Marta Cofini, Laura Lucchetti and Clelia Cipolla performed the literature search; Donato Rigante, Laura Penta and Susanna Esposito revised the draft and made a substantial scientific contribution. All the authors read and approved the final version of the manuscript.

Conflicts of Interest

The authors declare that they have no competing interests.

References

# data[].attributes.attachment_link.title
The Effect of Bisphenol A on Puberty: A Critical Review of the Medical Literature

# data[].attributes.attachment_link.description
Many scientific studies have revealed a trend towards an earlier onset of puberty and have disclosed an increasing number of children that display precocious puberty. As an explanation, some authors have considered the global socio-economic improvement across different populations, and other authors have considered the action of endocrine disrupting chemicals (EDCs).

# data[].attributes.headline
Kaila Robles: Effects of endocrine disruptors on fetal testis development, male puberty, and transition age

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Kaila Robles: Effects of endocrine disruptors on fetal testis development, male puberty, and transition age

# data[].attributes.body
This was a review of existing literature (at the time of publishing which was around 2021 so pretty recent) to find connections between endocrine disruptors and the development of fetal male testis, puberty in males, and male transition age. They concluded that there really isnt a lot of human studies done (for ethical reasons) but there are animal studies that have been done and those do show a negative effect of endocrine disruptors on the reproductive system. They note that further long term studies on humans need to be done in order to make any solid conclusions.

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Abstract

Keywords: Endocrine disruptors, Male reproductive health, Testicular function, Testicular cancer

Introduction

According to the WHO/IPCS 2002 definition, "an Endocrine Disruptor (ED) is an exogenous substance or mixture that alters functions of the endocrine system and consequently causes adverse effects in an intact organism, or its progeny." Over the past two decades, public health has focused on the identification of environmental EDs that are able to adversely affect hormonal function [ 1]. EDs mimic naturally occurring hormones like estrogens and androgens and exert their toxicity by interfering with the normal hormonal homeostatic mechanisms that promote growth and development of tissues. EDs usually interfere with the hormonal binding to the corresponding receptor, notably the androgen receptor (AR) or the estrogen receptor. Subsequently, EDs can trigger two types of response: agonistic and/or antagonistic effect. In addition, recent discoveries in molecular biology confirmed a possible interference by several compounds with the cell cycle, the apoptotic mechanisms, and the epigenetic regulation [ 2]. Epigenetic changes are able to modify activation and expression of genes, though not altering the genetic code sequence. Changes in DNA methylation, histone modifications, and noncoding RNAs are involved. Recent studies reported a transmission of epigenetic shifts from father to child, suggesting a transgenerational inheritance [ 3, 4]. Therefore, EDs may have negative effects not only in exposed individuals, but also in their offspring and in future generations. Main EDs characteristics are summarized in Table . In addition to exposure by direct contact with these materials, EDs are also released into the environment. Therefore, exposure may occur through food and water consumption, inhalation, or dermal contact. During fetal and neonatal life it could also occur through placenta and breast feeding. As a consequence, EDs may affect human pre and postnatal development. In fact, infants can be affected already at prenatal level due to maternal exposure to EDs [ 5]. The effects of EDs on the male reproductive system are usually attributed to the interactions of these chemicals with the normal production and/or function of steroid hormones that are responsible for the masculinization of the Wolffian ducts [ 6]. In males, reproductive disorders associated with impaired fetal testis development or function vary in both phenotype and time of manifestation. Often gathered under the "testicular dysgenesis syndrome" (TDS) hypothesis [ 7], these male disorders range from hypospadias and cryptorchidism in infants [ 8- 11] to low testosterone levels, infertility, and testicular cancer (TC) in adult men [ 12- 14]. TDS has been correlated with environmental factors during fetal life [ 7].

The aim of this review is to discuss the detrimental effects of EDs exposure on fetal testis development, male puberty, and transition age, the latter defined as age 18-25 years. This definition is consistent with the recent discoveries in the field of neurophysiology, in fact a maturation of prefrontal cortex until 25 years old has been pointed out [ 15, 16]. This review aims to provide an overview of animal, in vitro, and human studies, though human evidence-based reports were given priority. In particular, andrological parameters of male health such as anogenital distance (AGD), penile length, and testicular volume (TV) were investigated.

Effects of EDs on testis development

Testis development during fetal life is crucial for male reproductive function in adulthood [ 17]. Indeed, fetal period is critical for the regular development of the testis and is known as a period of high sensitivity to many EDs [ 18]. Both functions of testis (spermatogenesis and steroidogenesis) are set up early during fetal life [ 19]. Primordial gonads appear between the 4th and the 6th week post fertilization. They are rapidly colonized by primordial germ cells that migrate from extra-embryonic areas [ 20]. Six weeks post fertilization, the differentiation of the testis is due to Sertoli cells surrounding testicular cords [ 21]. Sertoli cells are central for germ cell development [ 22] and Leydig cells differentiation [ 23]. Indeed, at 6th week post fertilization, fetal Leydig cells start producing testosterone and insulin-like factor 3 (INSL3). Both hormones are involved in testicular descent [ 24]. Furthermore, testosterone is crucial for fetal masculinization. Therefore, perturbations to the function of fetal Leydig cells can predispose to the development of male reproductive disorders including TDS and other disorders of sex development [ 25].

In the last years, literature focused on possible association between EDs and testis development. Several experimental animal studies were conducted to investigate a possible correlation, especially in mouse models. Di-2-ethylhexyl phthalate (DEHP) and di-n-butyl phthalate (DBP) are the most abundant phthalates which, after ingestion, are hydrolyzed into the active monoesters monoethylhexyl phthalate (MEHP) and mono-n-butyl phthalate (MBP), respectively. In rodents, the effects of DEHP and DBP exposure in utero have been largely described. Numerous studies reported a disruption of normal fetal testis development and the subsequent development of male reproductive disorders [ 17, 26]. It is interesting to note that in mice phthalates can induce a positive effect on testosterone secretion by the cultured fetal testis [ 27, 28]. Surprisingly, such effect was not observed in the human testis. In this regard, both organ cultures and xenograft experiments revealed no marked change in testosterone production [ 29- 31], suggesting that exposure to environmental levels of DBP and DEHP is unlikely to result in effects on fetal testosterone production in humans. Notably, although no effects of phthalate exposure have been demonstrated in human fetal testes, antiandrogenic effects occur in adult human testis following in vitro culture. The different effect of exposure could depend on the developmental stage of the testis [ 32]. However, experimental studies in rodents and human fetal tissues are consistent regarding germ cells, showing a reduction in the gonocyte number following phthalate exposure [ 30, 33]. Concerning other EDs, inconsistent results on testosterone production and germ cell development have been identified in animal studies investigating the effects of BPA exposure on fetal testis development [ 34, 35]. In the same way, inconsistent results on association between BPA and clinical indicators of reduced fetal testosterone (cryptorchidism and hypospadias) have been reported in epidemiological studies [ 36, 37]. For human testicular tissue experiments, in vitro studies indicate the potential for BPA to reduce testosterone production in the fetal testicle, whereas xenotransplantation studies failed to demonstrate similar effects [ 34, 35]. In addition, in vivo human BPA exposure might be below the concentrations used for experimental studies involving animal or human tissues [ 38]. Regarding other EDs, PCBs have been associated with abnormal urogenital development in animal models [ 39, 40]. In particular, lactational exposure seems to affect histology of rat testis in both prepuberal and puberal F1 progeny [ 41]. In rats, perfluorooctanoic acid (PFOA) does not seem to affect fetal Sertoli cells but may increase tendency of apoptosis in fetal Leydig cells [ 42]. This damage seems to affect both proliferation and differentiation of stem Leydig cells or their progeny [ 43]. Regarding perfluorooctanesulfonic acid (PFOS), it seems to damage Sertoli cells by perturbing actin cytoskeleton in primary cultures of rodent and human [ 44, 45] and may directly inhibit pubertal development of rat Leydig cells [ 46]. In humans, prenatal PFOS exposure may increase fetal steroid hormone production, although no association with cryptorchidism or hypospadias has been observed [ 47]. Anti-Müllerian hormone and INSL3 have been recently recognized as optimal markers of Sertoli and Leydig cells function, respectively, in particular during the first years of life [ 48]. However, modifications of their levels after EDs exposure have not been widely investigated in human studies. These reports may eventually provide additional insight on the pathophysiology of endocrine disruption on testis development.

Effects of EDs on timing of puberty

The term puberty means a complex of psycho-neuro-endocrine changes that occur between the end of the childhood and the achievement of the complete sexual maturity. Usually, the normal length of puberty is about 5-6 years. Puberty usually begins later in males than in females, between 9.5 and 13.5 years old (on average 11.5 years old), and the first sign is the testes volume increase (>4 mL), followed by the pubarche within 6 months. After 12-18 months, the enlargement of penis is usually observed. All the clinical modifications that occur during the puberty are the consequence of the hypothalamus-pituitary-gonad axis activation, represented by the increase of the GnRH pulsatility and therefore of FSH, LH, and gonad steroids [ 49, 50]. The literature reports several studies investigating the relationship between potential endocrine-disrupting agents and the onset of puberty in boys and girls. Many cross-sectional and longitudinal human studies have evaluated the association between pubertal timing onset and prenatal or pubertal exposure to several chemical agents with plausible endocrine interference. Most studies were referred to girls, while few were committed for males' puberty. Den Hond et al. [ 51] evaluated 80 boys who were exposed, during the pubertal period, to PCBs and dioxin. They found a negative association between the elevated serum PCBs exposure and pubertal stages, particularly the genital maturation and the pubic hair presentation. On the contrary, they did not find negative effects of dioxin. Saiyed et al. [ 52] reported an association between pubertal exposure to the pesticide endosulfan and low level of pubic hair, testis, and penis maturation, suggesting a delay in sexual maturation. A work published in 2008 involved 18 girls and 15 boys who were exposed to dioxin-contaminated breast milk. The authors observed a delayed breast development in females and a delayed age at first ejaculation in males [ 53]. Considering more recent works, Ferguson et al. longitudinally analyzed prenatal and infantile effects of phthalates and BPA on 118 boys (aged 8-14). Prenatal exposure was negatively associated to the adrenarche and pubarche onset (with high SHBG levels), whereas the infantile exposure caused low testosterone levels but no association with puberty was reported [ 54]. A similar work evaluated the impact of in utero phthalate and BPA exposure on the sexual pubertal maturation of 109 males. In particular, first- and second-trimester pregnancy exposure to DEHP was linked to increased peripubertal serum estradiol levels, whereas third-trimester exposure was associated to a delay on the onset of pubarche, with increased SHBG levels [ 55]. A longitudinal study conducted on 516 boys considered the effects of many organochlorine chemicals, lead (Pb) and non-dioxin-like PCBs. The authors evaluated EDs concentrations at the age of 8-9 years, and successively annual visits were carried out until the age of 18-19. The endure of blood EDs negatively influenced the growth during the puberty; in particular, dioxin-like compounds, organochlorine pesticides and the Pb delay puberty onset, whereas non-dioxin-like PCBs tend to advance puberty beginning [ 56]. These studies are summarized in Table .

Effects of EDs on anogenital distance

The AGD refers to the distance between the anus and the external genitalia and it is approximately twice the length in male compared to female newborns [ 57]. AGD is considered a broad biomarker capable of both retrospectively determine early life androgen disruption and predict late-life reproductive disorders in male offspring [ 57- 59]. Prenatal androgen action determines reproductive organ size and AGD, and the action can be disrupted by EDs [ 60- 62]. Further studies have identified a fetal "masculinization programming window" (MPW), a period from the second to the third month after conception when androgen action could be remarkably affected. The difference of AGD between male and female may be explained by divergent androgen secretions in this period [ 63]. This sexual dimorphism is apparent in rodents as well as humans [ 64, 65]. Reproductive parameters at birth and in adulthood appear to be influenced by androgen action in the MPW, similar to their correlation with AGD in human studies [ 58]. Therefore, a short male AGD is considered a marker of disrupted androgen action. In rodents, a short male AGD largely predicts adverse effect outcomes and it had been used for decades as a marker of impaired fetal androgen action [ 57].

Animal studies

Compounds most frequently reported to affect male AGD are phthalate esters. Many have been tested in rats, with DBP and DEHP being the most prevalent. Fetal exposure to certain phthalates results in a short AGD in rat male offspring, without any significant effect on female AGD. It is, for the most part, a dose-dependent effect, where increasing dose levels result in progressively shorter AGD, as recently reported [ 66]. In mice, newborn males exposed to phthalates during the postnatal phase showed a significant short-term reduction of AGD as a possible result of prepubertal hormonal interference [ 67], although the exact mechanism is still unknown, given the embryonal determination of AGD.

Other substances with a clear antiandrogenic action can affect AGD in rat offspring. Prenatal exposure to high doses of certain AR antagonists, such as pesticide procymidone, vinclozolin, dichlorodiphenyltrichloroethane (DDT), and the non-steroidal prostate cancer drug flutamide, reduces male pup AGD up to 50% compared to controls [ 68- 72]. After exposure to these compounds, the male offspring also displays an increased rate of nipple retention, genital malformations, and severely reduced male reproductive organ weights [ 57, 68, 73- 75]. Fetal exposure to both the antimicrobial preservative butyl paraben [ 76, 77] and the industrial plasticizer BPA [ 78] has been shown to shorten male AGD around 7-16% in the male offspring, albeit there are studies reporting no effects on AGD for both butyl paraben [ 79] and bisphenol A [ 80- 84]. PCB exposure in female rats during lactation resulted in reduced AGD in male progeny aged 60 days, even at the lowest dose tested, possibly through a reduction of circulating androgens [ 85]. Therefore, all these studies suggest that exogenous chemicals can affect AGD in pre- and peri-natally exposed animals.

Human studies

Several epidemiological studies have investigated the effects of EDs on AGD, but the results have been controversial. Most studies focused on male infants, whereas less evidence is available to support a reduction in AGD following prenatal exposure to EDs even in puberty and transition periods. In fact, fetal exposure to different EDs has been frequently associated with a short AGD in newborn boys, in particular phthalates [ 86- 90], but also PFAS [ 91], dioxins [ 92], BPA [ 93, 94], and DDT [ 95]. Notably, several studies have not found significant correlations between exposure levels and short AGD in boys, including some phthalates [ 96, 97], DDT [ 98], triclosan [ 99], PFAS [ 100], and various pesticides [ 101]. These discrepancies do not necessarily diminish the cause for concern, but rather highlight the challenges of obtaining evidence for causal relationships from human epidemiological studies [ 57]. Swan et al. [ 89] examined AGD and other genital measurements in relation to prenatal phthalate exposure in 134 newborns aged 2-36 months. The results showed that urinary concentrations of four phthalate metabolites and the summary score were inversely related to AGD. The conclusion supported the hypothesis that prenatal phthalate exposure at environmental levels may adversely affect male reproductive development in humans [ 89]. Successively, the same group confirmed these results in 2015 [ 90]; moreover, they observed the same association with reduced AGD when the daily exposures were substantially lower than current US Environmental Protection Agency (EPA) reference doses [ 102]. Suzuki et al. [ 88] examined the relationship between prenatal exposure to seven urinary phthalate ester metabolites and AGD in 111 newborns. The results showed the MEHP was negatively associated with AGD. In a Swedish cohort of 196 infants aged 21 months, Bornehag et al. [ 86] reported a reduced AGD in relation to phthalates concentration in mothers. In a more recent study, the authors investigated phthalates and BPA prenatal exposure in 198 male infants aged 6 months. Surprisingly, the results showed that both MBP and the molar sum of low molecular weight phthalates were positively associated with AGD, although no mechanism to explain this association was suggested [ 103].

Regarding puberty age, a study on 153 male children aged 0-17 years examined the effect of maternal and paternal exposure to BPA on AGD. Although the cohort included mainly children <5 years old, after correction for age the negative association between either paternal or maternal BPA exposure and AGD remained significant, although the effect was greater when considering mothers' BPA levels [ 94]. Another study on young men aged 18-23 reported a reduced AGD associated with maternal exposure to pesticides during pregnancy [ 104], suggesting that the previously reported associations between EDs prenatal exposure and neonatal and perinatal AGD in male infants can be extended also to later ages. In the same way, these results were also recently confirmed for PFAS: in 212 young men aged 18-19 years, direct exposure to these chemicals resulted in a 10% reduction of AGD [ 91]. Although PFAS were measured directly in subjects, it could be considered a proxy of prenatal exposure, given the very long half-lives of PFAS [ 105] and the long-lasting pollution in the geographic area of the exposed subjects. Interestingly, the same study reported a significant negative association between PFAS exposure and another androgen-dependent parameter, penile length [ 91]. Human studies characteristics are summarized in Table .

Effects of EDs on penile length

Penile length is a parameter that positively correlates with postnatal androgen levels [ 74]. Several studies investigated this correlation, though in newborns. To date, there is only one study that evaluated penile length in pubertal boys in association with EDs exposure: in a cohort of 55 boys aged 11-14, maternal exposure to PCBs during pregnancy was associated with reduced penile length [ 106]. As previously mentioned, Di Nisio et al. observed similar results in young men aged 18-19, suggesting exposure to EDs can result also in reduced penis size in adolescence [ 91]. On the contrary, Leijs et al. reported no correlation in 15 young boys (14-19 years of age), exposed to PCBs and dioxin-like compounds, but results were not shown [ 53].

Effects of EDs on testicular volume

Only few studies investigated the association between EDs and TV in pubertal or transition age. In most studies, TV has been evaluated only for staging puberty [ 54- 56]. In addition, studies are extremely heterogeneous, investigating different EDs, considering different primary outcomes, and using different methods for testicular sizing. In fact, TV was evaluated using Prader orchidometer [ 51, 53, 107- 109], ultrasound [ 91], both [ 110- 112], or a digital caliper [ 104]. Moreover, TV may be affected by both in utero and adult exposure [ 113, 114], with no study investigating the one and the other at the same time. As said before, TV > 4 mL is considered the first pubertal sign, while a TV > 12 mL is considered normal at the end of puberty. Therefore, TV comparison was performed after adjustment for genital stage and in most studies was made according to tertiles or quartiles of exposure, without a control group. Mol et al. [ 110] observed no significant differences in mean TV, hormonal concentration, and Tanner stage after prenatal PCB exposure in a cohort of children examined at mid puberty. Grandjean et al. [ 112] evaluated from the same birth cohort 438 adolescent boys, at age 14 years, confirming the same results. On the contrary, Den Hond et al. evaluated postnatal PCB and dioxin-like compounds exposure in 80 boys coming from three different areas of Belgium. The authors reported differences in TV between the areas, even after adjustment for genital stage. However, TV did not correlate with any of the investigated biomarkers of exposure, suggesting it may be a consequence of maternal exposure or other not explored factors [ 51]. Leijs et al. reported the same no association, but results were not shown [ 53]. Different results were observed by Cremonese et al., though they used a questionnaire to investigate occupational exposure to pesticides in young Brazilian men. Testicles were larger in rural men, in men using pesticides for more than 1 year, and also among men born to women who worked in agriculture during pregnancy [ 104]. This is the only study reporting an increase in TV. Authors suggested two possible mechanisms: fetal life exposure with prevalent androgen effect or adult exposure with inflammation of the testicles. Concerning other EDs, Vested et al. [ 107] recruited 169 young male, aged 19-21 years, whose mothers' blood had been previously collected during pregnancy. No correlation between PFOA, PFOS, and self-measured TV was observed [ 107]. Same results were recorded by Joensen et al., who investigated PFAS postnatal exposure in 247 randomly selected healthy young Danish men [ 111]. On the contrary, Di Nisio et al. reported smaller mean TV in exposed PFAS group (mean age 18.5 ± 0.8 years). Interestingly, both serum and semen PFOA, but not PFOS, were associated with reduced TV [ 91]. Regarding phthalates, Axelsson et al. investigated prenatal exposure in 112 young Swedish men, aged 17-20 years. Maternal blood samples were recovered in a biobank. The only significant result was that men in the highest tertile of prenatal mono(carboxy-isooctyl) phthalate exposure had smaller TV than men in the lowest [ 108]. On the contrary, Durmaz et al. found plasma levels of DEHP and MEHP significantly higher in 40 newly diagnosed pubertal gynecomastia cases, aged 11-15 years. However, there was no significant association between their concentration and TV [ 109]. Studies characteristics are summarized in Table .

Effects of EDs on testicular cancer

Few studies investigated a possible correlation between EDs exposure and TC. None of these was performed specifically in adolescents or in transition age. Some papers did not report the age of the studied group or all included patients were older than transition age [ 115- 119]. However, TC is the most common tumor diagnosed in men aged 14-44 years [ 120]. Most studies evaluated a possible correlation between TC and PCBs. Indeed, the International Agency for Research on Cancer (IARC) rendered PCBs as definite carcinogens in humans [ 121], while according to the US EPA PCBs cause cancer in animals and are probable human carcinogens ( http://www.epa.gov/epawaste/hazard/tsd/pcbs/pubs/effects.htm). However, results regarding TC, as well as other neoplasms, are limited and often discordant. The only study taking into consideration a subgroup of adolescents was performed by Koifman et al. An epidemiological study was carried out by comparing the total amount of pesticides sales in 1985 and health data traced in a National database. A non-statistically significant correlation was observed for TC hospitalization in both age groups (0-14, 15-49 years old) [ 122]. However, authors did not quantify either the exact amount of pesticides used or the exact area where they were used. In a similar registry-based study, Le Cornet et al. showed no evidence of an association between parental exposure to pesticides and TC [ 123]. In a cohort of US soldiers, McGlynn et al. reported an increased TC risk in patients with higher plasma levels of DDE and chlordane components [ 124]. Surprisingly, postnatal PCBs exposure was associated with decreased TC risk in the same cohort [ 125]. At the same way, Biggs et al. evaluated 246 TC patients after the first course of cancer treatment; however, only 33 were aged 18-24 years (13,4%). TC risk was similar across groups and no trend with increasing serum pesticide levels was observed [ 126]. Different results were reported by Paoli et al., who observed a statistically significant increase in TC risk in cases with detectable values of total PCB [ 127]. On the contrary, Hardell et al. investigated both prenatal and postnatal PCB exposure. In patients, only the concentration of cis-nonachlordane was significantly increased, whereas their mothers showed significantly increased concentrations of the sum of PCBs, hexachlorobenzene, trans- and cis-nonachlordane, and the sum of chlordanes [ 128, 129]. Studies characteristics are summarized in Table .

Limitations

Several studies have focused on the effects of EDs, especially on testis development; however, they used different animal models, different EDs doses, and different methodologies, with results not easily comparable among them. In humans, there are only few original papers on the effects of EDs on puberty in males, as the majority focus on female puberty. This aspect could find an explanation in the fact that females' puberty is more easily detectable, since menarche represents an undisputed sign of sexual maturation. Given the lack of homogeneity across the different studies, because of the wide spectrum of EDs and different exposure sources, age of subjects, and analytical measurements, it is difficult to infer a comprehensive conclusion on the effect of EDs on puberty and transition period. Moreover, we should consider the lack of longitudinal studies in the literature, and in most cases the quantification of EDs levels was performed only in mothers during pregnancy or only in children after birth, thus not providing a direct assessment of mother-fetus exposure levels. In other cases, the analytical measurement of chemicals is missing and it is only presumed by occupational exposure. In addition, humans are exposed to several chemicals, which could affect male health in a dose-additive manner. In fact, testicular toxicity was reported in rats exposed to a mixture of phthalates, though dosage of each was below the adverse effect threshold [ 130]. Therefore, bioaccumulation in adipose tissue of several EDs may be responsible of a "cocktail effect," a possible sum of effects with unknown outcomes that may unexpectedly appear after years of exposure to low dosages [ 131]. Moreover, as said before, recent data suggest that EDs detrimental effects may even be inherited by future generations [ 132]. All these factors may partially contribute to the heterogeneity of results reported in literature. In the same way, lifestyle factors in young adults should not be neglected. An increasing trend of health risk behaviors, such as smoking, use of illegal drugs and alcohol, has been recently reported in adolescents. All these behaviors have been associated with andrological disorders and could impair testicular development [ 133].

Conclusions

Current evidence does not clarify the impact of EDs on human male reproductive health. In animal models, severe harmful effects were observed. However, human studies have shown controversial results. This discrepancy may be due to several factors, as said above. Despite the lack of consistency in the results, overall conclusion points toward a positive association between exposure to EDs and reproductive system damage. Among the studies based on the consequences of EDs exposure on males' puberty, the main findings concern on delayed puberty, probably associated to the xeno-estrogens effects of PCBs, polychlorinated dibenzofurans, and endosulfan. In the same way, by using AGD as a proxy of fetal exposure to endocrine-disrupting chemicals, most studies agree on an antiandrogenic effect of different classes of EDs (phthalates, BPA, pesticides, PFAS), which results in reduced AGD after birth, as measured from newborns until transition period. On the contrary, the correlation between EDs and TV, as well as that with testicular cancer, is more uncertain. EDs mechanisms of damage and the adverse consequences on male puberty and andrological health are summarized in Fig. . Although the observed effects may be subtle on an individual level, the biological link between them (i.e., TDS: decreased androgen levels contributing to cryptorchidism, reduced penile length, reduced TV) should raise concern about the effects of EDs at population levels in young men. Further long-term studies performed on a wide number of subjects are necessary in order to identify damaging compounds, clarify sources of exposure, and replace them with harmless substances.

Acknowledgements

This study has been proposed and scientifically supported by the TALENT Study Group, Sapienza University of Rome, Italy. Open access funding provided by Università degli Studi di Roma La Sapienza within the CRUI-CARE Agreement.

Author contributions

All authors were involved in the design of this study and made substantial contributions to this paper. All authors critically revised and approved the final version of the paper.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Change history
1/4/2021

A Correction to this paper has been published: 10.1007/s12020-020-02581-1

References

# data[].attributes.attachment_link.title
Effects of endocrine disruptors on fetal testis development, male puberty, and transition age

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Keywords: Endocrine disruptors, Male reproductive health, Testicular function, Testicular cancer According to the WHO/IPCS 2002 definition, "an Endocrine Disruptor (ED) is an exogenous substance or mixture that alters functions of the endocrine system and consequently causes adverse effects in an intact organism, or its progeny."

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High DDT and PCB exposure during adolescence and adulthood is associated with abnormal chromosomes in sperm.

# data[].attributes.attachment_link.readable_text
Men exposed to certain banned but long-lived chemicals at high levels as teenagers are more likely to have defective sperm later in life, according a new study.

Researchers report today that organochlorine chemicals-specifically DDT and polychlorinated biphenyls (PCBs)-may affect how testicles mature and function. It is the first study to examine men's exposure to the chemicals during the teenage years and abnormal sperm later in life, and suggests that the chemicals-banned in the United States but still lingering in soil, water and people-may contribute to male infertility.

"These chemicals continue to persist in our environment. Levels are going down over the past 30 years, but all of us still have levels in our bodies," said lead author Melissa Perry, a professor and researcher at George Washington University's Milken Institute School of Public Health.

Perry and colleagues examined sperm and blood samples from 90 men from the Faroe Islands in the North Atlantic Ocean. They looked at organochlorine chemicals in their blood as adults, and checked their sperm for abnormal amounts of chromosomes. For 33 of the men, they also had blood samples at age 14.

Men with higher levels of DDE-a breakdown product of the pesticide DDT-and polychlorinated biphenyls (PCBs), which were used in transistors and electronics, at 14 years old had higher rates of abnormal sperm. The same link was found for levels of the chemicals in the men as adults, according to the study published today in Environmental Health Perspectives.

"It's another one of those studies that helps us understand why male fertility is in decline in certain areas of the world," said Thomas Zoeller, a professor and researcher at the University of Massachusetts, Amherst who was not involved in the study.

Previous research suggests sperm with an abnormal amount of chromosomes may lead to failed pregnancies and birth defects.

The study doesn't prove that the chemicals hamper sperm but both DDT and PCBs are known to disrupt the endocrine system. And sperm production is a "very hormone dependent" process, Perry said.

With many endocrine disrupting chemicals, the major risk is exposure as a fetus or young child. However, sperm production is a "continuous process" in men into adulthood, said Zoeller, who is a leading researcher on endocrine disrupting chemicals.

Organochlorine pesticides were used heavily in the U.S. from the 1940s to the 1960s. Concern over their potential to impact health was stoked in the 1960s, in large part due to Rachel Carson's 1962 book, Silent Spring, which focused on DDT's harm to birds and the world's food supply.

DDT and PCBs were banned in the U.S. in 1973 and 1977, respectively, and the International Stockholm Convention on Persistent Organic Pollutants banned their use and production in 2001, with 170 countries signing on. The bans were in response to increasing evidence of the chemicals' harm: including effects on fertility, immune systems, hormones and brain development.

However, these chemicals don't just go away. They are extremely persistent in the environment and the human body. Most people are exposed through diet, from eating food such as meat, fish and some dairy.

Also some countries still use DDT to control for disease-spreading insects. A 2012 report found that "global use of DDT has not changed substantially" since the Stockholm Convention went into effect.

"If other countries use organochlorines and we [U.S] import food from them it can be contaminated," Perry said.

While the researchers in the current study can't pinpoint exposure, people in the Faroe Islands eat a lot of seafood, such as whale meat and blubber-which act like storage containers for these persistent chemicals. The men in the study had much higher concentrations of DDE and PCBs than the U.S. average.

Human health scientists have for years suspected organochlorines may impact male fertility but studies have had mixed results. However, in a 2002 Netherlands study, researchers reported PCBs in adult mens' blood was related to decreased sperm counts. And a 2004 study of Greenland men suggested that regional differences in sperm quality may be related to organochlorine exposure.

Zoeller said Perry's study has relevance beyond the banned chemicals and should inform today's chemical regulation.

"With PCBs ... we know they're bad, but it's too late. Forty years after they're banned we're still contaminated and they're still having an impact on us," he said. "That knowledge, seems to me, should make us more cautious when we produce new chemicals, especially those very similar to these [PCBs and DDT]."
This article originally ran at Environmental Health News, a news source published by Environmental Health Sciences, a nonprofit media company.

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Brian Bienkowski, Environmental Health News

# data[].attributes.attachment_link.description
Men exposed to certain banned but long-lived chemicals at high levels as teenagers are more likely to have defective sperm later in life, according a new study. Researchers report today that organochlorine chemicals-specifically DDT and polychlorinated biphenyls (PCBs)-may affect how testicles mature and function.

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Elaborates on the environmental, chemical and other possible exposures that impact the development of children. The piece was written in 2013 and the research is primarily concentrated on the change of development within the last couple decades expanding on specific genders, race, ethnicity and age.

https://www.scientificamerican.com/article/rises-in-early-puberty-may-have-environmental-roots/

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EarthTalk®
E - The Environmental Magazine

Dear EarthTalk: Is it true that American kids are going through puberty earlier today than in previous generations, and are there any environmental causes for this? - Paul Chase, Troy, NY

Research indicates that indeed Americans girls and boys are going through puberty earlier than ever, though the reasons are unclear. Many believe our widespread exposure to synthetic chemicals is at least partly to blame, but it's hard to pinpoint exactly why our bodies react in certain ways to various environmental stimuli.

Researchers first noticed the earlier onset of puberty in the late 1990s, and recent studies confirm the mysterious public health trend. A 2012 analysis by the U.S. Centers for Disease Control and Prevention (CDC) found that American girls exposed to high levels of common household chemicals had their first periods seven months earlier than those with lower exposures. "This study adds to the growing body of scientific research that exposure to environmental chemicals may be associated with early puberty," says Danielle Buttke, a researcher at CDC and lead author on the study. Buttke found that the age when a girl has her first period (menarche) has fallen over the past century from an average of age 16-17 to age 12-13.

Earlier puberty isn't just for girls. In 2012 researchers from the American Academy of Pediatrics (AAP) surveyed data on 4,100 boys from 144 pediatric practices in 41 states and found a similar trend: American boys are reaching puberty six months to two years earlier than just a few decades ago. African-American boys are starting the earliest, at around age nine, while Caucasian and Hispanics start on average at age 10.

One culprit could be rising obesity rates. Researchers believe that puberty (at least for girls) may be triggered in part by the body building up sufficient reserves of fat tissue, signaling fitness for reproductive capabilities. Clinical pediatrician Robert Lustig of Benioff Children's Hospital in San Francisco reports that obese girls have higher levels of the hormone leptin which in and of itself can lead to early puberty while setting off a domino effect of more weight gain and faster overall physical maturation.

Some evidence suggests that "hormone disrupting" chemicals may also trigger changes prematurely. Public health advocates have been concerned, for example, about the omnipresence of Bisphenol A (BPA), a synthetic chemical in some plastics, because it is thought to "mimic" estrogen in the body and in some cases contribute to or cause health problems. BPA is being phased out of many consumer items, but hundreds of other potentially hormone disrupting chemicals are still in widespread use.

Dichlorobenzene, used in some mothballs and in solid blocks of toilet bowl and air deodorizers, is also a key suspect in triggering early puberty. It is already classified as a possible human carcinogen, and studies have linked prenatal exposure to it with low birth weight in boys. The U.S. Environmental Protection Agency (EPA) has recently made screening Dichlorobenzene for hormonal effects a priority.

Parents can take steps to reduce our kids' so-called "toxic burden": Buy organic produce, hormone- and antibiotic-free meat and dairy and all-natural household cleaners. And keep the dialogue going about healthy food and lifestyle habits so kids learn how to make responsible, healthy choices for themselves.

CONTACTS: CDC, www.cdc.gov; AAP, www.aap.org.

EarthTalk® is written and edited by Roddy Scheer and Doug Moss and is a registered trademark of E - The Environmental Magazine (www.emagazine.com). Send questions to: earthtalk@emagazine.com. Subscribe: www.emagazine.com/subscribe. Free Trial Issue: www.emagazine.com/trial.

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centers for disease control and prevention

# data[].attributes.attachment_link.title
Rises in Early Puberty May Have Environmental Roots

# data[].attributes.attachment_link.description
EarthTalk® E - The Environmental Magazine Dear EarthTalk : Is it true that American kids are going through puberty earlier today than in previous generations, and are there any environmental causes for this? - Paul Chase, Troy, NY Research indicates that indeed Americans girls and boys are going through puberty earlier than ever, though the reasons are unclear.

# data[].attributes.headline
Jon Peterson: Prenatal exposure to mixtures of persistent endocrine disrupting chemicals and early menarche in a population-based cohort of British girls

# data[].attributes.subject
Jon Peterson: Prenatal exposure to mixtures of persistent endocrine disrupting chemicals and early menarche in a population-based cohort of British girls

# data[].attributes.headline
Erin Templeton: BPA can disrupt hypothalamus signaling causing an early onset of puberty in female rats.  https://doi.org/10.1095/biolreprod.112.100826

# data[].attributes.subject
Erin Templeton: BPA can disrupt hypothalamus signaling causing an early onset of puberty in female rats.  https://doi.org/10.1095/biolreprod.112.100826

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Abstract

Introduction

Materials and Methods

Animals

A transgenic strain of enhanced green fluorescent protein (EGFP)-GnRH Wistar rats was used. These animals, which express EGFP driven by the GnRH promoter were generated and previously characterized by Fujioka et al. [ 62] and Kato et al. [ 63] from Nippon Medical School, Tokyo, Japan. Our colony was established from a kind gift from founder Susan Smith at the Oregon National Primate Research Center, Beaverton, OR [ 64]. Animal care and maintenance were conducted in accordance with the applicable portions of the Animal Welfare Act and the U.S. Department of Health and Human Services publication Guide for the Care and Use of Laboratory Animals and was approved by the North Carolina State University (NCSU) Institutional Animal Care and Use Committee. All litters were born to animals bred in-house. The sire was removed on the day of birth (defined as Postnatal Day zero [PND 0]). Dams (n = 17) were maintained on a 12L:12D light cycle (lights on from 700 to 1900 h) at 23°C and 50% average relative humidity at the Biological Resource Facility at NCSU and maintained on a semipurified, phytoestrogen-free diet ad libitum for the duration of the study (product no. AIN-93G; Test Diet) in thoroughly washed polysulfone caging with glass water bottles (rubber stoppers and metal sippers) and woodchip bedding to minimize exposure to exogenous EDCs [ 65]. On PND 21, all pups were weaned into same-sex littermate groups of up to 4 and housed under the same conditions as the dams.

Neonatal Exposure and Tissue Collection

All compounds were dissolved in 100% ethanol (EtOH; Pharmaco) and then sesame oil (Sigma) at a 10% EtOH:90% oil ratio as we have done previously [ 2, 48]. Starting on the day of birth, we injected pups s.c. with 0.05 ml of vehicle (oil), 10 μg of 17β-estradiol (E2; Sigma), and 50 μg/kg body weight (bw) BPA (low BPA; Sigma) or 50 mg/kg bw BPA (high BPA). The high dose corresponds to the lowest observed adverse effect level for chronic oral exposure, and the low dose corresponds to the ''safe,'' or reference dose, for human oral exposure. Injections were administered every 24 h for 4 days (PND 0-3). E2 was used as a positive control, at a dose sufficient to induce hypothalamic masculinization, as we have done previously [ 66]. Although we recognize that injection does not model a typical human oral exposure, this route was selected for the purposes of these largely mechanistic studies to ensure that every animal received an identical dose. Injection likely results in a higher internal dose than oral exposure [ 67], although at least one study has shown that this difference is not significant in neonatal mice [ 68]. Based on that previous work, we estimated that the average plasma concentration over a 24-h period would be approximately 0.3 ng/ml for the low BPA group and 330 ng/ml for the high BPA group [ 68].

Female pups were euthanized on PNDs 17, 21, 24, 28, or 33 and males on PND 21 or 33 at between 0930 and 1400 h by transcardial perfusion as described previously [ 48]. To experimentally control for potential litter effects, we used no more than two pups per litter in each age group. Therefore, within each age group, pups came from a minimum of three dams. At the time they were euthanized, females were weighed and checked for vaginal opening, a hallmark of pubertal onset in the rat (occurring on PND 32-36 in unexposed animals) [ 69]. Vaginal lavage confirmed that none of the animals had started their estrous cycle. Plasma was extracted from trunk blood by centrifugation and stored at −80°C.

Immunohistochemistry

Perfused brains were sliced into 35-μm coronal sections and stored free floating in antifreeze (20% glycerol, 30% ethylene glycol in potassium phosphate buffer solution) at −20°C [ 70]. Two sets of hypothalamic sections, one containing the organum vasculosum of the lamina terminalis (OVLT) through the caudal border of the medial preoptic area (MPOA), and the other consisting of the rostral borders of the ARC were immunolabeled for Kp, using immunohistochemistry techniques detailed previously [ 43, 48].

Two more sets of sections (from PND 28 and PND 33 animals only), one containing the OVLT through the caudal border of the MPOA and the other comprising the rostral-to-caudal borders of the DMN, were immunolabeled for RFRP3, using immunohistochemistry techniques similar to those used for Kp labeling. RFRP3 was detected using a rabbit-derived GnIH antibody (1:4000 dilution), a generous gift from Kazuyoshi Tsutsui, Waseda University [ 26, 71]. Limited quantities of primary antibody required us to restrict our analysis of RFRP3-ir to two time points. We chose PND 28 and 33 because they most closely corresponded to time points at which the low BPA group and the controls underwent vaginal opening, respectively.

Quantification of Kp-ir Fiber Density in the AVPV and ARC

The tissue was visualized with a Leica model TCS SPE confocal microscope using a 40× corrective objective lens. Image stacks (z-step distance of 1 μm) were analyzed using Image J software (National Institutes of Health, Bethesda, MD), as detailed previously [ 43, 73], to obtain the average number of immunoreactive voxels within the region of interest. The quantification process was completed independently by two people, both blind to the exposure groups. The values obtained by each were then averaged to create the data set used for analysis [ 43, 73].

Quantification of RFRP3-ir Perikarya and Fiber Density

To quantify RFRP3-ir perikarya, 1-2 sections containing the DMN were selected from PND 28 females and PND 33 males and females. A set of serial image planes (z-step distance of 3.5 μm) was collected with confocal microscopy at a magnification of 40×. Image stacks were visualized using ImageJ software, and the number of RFRP3-ir perikarya were manually counted by two people, and results were averaged across observers. RFRP3-ir fibers were quantified in PND 28 females, and PND 33 males and females, using the same methods applied for Kp-ir fiber density (detailed above). For this analysis, 2-4 sections containing the OVLT were selected based on the presence of EGFP-positive GnRH neurons in the area of interest.

Quantification of Putative RFamide Contacts on GnRH Neurons

Putative contacts on OVLT GnRH neurons by Kp-ir fibers (PND 33 animals) and RFRP3-ir fibers (PND 28 and 33 animals) were evaluated using confocal microscopy. For every animal, 5-6 GnRH neurons were scanned at ×40 magnification, using a 2.5× digital zoom and a 1.5-μm-step distance, and were assessed for close appositions of RFamide fibers. The percentage of GnRH neurons with contacts was calculated for every group and recorded.

Assessment of Serum Hormone Levels

Serum luteinizing hormone (LH) levels for PND 28 and 33 females (n = 4-7 per group) were assessed in collaboration with Jerome Goldman and Ashley Murr at the U.S. Environmental Protection Agency. LH levels were determined using iodination preparation I-10, reference preparation RP-3, and antisera S-11, all supplied by the National Institute of Diabetes and Digestive and Kidney Diseases. Iodination materials were radiolabeled with iodine-125 ( ¹²⁵I; New England Nuclear), using the chloramine-T method [ 74], and labeled hormone was separated from unreacted iodide by using a P-60 gel column. Assays were carried out according to recommendations provided in the kit, with an increase in sensitivity due to a 24-h incubation of sample and first antibody prior to the addition of ¹²⁵I-labeled tracer. Goat anti-rabbit gamma globulin (Calbiochem) was used as the second antibody [ 75]. Assay sensitivity was 0.115 ng/tube, and all samples were run in duplicate with an intra-assay coefficient of variation of 7.75%. Serum levels of total E2 for PND 17, 21, 24, 28, and 33 females (n = 4-6) were quantified in-house by radioimmunoassay using the Coat-A-Count estradiol kit (Siemens). All samples were run in duplicate, and the intra-assay coefficient of variation was 6.50%. An insufficient volume of blood was available from PND 28 vehicle control females, so they were not included in the analysis.

Statistical Analysis

Statistical analysis followed established guidelines for low-dose endocrine disruptor exposure [ 76]. Generally, AVOVA was used to compare effects between groups of females, and t-tests were used to explore sex differences. Bodyweight, estrogen levels, and Kp-ir fiber densities in the AVPV and ARC were analyzed first by three-way ANOVA with litter, age, and exposure as factors. No main effects of litter nor any significant interactions with litter were identified, hence, data were subsequently analyzed by two-way ANOVA with age and exposure as factors. This was followed with a one-way ANOVA for each age examined and Fisher least significant difference (LSD) post hoc test when ANOVA was significant. On PND 21 and 33, a t-test comparing males to control females was conducted to confirm the expected sex difference. RFRP3 fiber density was analyzed at PND 28 and PND 33 by one-way ANOVA (for PND 33, males were included because it was not known whether a sex difference existed) and then followed with a Fisher LSD post hoc test. LH levels were analyzed by one-way ANOVA for each age group, as only two ages were examined. Percentages of GnRH neurons with Kp contacts, RFRP3 cell numbers, and percentages of GnRH neurons with RFRP3 contacts were analyzed with a hypothesis-driven (based on fiber density results) one-tailed Dunnett test [ 76]. We hypothesized that Kp contacts on GnRH neurons would be upregulated, while RFRP3 cell numbers and contacts on GnRH neurons would be downregulated.

Results

Vaginal Opening, Body Weight, and Circulating Hormone Levels

Peripubertal Kp-ir Fiber Density and GnRH Contacts

Main effects of exposure and age on female AVPV Kp-ir fiber density were identified (F[ 3,103] = 6.403, P ≤ 0.001; and F[ 4,103] = 2.565, P ≤ 0.04, respectively). As anticipated, Kp-ir fiber density increased with age in the vehicle control group, becoming sexually dimorphic by PND 33 ( Fig. 2, A and G) [ 42, 45, 48]. Kp-ir fiber density in the E2-exposed animals, however, remained relatively low and flat and thus more reminiscent of the male pattern ( Fig. 2 G). No significant effect of BPA exposure was detected.

There was a significant effect of exposure (F[ 3, 113] = 14.977; P ≤ 0.001) but not age, on Kp-ir fiber density in the ARC ( Fig. 2, D and H). Consistent with our what we have reported previously [ 48], Kp-ir fiber density in the ARC was sexually dimorphic ( P ≤ 0.001), with females having a denser plexus ( Fig. 2H). A significant effect of exposure was present on all 5 days examined ( P ≤ 0.05). E2-exposed females had fewer Kp-ir fibers than vehicle controls ( P ≤ 0.05 on all days except PND 24, where P ≤ 0.07), with levels remaining relatively low and flat across peripuberty ( Fig. 2H) and statistically indistinct from male levels. No significant effect of low BPA exposure was observed. By PND 33, high BPA-exposed females had Kp-ir levels in between those of control females ( P ≤ 0.05) and males ( P ≤ 0.03) ( Fig. 2H). Prior work from our laboratory demonstrated that this effect persists into adulthood [ 43].

Kp appositions on GnRH neurons ( Fig. 2I) were readily distinguishable from situations where the two were in close proximity but not actually in contact ( Fig. 2J). As expected, the percentage of GnRH neurons in close apposition to Kp-ir fibers was sexually dimorphic on PND 33, with males having fewer contacts than females ( P ≤ 0.02) [ 42]. Results of the Dunnett test comparing control females to exposed females revealed no significant effect of exposure ( Fig. 2 K), demonstrating that this aspect of the Kp/GnRH signaling pathway is resistant to disruption by BPA but also by E2.

RFRP3 Fiber Density, Cell Numbers, and GnRH Contacts

In females, a main effect of exposure on RFRP3 fiber density was detected on PNDs 28 and 33 (F[ 3, 17] = 4.976, P ≤ 0.02; and F[ 4, 16] = 3.447, P ≤ 0.03, respectively) ( Fig 3, A-C) with low BPA and E2-exposed animals having significantly fewer fibers than controls ( P ≤ 0.05). Similarly, low BPA-exposed animals had significantly fewer RFRP3-ir cells on PNDs 28 and 33 ( P ≤ 0.001 and P ≤ 0.05, respectively) ( Fig. 3, D-F) than unexposed controls. On PND 28, a main effect of exposure on the percentage of GnRH with putative RFRP3 contacts was detected (F[ 3, 15] = 7.464, P ≤ 0.003) ( Fig. 3, G and H), but this did not persist to PND 33 ( Fig. 3I). The percenage of GnRH neurons with RFRP3 appositions on PND 28 was significantly lower in the low BPA and E2-exposed animals than the control females ( P ≤ 0.03 and P ≤ 0.01, respectively). None of these features was sexually dimorphic in peripuberty ( Fig. 3, C, F, and I).

Discussion

Advanced vaginal opening and compromised estrous cyclicity following estrogen or low-dose perinatal BPA exposure has been reported numerously by our group and others [ 1- 3, 77- 79], but the mechanisms underlying these effects remain elusive. Collectively, our data indicate that accelerated female pubertal onset following neonatal exposure to low dose BPA may result from decreased inhibition of GnRH activity by RFRP3 neurons. The alternative hypothesis stating that neonatal BPA exposure accelerates the ontogeny of Kp projections to GnRH neurons was not supported by the data. Instead, evidence for masculinization at the high dose of BPA was observed in the ARC, an effect which may contribute to irregular or persistent estrus. These data provide evidence that low dose BPA can alter the organizational tempo and sexual differentiation of RFamide pathways in the female rat and suggest a novel mechanism by which the timing of pubertal onset and female reproductive physiology may be altered by early life exposure to EDCs.

Although neonatal exposure to low BPA resulted in fewer RFRP3 cell numbers on PND 28 and 33, neonatal E2 exposure did not. One possible explanation is that vaginal opening occurred a few days earlier in the E2-exposed group, and thus appreciable differences in cell numbers occurred at a younger age. Prior studies have found conflicting effects of E2, depending on the timing and context of administration. For example, E2 has been shown to downregulate prepro-RFRP mRNA expression in ovariectomized adult mice [ 85], but a recent RT-PCR study revealed that peripubertal E2 administration increases RFRP3 mRNA levels within the whole hypothalamus of female rats [ 56]. We surmise that the sensitivity of RFRP3 neurons to E2 and, by extension, EDCs depends on dose, exposure window, and duration of exposure. This differential sensitivity could underlie the biphasic effect of BPA on RFRP3 cell numbers and the density of OVLT efferents. An alternative possibility is that the BPA effect is not classically "estrogenic" and that two different mechanisms drive early vaginal opening in BPA and E2-exposed females. In addition to a change in cell numbers, we also found that neonatal low BPA exposure resulted in a decreased percentage of GnRH neurons with RFRP3 appositions on PND 28, a difference that was recapitulated by E2 exposure. By PND 33, this difference was no longer statistically significant, suggesting that it is a transient effect accompanying vaginal opening. These results could reflect a lower density of RFRP3 projections to GnRH neurons or reduced protein content within these projections at the time of vaginal opening. Collectively, these data reveal a novel mechanism by which EDC exposure may modulate the tempo of reproductive development.

Information about the neuroanatomical organization of female peripubertal RFRP3 signaling pathways is sparse but rapidly emerging, and the observations reported here are consistent with those of prior work revealing the fact that these pathways are dynamic and hormone sensitive but not sexually dimorphic in peripuberty [ 57]. A recent study reported that in female rats, hypothalamic RFRP3 expression increased with age but remained relatively flat between PNDs 28 and 35 [ 56]. Similarly, hypothalamic expression levels of the putative RFRP3 receptor GPR147 also increased as puberty approached and then declined. Because these assessments were made using the whole hypothalamus, it is unclear whether a similar expression pattern occurs specifically in GnRH neurons. In the present study, RFRP3 fiber density in the vicinity of GnRH neurons rose with age, an effect that is consistent with RFRP3 expression data. A notable caveat, however, is that the fiber density data obtained from the two time points cannot be statistically compared because they were not processed simultaneously.

Finally, it has been suggested that increased bw could be a contributing factor for early puberty in humans and rodents [ 1, 93, 94], but it does not appear to have played a significant role here. Only the high dose of BPA resulted in significantly increased bw, and this effect did not manifest until PND 33. While this suggests that increased bw is not driving advanced vaginal opening, it could indicate that energy balance is disrupted. It has previously been shown that RFamides, including Kp and RFRP3, play an important role in energy balance and metabolism [ 58, 59]. In addition to the effects of RFamides on GnRH release, they also have opposing effects on pro-opiomelanocortin (POMC) cells, with Kp being stimulatory and RFRP3 being inhibitory on these anorexigenic neurons [ 95]. Therefore, disruption of these signaling pathways could be a contributing factor for the observed changes in energy balance and bw following EDC exposure.

Overall, data indicate that early vaginal opening following neonatal low dose BPA exposure results from the premature release of GnRH neurons from RFRP3 inhibition rather than from premature stimulation from Kp neurons. Considering that the RFamide system is highly conserved across species [ 96], these findings may give further insight into the neuroendocrine systems that coordinate pubertal timing and maintenance of normal estrous cyclicity in all mammalian species, including humans. Numerous xenoestrogens have been reported to affect pubertal timing [ 65, 97- 99]. Understanding the mechanisms by which these compounds can alter the tempo of pubertal development is fundamental for making predications about how exposure to these compounds may impact human reproductive health.

Acknowledgment

The authors thank Barbara Welker, Kay Coole, and Linda Hester for their assistance with animal husbandry. We also appreciate the help of Linwood Joyner, an undergraduate student and NCSU Park Scholar, with cell counting. We gratefully acknowledge Susan Smith at Oregon Health and Science University for providing us with EGFP-positive GnRH rats to start our colony and Jerome Goldman and Ashley Murr at the U.S. Environmental Protection Agency (Research Triangle Park, NC) for analyzing serum LH levels. Thanks also to Emily Sluzas for critical reading of the manuscript.

References

Author notes

# data[].attributes.attachment_link.title
Disrupted Organization of RFamide Pathways in the Hypothalamus Is Associated with Advanced Puberty in Female Rats Neonatally Exposed to Bisphenol A1

# data[].attributes.attachment_link.description
A transgenic strain of enhanced green fluorescent protein (EGFP)-GnRH Wistar rats was used. These animals, which express EGFP driven by the GnRH promoter were generated and previously characterized by Fujioka et al. [ 62] and Kato et al. [ 63] from Nippon Medical School, Tokyo, Japan.

# data[].attributes.headline
Addison Thompson: Pre-pubescent and early Tanner stage exposure to some phenols and phthalates could contribute to the early onset of menarche in Latinas of global south.

# data[].attributes.subject
Addison Thompson: Pre-pubescent and early Tanner stage exposure to some phenols and phthalates could contribute to the early onset of menarche in Latinas of global south.

# data[].attributes.attachment_link.readable_text
Environmental Health 17, Article number: 32 (2018) Cite this article

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Abstract

Background

The age of menarche has been associated with metabolic and cardiovascular disease, as well as cancer risk. The decline in menarcheal age over the past century may be partially attributable to increased exposure to endocrine disrupting chemicals (EDCs).

Methods

We assessed the influence of 26 phenol and phthalate biomarkers on the timing of menarche in a longitudinal cohort of Chilean girls. These EDCs were quantified in urine collected prior to the onset of breast development (Tanner 1; B1), and during adolescence (Tanner 4; B4). Multivariable accelerated failure time (AFT) models were used to analyze associations between biomarker concentrations and the age of menarche adjusting for body mass index (BMI) Z-score and maternal education, accounting for within-subject correlation.

Results

Several biomarkers were significantly associated with the age at menarche; however, these associations were dependent on the timing of biomarker assessment. A log(ng/ml) increase in B1 concentrations of di(2-ethylhexyl) phthalate biomarkers was associated with later menarche (hazard ratio (HR): 0.77; 95% CI: 0.60, 0.98), whereas higher B1 concentrations of 2,5-dichlorophenol and benzophenone-3 were associated with earlier menarche (HR: 1.13; 95% CI: 1.01, 1.27; HR: 1.17; 95% CI: 1.06, 1.29, respectively). Elevated B4 concentrations of monomethyl phthalate were similarly associated with earlier menarche (HR: 1.30; 95% CI: 1.10, 1.53). The impact of monoethyl phthalate and triclosan concentrations on pubertal timing were significantly modified by BMI Z-score. Higher monoethyl phthalate and triclosan concentrations were associated with earlier menarche among overweight or obese girls, but not among those that were normal weight.

Conclusions

This study identifies modulation of sexual maturation by specific EDC biomarkers in Latina girls.

Peer Review reports

Background

Determinants of pubertal timing are of public health concern because of the association between the age of onset and adult disease susceptibility. Among females, early menarche has been associated with an increased risk of type 2 diabetes, cardiovascular disease, and reproductive cancers, including breast [ 1, 2, 3]. The average age of menarche has decreased from 16-17 years at the end of the nineteenth century, to less than 13 years across Europe and the United States, likely due to improvements in nutrition and general health [ 4, 5]. While the age of menarche was thought to have stabilized in the past 50 years, more recent studies have suggested that the age of menarche has continued to decline in South America, the United States, and many countries in Europe [ 6, 7, 8, 9]. The secular trend towards earlier menarche has been observed across race/ethnicity groups, despite differences in timing [ 7, 10, 11, 12]. Within the United States, the average age of menarche is earlier among Hispanic and black females relative to white females, adjusting for BMI, as well as social and economic indicators [ 7, 13]. Across Europe and South America, the fastest decline has been among impoverished girls, in spite of earlier menarche being previously more common among affluent families [ 6, 8, 9, 14, 15].

The two primary hypotheses for this shift towards earlier menarche are the growing childhood obesity epidemic and increasing exposure to endocrine disruptor chemicals (EDCs) [ 16, 17, 18]. EDCs can influence the endocrine system through a multitude of mechanisms, including competitive binding to hormone receptors to modulation of hormone synthesis and metabolism [ 19]. There is building evidence that EDCs can affect proper function across a number of different tissues, including breast tissue and female reproductive organs [ 19], as well as influencing the neuroendocrine control of reproduction. Results of both in vivo studies and a few longitudinal human cohorts suggest some of these compounds may be obesogenic, with early life exposure associated with childhood body size [ 20, 21, 22, 23]. Phenols and phthalates are two classes of EDCs of particular concern due to their use in an extensive array of products, including plastics, building materials, personal care products, insecticides, and herbicides [ 24]. In this study, we analyzed the association between pre-pubescent and pubescent biomarkers of exposure to select phenols and phthalates and the timing of menarche within a longitudinal cohort of Chilean girls born in 2002-2003. Two cross-sectional studies and four longitudinal studies have analyzed the influence of childhood EDC exposure on the age of menarche [ 25, 26, 27, 28, 29, 30]. The results of these studies have been largely discordant, potentially reflecting regional and socioeconomic influences on exposure profile, differences in the age of biomarker measurement, and possible effect modification by race/ethnicity. For this study, we quantified a broad panel of 26 phenol and phthalate urinary biomarker concentrations prior to the onset of breast development (Tanner 1; B1) and at Tanner 4 (B4). This study provides unique insight into the influence of these compounds on pubertal timing in Latina girls.

Methods

Study population

Our study population was a random subset of 200 girls part of the longitudinal Growth and Obesity Cohort Study (GOCS) with urine samples collected at B1 (ages 6.7 to 9.6 years; median age: 7.9 years) and B4 (ages 9.4 to 13.1 years; median age: 11.2 years). The GOCS children were born in 2002-2003, and are representative of the low to middle-income families in Santiago, Chile. A description of the cohort has been provided elsewhere [ 31]. Starting in 2009, breast development was assessed by two trained dietitians (kappa with pediatric endocrinologist = 0.85) by visual inspection using Tanner's rating scale approximately every 6 months [ 32]. Palpation was additionally used to distinguish breast Tanner 1 from Tanner 2. Sex and age-adjusted BMI Z-score at each visit were calculated based on the Centers for Disease Control and Prevention (CDC) growth charts. Girls were categorized as having excess weight (overweight or obese) if their BMI Z-score was equal or above the 85th percentile. Informed consent was obtained from all parents or guardians of children before the start of data collection.

Biomarker measurements

Fasting spot urine samples were collected between 10 AM and 12 PM in polypropylene sterile cups, and were immediately vortexed and aliquoted. Concentrations of 26 phenol and phthalate biomarkers were quantified in urines collected at breast Tanner 1 (B1) and Tanner 4 (B4) from 200 girls (400 samples). The analytical measurements were performed at the CDC National Center for Environmental Health Laboratory using previously described analytical methods [ 33, 34]. Concentrations below the limit of detection (LOD) were given an imputed value equal to LOD/sqrt(2) [ 35]. Biomarker concentrations (ng/ml) were corrected for specific gravity. Dilution adjustment was performed using the formula P c = P[(1.015-1)/(SG-1)], where P c is the specific gravity-corrected biomarker concentration, P is the observed biomarker concentration, SG is the specific gravity of the urine sample, and 1.015 is the median SG of the study population [ 36, 37]. To calculate the summation of di(2-ethylhexyl) phthalate (DEHP) metabolites (⅀DEHP), concentrations of MEHP, MEOHP, MEHHP, and MECPP were converted to nmol/L before being added together. The analysis of blinded specimens by the CDC laboratory was determined not to constitute engagement in human subjects' research.

Age of menarche

Prior to the onset of B4, girls were asked to report the date of their first menstrual bleeding at each 6-month visit. After achieving B4, girls were contacted by study dieticians every 3 months to survey whether the girl had reached menarche. During this phone interview, a questionnaire was used to differentiate menarche from other potential causes of vaginal bleeding, such as vaginal infection, urinary infection or trauma. Longitudinal follow-up of participants enabled the confirmation of menarche onset.

Statistical methods

Multivariable accelerated failure time (AFT) models were used to assess the influence of individual phenol and phthalate biomarker concentrations on time to menarche, assuming a Weibull distribution. To account for possible confounding by body size, models were adjusted for BMI Z-score at EDC measurement [ 16, 17, 18, 38, 39]. Models were additionally adjusted for maternal education as an indicator of socioeconomic status, which is related to both exposure profile and age of menarche [ 6, 8, 9, 14, 15, 40]. Sensitivity analyses were conducted further adjusting for the mother's age of menarche (recall; ≤11, 12, 13,≥14 years), which may capture confounding by transgenerational exposures correlated with socioeconomic status. These analyses were restricted to the subset of the families that reported mother's age of menarche ( N = 181), assuming recall data was missing completely at random. A cluster statement was used to account for within-subject correlation between B1 and B4 biomarker measurements. Accordingly, inference was based on robust standard errors estimated using the Huber sandwich estimator. For incident cases, survival time was the age at menarche, estimated based on time between the self-reported date of first menses and date of birth. Survival time for right censored individuals was the age at last clinic visit, based on the time between date of last visit and date of birth. Time-varying associations between biomarker concentration (log(ng/ml)) and the age of menarche were investigated by adding Tanner stage as well as an interaction between biomarker concentrations and Tanner stage to our models. Association estimates from the AFT models were reported as the relative change in hazard of menarche (hazard ratio; HR) associated with a log(ng/ml) increase in biomarker concentration, which generally corresponded to a change from the lowest concentration quartile to the highest quartile. If the interaction between Tanner stage and concentration on timing of menarche was significant (Wald test; p < 0.05), the reported associations were stratified by Tanner stage. Similarly, a Wald test was used to assess effect modification by BMI Z-score. If the interaction term significantly improved model fit (p < 0.05), the influence of the biomarker on the timing of menarche was reported separately for overweight/obese girls and normal weight girls. To identify potential non-monotonic dose-response relationships, we additionally modeled tertiles of biomarker concentration, with tertile cut-points stratified by Tanner stage. Significant trends across categories were evaluated by modeling the log(median) concentration within tertiles as a continuous variable. To visualize the change in the timing of menarche, we computed median age at menarche for tertiles of urinary concentrations using the baseline survivor function of multivariable adjusted AFT models. All statistical analysis was performed in R Version 3.3.1 and figures were generated using ggplot2 [ 41].

Results

The influence of EDC exposure across childhood and adolescence on the age of menarche was assessed among 200 Chilean girls born in 2002-2003. The median age at urine collection prior to the onset of breast development (B1) was 7.9 years and 11.2 years at Tanner 4 (Table 1). Proportion of samples with measurements above the limit of detection, as well as the geometric mean (95% CI) biomarker concentrations at B1 and B4 are reported in Additional file 1: Tables S1a and S1b. Analysis was restricted to the subset of 21 biomarkers with detectable concentrations at Tanner 1 and Tanner 4 in at least 75% of the girls. These 21 biomarkers included: 2,4-dichlorophenol, 2,5-dichlorophenol, benzophenone-3, BPA, MBP, MBzP, MCNP, MCOP, MCPP, MECPP, MEHHP, MEHP, MEOHP, MEP, methyl paraben, MHBP, MHiBP, MiBP, MMP, propyl paraben, triclosan; excluded were: MNP, B-PB, BPF, BPS, and E-PB (see Abbreviation List; Additional file 1: Table S1a and S1b). For reference, geometric mean (95% CI) phenol and phthalate creatine-adjusted urinary biomarker concentrations among U.S. females of a similar age range from the 2011-2012 National Health and Nutrition Examination Survey are reported in Additional file 1: Table S2. In both the U.S. and Chilean populations, most biomarker concentrations decreased with age (Additional file 1: Table S1a and S1b; Additional file 1: Table S2). The intra-individual correlation between phenol and phthalate biomarkers at B1 and B4 was moderate to low (Additional file 1: Table S3). The mean age of menarche was 12.0 years; the median age was similarly 12.0 years (95% CI: 11.9-12.2 years). At the time of analysis, 18 girls had not reached menarche or had dropped out of the cohort. Of those that achieved menarche during the course of the study, 10.5% ( n = 21) reached menarche prior to the first detection of B4. The occurrence of menarche prior to B4 did not significantly modify the linear association between any biomarkers and the age of menarche in adjusted models. Therefore, B4 biomarker measurements for these 21 girls were included in all subsequent models. Biomarker concentrations at B1 were not significantly correlated with BMI Z-score. Similarly, most B4 biomarker concentrations were not correlated with B4 BMI Z-score, with the exception of monoethyl phthalate (MEP) and 2,5-dichlorophenol, which had moderate positive associations with BMI Z-score (ρ = 0.21 and ρ = 0.22, respectively). Additional study population characteristics are outlined in Table 1.

Table 1 Study population characteristics

We did not detect a significant linear impact of average concentration across Tanner stages on the age of menarche for any biomarker (Table 2). However, the association between urinary biomarker concentrations and the timing of menarche was significantly modified by the Tanner stage at biomarker measurement for 2,5-dichlorophenol, benzophenone-3, monomethyl phthalate (MMP), and all of the metabolites of DEHP after adjusting for BMI Z-score and maternal education (Table 2). Further adjustment for mother's age of menarche (≤11, 12, 13, ≥14 years) did not change the shape or strength of the associations between EDC biomarker levels and daughter's age of menarche (Additional file 1: Tables S4 and S5). For ease of interpretation, and to identify potential non-linear influences, we evaluated the association between tertiles of each biomarker and the timing of menarche in adjusted models (Table 3; Fig. 1; T1=lowest tertile, T2=middle tertile, T3=highest tertile). Among the subset of biomarkers for which we did not detect significant effect modification by Tanner stage at measurement, we found a non-monotonic change in the timing of menarche between tertiles of monobenzyl phthalate (MBzP) across puberty (Table 3; Fig. 1). While there was a later age of menarche among the second tertile of MBzP relative to the first (HR: 0.70; 95% CI: 0.54, 0.93), the highest tertile was not significantly different from the lowest (Table 3; Fig. 1).

Table 2 Menarche hazard ratio (95% CI) associated with log(ng/ml) increase in each EDC biomarker across puberty

Median menarcheal age (95% CI) between tertiles of EDC biomarker concentrations across Tanner stages. Significant difference ( p  < 0.05) between: tertiles 1 and 2 indicated by A, tertiles 2 and 3 indicated by B, and tertiles 1 and 3 indicated by C

Increased B1 concentrations of 2,5-dichlorophenol and benzophenone-3 were associated with decreased time to menarche (Table 4). In contrast, higher B1 concentrations of the DEHP biomarkers mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), and mono(2-ethylhexyl) phthalate (MEHP) were associated with later menarche (Table 4). Girls in the highest tertile of B1 2,5-dichlorophenol concentration had an earlier age of menarche compared to those in the lowest tertile (Table 5; HR: 1.58; 95% CI: 1.09, 2.29), corresponding to 4.09 months earlier median age of menarche (Fig. 2). The influence of benzophenone-3 and the DEHP metabolites on the relative time to menarche was less linear (Table 5; Fig. 2). The median age of menarche was 4.10 months earlier among girls in the second tertile of B1 benzophenone-3 concentration compared to the lowest tertile (HR: 1.58; 95% CI: 1.12, 2.22), but the change in timing was not significant between the lowest and highest tertiles (Table 5). For all of the secondary oxidative DEHP biomarkers quantified (MEHHP, MECPP, and MEOHP), the median age of menarche was 6.57 to 7.37 months later among girls in the middle tertile relative to those in the lowest tertile of B1 concentration (Table 5). For MEHHP, girls in the highest tertile at B1 also had a significantly later age of menarche, but the median delay was not as great as that among girls in the second tertile (Table 5; Fig. 2).

Median menarcheal age (95% CI) between tertiles of EDC biomarker concentrations stratified by Tanner stage

A log(ng/ml) increase in B4 concentrations of MMP was associated with a faster time to menarche (Table 4; HR: 1.30; 95% CI: 1.10, 1.53). The influence of B4 concentrations of MMP on the age of menarche was relatively linear. Among girls in the highest B4 concentration tertile, the median age of menarche was 3.15 months earlier than girls in the lowest MMP tertile (Table 5; HR: 1.42; 95%: 1.02, 1.99).

For MEP and triclosan, the influence on the age of menarche was significantly modified by age and sex-adjusted BMI Z-score (Table 2). The average effect of triclosan across various body sizes was negligible, whereas there was a shift towards earlier menarche among girls with higher concentrations of MEP (Table 2). Stratifying by BMI category, a log(ng/ml) increase in triclosan concentration was associated with earlier age of menarche (Table 6; HR: 1.16; 95% CI: 1.01, 1.34) among girls that were either overweight or obese (≥85th percentile BMI Z-score). Similarly, a log(ng/ml) increase in MEP among overweight or obese girls was associated with earlier menarche (HR: 1.24; 95%CI: 1.05, 1.47), with the median age of menarche 5.03 months earlier in the highest compared to the lowest tertile (Fig. 3).There was no significant association with either triclosan or MEP among girls that were normal weight.

Median menarcheal age (95% CI) between tertiles of EDC biomarker concentrations stratified by BMI percentile

Discussion

In this study, we report changes in the timing of menarche associated with earlier childhood and adolescent biomarker urinary concentrations of several phthalates and phenols. For most of these biomarkers, the strength of the association was dependent on the timing of biomarker measurement, suggesting specific periods of susceptible hormone-driven development. High childhood (Tanner 1) urinary concentrations of DEHP metabolites were associated with later menarche. In contrast, increased childhood levels of 2,5-dichlorophenol and benzophenone-3 were associated with early menarche. Increased adolescent (Tanner 4) concentrations of MMP were similarly associated with earlier menarche. This sensitivity to exposure window may partially explain some discordance between prior studies of childhood EDC exposure and the age of menarche, which evaluated biomarker concentrations at various ages [ 25, 26, 27, 28].

Among this cohort of Latina girls, increased urinary concentrations of DEHP biomarkers during childhood were associated with later menarcheal age. High pre-pubertal concentrations of these biomarkers have been associated with decreased concentrations of adolescent androgens, suggesting DEHP may inhibit adrenal androgen synthesis in humans [ 29]. This anti-androgenic influence of DEHP is further supported by the later age of pubarche among girls with relatively high concentrations of DEHP metabolites [ 42, 43]. Another mechanism by which childhood DEHP exposure may influence pubertal timing is through its influence on concentrations of IGF-1 and thyroid hormones [ 44, 45, 46, 47]. DEHP may also act by modifying the production of luteinizing hormone and follicle-stimulating hormone, reducing estrogen formation, or through the activation of peroxisome proliferation-activated receptors [ 48]. Similar to our findings, one longitudinal study of German adolescents recently reported that higher urinary concentrations of DEHP metabolites from ages 8-10 years were associated with decreased odds of having reached menarche at subsequent visits [ 30]. Other studies have not reported the same association between urinary DEHP biomarker concentrations and age of menarche. A cross-sectional study of U.S. girls 12-16 years reported the summation of phthalate urinary biomarker concentrations was not associated with the onset of menarche [ 27]. A smaller cross-sectional study of Hispanic girls in Mexico City also did not report a significant influence of these exposures [ 28]. In contrast, greater concentrations of DEHP biomarkers between 7 and 15 years were associated with increased odds of earlier menarche within a cohort of Chinese girls [ 26]. Our study results suggest that the influence of the biomarkers was modified by pubertal stage at the time of exposure assessment, and suggested the correlation in DEHP biomarker concentrations across puberty was weak. Therefore, these disparate findings may partially reflect differences in exposure window.

In contrast to the influence of the DEHP biomarkers, increased concentrations of 2,5-dichlorophenol, benzophenone-3, and MMP were associated with earlier menarche. 2,5-dichlorophenol is the major biomarker of para-dichlorobenzene, a chemical used in moth repellants, room deodorants, and fumigants. Two prior studies of U.S. girls reported a similar association between 2,5-dichlorophenol and age of menarche, which is consistent with the estrogenic activity 2,5-dichlorophenol has demonstrated in vitro and in vivo [ 25, 27, 49]. The sunscreen agent benzophenone-3 has also been shown to have estrogenic characteristics, including exerting uterotrophic effects in rats, simulating proliferation of breast cancer cell lines, as well as binding and activating estrogen receptors [ 50, 51]. While these compounds may have a similar mechanism of action, prior studies did not report a significant association between benzophenone-3 and menarcheal age [ 25, 27]. In our study, only pre-pubertal concentrations of 2,5-dichlorophenol and benzophenone-3 were associated with earlier menarche. Conversely, MMP concentrations close to the onset of menarche were associated with earlier timing. Two studies of Chinese girls reported conflicting associations between MMP concentrations and pubertal timing [ 52, 53]. The parent compound of MMP, dimethyl phthalate (DMP), can be found in insect repellants and plastics. While no estrogenic effect of DMP has been demonstrated in vivo, concentrations of MMP among children 5-7 years have been inversely associated with concentrations of IGF-1, with no significant impact on thyroid function [ 54]. The potential influence on the regulation of growth and development suggests the need for further investigation into the impact of MMP, or its precursor DMP, on adolescent development.

BMI significantly modified the influence of both MEP and triclosan on age of menarche. An increase in either biomarker was only associated with earlier menarche among girls that were either overweight or obese. Among U.S. children, neither association was significantly modified by BMI; however, the association with MEP was in a similar direction among all girls [ 25, 27]. It is possible that these differences in effect modification by BMI are due to race/ethnicity disparities in adolescent body composition [ 55]. Compared to white U.S. girls, Asian and Hispanic adolescents have greater trunk fat placement independent of total body fat [ 55]. Furthermore, Hispanic girls tend to have higher percent body fat than white girls, adjusting for body size [ 56]. It is possible the action of MEP and triclosan may be modified by elements of metabolic profile shaped by fat distribution, such as modulations in insulin resistance or leptin production [ 18, 57]. Alternatively, the observed statistical interaction with BMI may reflect an influence of MEP and triclosan on both adiposity and menarcheal age. Prenatal exposure to MEP has been associated with decreased childhood BMI Z-score [ 23], but this association has been inconsistent across populations [ 22]. In one cross-sectional study, triclosan exposure was similarly associated with a decrease in BMI and waist circumference among children [ 58], but this correlation was not observed in a second cross-sectional study [ 38]. Given the impact of childhood adiposity on pubertal timing [ 59, 60, 61, 62, 63], the potential influence of EDCs on adiposity raises important consideration of the direction of causation. Specifically, EDC exposure could have a direct effect on menarcheal age, or influence adiposity to impact pubertal timing, or influence pubertal timing to impact adiposity. These theories are not mutually exclusive, and should be addressed by follow-up studies.

One limitation to this study is that we cannot account for the influence of earlier EDC exposure on pubertal timing that may have partially confounded the observed associations due to consistent home environment. For example, there is indication that exposure to certain EDCs in utero, such as DEHP, may influence both childhood adiposity and pubertal timing [ 21, 22, 23, 28]. A second limitation is that while this study did measure urinary biomarkers at two developmental time points, concentrations in a single spot urine may not be representative of average exposure. Nonetheless, prior longitudinal assessments of EDC concentrations suggest that spot measurements can estimate relative EDC exposures over the course of several months to a year due to recurrent exposures [ 64, 65, 66, 67]. A third important consideration is that these girls were primarily from low socioeconomic status families, which may have an exposure profile that is not representative of all children and adolescents. However, given the faster decline in menarcheal age among impoverished girls [ 6, 8, 9, 14, 15], the determinants of pubertal timing within this subset of the population is of particular public health concern. Finally, this study did not account for potential synergistic or antagonistic impacts of EDC co-exposures on pubertal timing. In addition to the studied biomarkers, exposure to other types of unmeasured EDCs, such as persistent organic pollutants, may have modified our observed associations with pubertal development. These interactions may contribute to the lack of reproducibility in the associations between EDC biomarkers and the onset of menarche across populations. Given the concurrence of EDCs in consumer products and the home environment [ 68], it is difficult to disentangle the independent influence of any single chemical on menarcheal age. However, we hope these findings spur additional research into modifiable sources of EDC exposure during childhood and adolescence.

Conclusions

Exposure to certain phenols and phthalates during specific stages of pubertal development was associated with menarcheal age in Chilean girls. This study highlights the necessity of capturing critical windows of exposure during periods of rapid physiological change. Additionally, the unique associations observed in this cohort emphasize the need to integrate investigations from across the world to identify population-specific sensitivities, which can guide global health recommendations. As data collection in this longitudinal cohort continues, we plan to explore the impact of EDC exposure on additional facets of pubertal development, such as the onset of regular cycling. These shifts in pubertal timing may have both immediate psychological implications for these girls, and may indicate changes in the predisposition to adverse health in adulthood. Girls with early puberty have been shown to suffer from higher rates of adolescent depression, anxiety, and delinquent behavior [ 69, 70, 71, 72]. Early onset of menarche has also been associated with increased risk of type-2 diabetes, cardiovascular disease, and cancer risk. Future studies will be necessary to directly link the variation in pubertal timing associated with early life EDC exposure to adult health, as well as the potential direct effect of childhood/adolescent EDC exposure on adult health independent of age of menarche.

Abbreviations

24-DCP:

2,4-dichlorophenol

25-DCP:

2,5-dichlorophenol

AFT:

Accelerated failure time

B1:

Tanner 1

B4:

Tanner 4

BP-3:

Benzophenone-3

BPA:

Bisphenol A

B-PB:

Butyl paraben

BPF:

Bisphenol F

BPS:

Bisphenol S

DEHP:

of di (2-ethylhexyl) Phthalate

EDC:

Endocrine disrupting chemical

E-PB:

Ethyl paraben

MBP:

Mono-n-butyl phthalate

MBzP:

Monobenzyl phthalate

MCNP:

Mono carboxyisononyl phthalate

MCOP:

Mono carboxyisooctyl phthalate

MCPP:

Mono-3-carboxypropyl phthalate

MECPP:

Mono(2-ethyl-5-carboxypentyl) phthalate

MEHHP:

Mono(2-ethyl-5-hydroxyhexyl) phthalate

MEHP:

Mono(2-ethylhexyl) phthalate

MEOHP:

Mono(2-ethyl-5-oxohexyl) phthalate

MEP:

Monoethyl phthalate

MHBP:

Mono-hydroxybutyl phthalate

MHiBP:

Mono-hydroxyisobutyl phthalate

MiBP:

Mono-isobutyl phthalate

MMP:

Monomethyl phthalate

MNP:

Mono-isononyl phthalate

M-PB:

Methyl paraben

P-PB:

Propyl paraben

TCS:

Triclosan

References

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Acknowledgements

We acknowledge Manori Silva, Ella Samandar, Jim Preau, Prabha Dwivedi, Xiaoliu Zhou, and Tao Jia who performed the measurements of phthalates and phenols biomarkers.

Funding

This work was supported by Public Health Service grant R01CA158313 from the National Cancer Institute, National Institutes of Health, U.S. Department of Health and Human Services (to KBM).

Availability of data and materials

Please contact author for data requests.

Disclaimer

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Use of trade names is for identification only and does not imply endorsement by the CDC, the Public Health Service, or the US Department of Health and Human Services.

Author information

Affiliations

1. Department of Epidemiology, Fielding School of Public Health, University of California, Los Angeles, CA, USA

Alexandra M. Binder & Karin B. Michels

2. Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile

Camila Corvalan & Ana Pereira

3. Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, USA

Antonia M. Calafat & Xiaoyun Ye

4. Institute of Maternal and Child Research, University of Chile, Santiago, Chile

Verónica Mericq

Contributions

AMB, CC, AP, and KBM designed the research plan; CC, VM, and AP acquired the data; AMC and XY measured the urinary biomarkers; AMB, CC, AP, and KBM interpreted the data; AMB performed the statistical analysis and wrote the paper; AMB, AMC, CC, VM, AP, and KBM provided critical revision of the manuscript for important intellectual content; AMB and KBM had primary responsibility for final content. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

Corresponding author

Correspondence to Karin B. Michels.

Ethics declarations

Ethics approval and consent to participate

The study protocol was approved by the Ethics Committee of the Institute of Nutrition and Food Technology, University of Chile. The study was also reviewed and approved by the IRB of the University of California, Los Angeles. Informed consent was obtained from all parents or guardians of children before the start of data collection.

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Not applicable.

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The authors declare that they have no competing interests.

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Binder, A.M., Corvalan, C., Calafat, A.M. et al. Childhood and adolescent phenol and phthalate exposure and the age of menarche in Latina girls. Environ Health 17, 32 (2018). https://doi.org/10.1186/s12940-018-0376-z
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Keywords

Menarche

Puberty

Phenols

Phthalates

Endocrine disrupting chemicals

# data[].attributes.attachment_link.title
Childhood and adolescent phenol and phthalate exposure and the age of menarche in Latina girls - Environmental Health

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Background The age of menarche has been associated with metabolic and cardiovascular disease, as well as cancer risk. The decline in menarcheal age over the past century may be partially attributable to increased exposure to endocrine disrupting chemicals (EDCs).

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This article talks about how because puberty is such a susceptible window of time to EDC's, they can definitely influence the timing of puberty by "accelerating and anticipating the processing of maturation of secondary sexual characteristics." The article also mentions how exposure to EDC's during puberty can increase the risk of cancer.

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Madison McRobert- This article talks about all the different types of EDC’s that everyone can experience. But this focuses on how during puberty their is a chance that breast during puberty. Which can include the chance of getting breast cancer, while growing and going through puberty.

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Abstract

Puberty is the process of physical changes between childhood and adulthood during which adolescents reach sexual maturity and become capable of reproduction. It is considered one of the main temporal windows of susceptibility for the influence of the endocrine-disrupting chemicals (EDCs). EDCs may act as single chemical agents or as chemical mixtures; they can be pubertal influencers, accelerating and anticipating the processing of maturation of secondary sexual characteristics. Moreover, recent studies have started to point out how exposure to EDCs during puberty may predispose to breast cancer later in life. In fact, the estrogen-mimicking endocrine disruptors (EEDs) may influence breast tissue development during puberty in two main ways: the first is the action on the proliferation of the breast stromal cells, the second concerns epigenetic mechanisms. The aim of this mini-review was to better highlight what is new and what is not completely known regarding the role of EDCs during puberty.

Keywords: puberty, Endocrine disrupting chemicals (EDCs), Window of susceptibility, Early Puberty, Estrogen-mimicking Endocrine Disruptors (EEDs), breast development, breast cancer

1. Background

Endocrine-disrupting chemicals (EDCs) influence one or more functions of the endocrine system and cause several adverse health effects in human beings. They are ubiquitous in nature, being found in industrial chemicals, drugs, plastics, and other materials common in everyday life [ 1].

EDCs may represent a risk for our health, working through many different mechanisms of action. In fact, they may take several routes of absorption (oral, dermal, etc.), affect different temporal windows (prenatal and postnatal life, puberty), and be made up of many combinations of chemicals (mixtures), leading to extremely difficulty in interpreting their influence on human wellbeing.

Moreover, their effect seems to be transgenerational, occurring over at least two or three generations. As the window of susceptibility, puberty is considered one of the hot spots in the lifetime when EDCs may exert their effects [ 2].

The main aims of this mini-review were (1) to provide an update of what we know about the relationships between EDCs and pubertal trends, and (2) to highlight the new role of EDCs as influencers of breast cancer predisposition later in life.

2. Puberty and Pubertal Onset

Puberty is the process of physical change that occurs between childhood and adulthood, during which adolescents reach sexual maturity and become capable of reproduction.

The main clinical manifestations of puberty are the growth spurt, progression of secondary sexual characteristics, development of gonads, and changes in body composition. These features lead to an increase in strength and endurance [ 3].

During pubertal development, changes in the brain activity are also detectable: the frontal lobes increase their volume and activity, leading to more complex psychological understanding [ 4]. The whole process involves years of development on both a mind and a body level.

In the last two centuries, pubertal timing has changed dramatically. Data show that the age of the menarche is experiencing a declining trend, from 17 years old at the beginning of nineteenth century to 13 years old by the 50s [ 5]. In the United States, the onset of puberty in girls is occurring earlier than previous studies have documented, with breast and pubic hair development appearing on average 1 year earlier in white girls and 2 years earlier in African-American girls. Beyond these data, it is also underlined how this early onset of puberty seems not to be correlated with a parallel anticipation of the menarche age. According to the most recent studies, the age of menarche has decreased by only few months [ 6].

As far as timing is concerned, in the 60s, Tanner et al. identified the normal range of pubertal onset as between 8 and 13 years in girls, and between 9 years and 6 months and 13 years and 8 months in boys [ 3, 4]. Therefore, the development of secondary sexual characteristics is considered precocious when it happens in girls under the age of 8 years old and in boys younger than 9 years old.

The timing of pubertal onset depends on genetic and environmental factors. The genes involved in this process are the ones that control GnRH hypothalamic secretion, pituitary development and its functioning, hormone synthesis and bioactivity, energy homeostasis and growth, and potential peripheral feedback from sex steroids [ 7].

Studies show that some populations are much more likely to develop precocious puberty: girls; African-Americans; individuals presenting other medical conditions (McCune-Albright syndrome, congenital adrenal hyperplasia) or experiencing or having experienced radiation therapy of the central nervous system; those exposed to sex hormones; children that may have been in contact with estrogen or testosterone cream or ointment, or with other substances such as adults' medications, dietary supplements, or food containing phytoestrogens.

The nutritional status of the pediatric population influences the age of pubertal onset. Adolescents with anorexia nervosa usually reach menarche later [ 8], highlighting a correlation between nutritional state and development. Girls who are overweight or obese have a higher probability of being diagnosed with central precocious puberty or early puberty compared to the general population [ 9]. Genomic studies and human epidemiological data suggest a correlation between early infancy weight gain, early pubertal onset, and later obesity in adulthood, which can be explained by the overlapping of genes involved in pubertal onset and obesity [ 10]. Moreover, obesity induces hyperinsulinemia and hyperleptinemia, which can affect linear growth and advanced puberty [ 11].

Both events, pubertal timing and obesity, are influenced by the environment, and there is a strong interaction between genes and environments during critical periods of development such as puberty. This interaction is due to substances, EDCs, which derail the normal hormonal process.

3. EDCs and Puberty

In 2015, the Endocrine Society defined an EDC as "an exogenous chemical or mixture of chemicals, which interferes with any aspect of hormone action". The EDCs affect the control of energy balance, reproduction, hormone-sensitive cancer, thyroid, neurodevelopment, and the neuroendocrine system. They can act at any level in the hypothalamic-pituitary-gonadal system or the peripheral tissue. During a lifespan, there are some critical points (windows of development) that are important because during them, cells are rapidly proliferating and epigenetic changes are more likely to occur. These periods are fetal life, neonatal life, and puberty [ 12].

Moreover, EDCs act at any level in the hypothalamic-pituitary-gonadal-peripheral tissue-endocrine axis. Indeed, they can act not only as agonists or antagonists of estrogen receptors, but can also target and act against androgen and progesterone receptors. They may also mimic the natural occurrence of estrogens and androgens. Furthermore, EDCs bind to cell receptors and block the functions of endogenous hormones, thus acting as anti-estrogens and anti-androgens [ 13].

The most important effect of EDCs is considered to be the anticipation of or, more generally, impact on the timing of female puberty [ 14]. Nevertheless, this effect is difficult to demonstrate since low-level exposures to a mixture of EDCs may mask the effects of some of them. Moreover, inter-individual variation in susceptibility might interfere with an objective analysis. Finally, the effects on humans to EDCs exposure may show up with a considerable delay.

We analyzed the most important and well-known EDCs that might interfere with natural pubertal timing ().

Table 1

Atrazine

Herbicide

<1 day

Dermal absorption, inhalation

Prenatal life

ER agonist

Early puberty

↑incidence of breast cancer

BPA

Polycarbonate plastics, epoxy resins, plastic toys and bottles, lining of food cans

4-5 h

Ingestion, dermal absorption, inhalation

Prenatal life
Puberty

ER agonist
Antiandrogen

Premature thelarche

Induce neoplastic transformation

DES

Nonsteroidal estrogen medication used in the past

3-6 h

Ingestion

Prenatal life
Puberty

ER agonist
Epigenetic silencing of mRNA

Not known

↑breast cancer

DDT/DDE

Contaminated water, soil crops, fish

6-10 years

Ingestion, dermal absorption, inhalation

Prenatal life and lactation period

ER agonist
Antiandrogen
Antiprogestin
Induction of aromatase

Precocious puberty
Anticipated menarche
Later onset of puberty

↑cells proliferation index and mammary tumor cells proliferation

Dioxin

By-product of chlorinated herbicide production, smelting, chlorine bleaching of paper

7-11 years

Ingestion, inhalation

Prenatal and lactation
Puberty

ER agonist
Antiandrogen
Interfere with sex-steroid synthesis

Delayed breast development

Induce epigenetic silencing of key tumor suppressor (TCDD)

Parabens

Cosmetics and pharmaceutical products, toothpaste and food preservatives.

<24 h

Dermal absorption, ingestion

Pre- post-natal life and puberty

ER agonist

Not known

↑incidence of breast cancer

PBDEs/PBB

Furniture, mattresses, carpet pads, automobile seats, flame-retardant textiles

2 days-3 months

Ingestion, dermal abruption, inhalation

Prenatal life, perinatal period, peripuberty

ER agonist/antagonist
Antiandrogen

Early pubic hair in boys
Premature thelarche
Early/late menarche
Early puberty

Not known

PCBs

Contaminated air and food, skin contact with old electrical equipment

12 days-16 years

Ingestion, dermal absorption, inhalation

Prenatal life

ER agonist/antagonist
Antiandrogen

Early onset of menarche
Delayed pubertal development

Accumulates in breast adipose tissue

PFOA

Carpeting, upholstery, floor wax, firefighting foam, sealants

3.8 years

Ingestion, inhalation

Prenatal life
Puberty

Induced estrogen

Delayed pubertal development

↑susceptibility to carcinogenesis in future generations

Phthalates

Contaminated food, PVC plastics and flooring, personal care, medical devices and tubing

12 h

Ingestion, dermal absorption, inhalation

Prenatal life

ER agonist/antagonist
Antiandrogen
Interfere with androgen synthesis

Early puberty
Premature thelarche
Delayed pubic hair development

↑cells'proliferation in the breast

Pyrethroids

Contaminated water, soil, food

10 h

Ingestion, dermal abruption, inhalation

Androgen agonist

Delayed puberty

Not known

Vinclozolin

Fungicide

1-3 months

Ingestion, dermal absorption, inhalation

Prenatal life
Puberty

ER agonist
Antiandrogen
Epigenetic alteration

Not known

Dysregulates the gland development
↑formation of mammary tumor

3.1. BPA

Bisphenol A (BPA) is a precursor of plastics, polycarbonates, and epoxy resins. BPA-based plastic is clear and tough, and it is used in a lot of common objects such as plastic bottles, food storage containers, baby bottles, and CDs. Epoxy resins are utilized to line water pipes, as coating on the insides of many food and beverage containers, and in making thermal paper. This chemical is almost ubiquitous, which means that people are at great risk for exposure and, even if the estrogen receptor agonist activity is weak, its potential should not be underestimated. BPA also acts as an anti-androgen.

Studies demonstrate how this EDC is associated with premature thelarche, precocious puberty and pubertal development [ 15, 16, 17].

3.2. DDT/DDE

Dichlorodiphenyltrichloroethane, commonly known as DDT, is an organochlorine. It was originally developed as an insecticide for use in agriculture and it is colorless, tasteless, and odorless. Because of these features, exposure to DDT is imperceptible, but being exposed during fetal life and lactation can affect sexual development. Even if DDT is banned from the market, it will remain present in the environment as a persistent organic pollutant (POP). Moreover, in some low-income countries, it is still widely used.

DDT and its metabolite dichlorodiphenyldichloroethane (DDE) are the most studied of the pesticides that have been identified as EDCs. Both have estrogenic, anti-androgenic, and antiprogestin effect and induce aromatase. According to Vasiliu et al. (2004), there is an association between exposure to these chemicals and precocious puberty, delayed puberty, and earlier age of menarche [ 18, 19].

3.3. Dioxins

Dioxins are byproducts of burning or various industrial processes such as chlorinated herbicide production, smelting, or chlorine bleaching of paper. They have an estrogenic and anti-androgenic effect, and they interfere with sex steroid synthesis. Exposure to dioxins during the prenatal and lactation period is associated with delayed breast development. On the other hand, exposure to these chemicals has not been correlated with age of menarche or pubic hair development [ 20].

3.4. PBDEs and PBB

Flame retardants are a group of chemicals added to manufactured materials (plastics, textiles, surface finishes, and coatings). They are intended to prevent or slow down the further development of ignition. Some examples of such chemicals are polybrominated diphenyl ethers (PBDEs) and polybrominated biphenyl (PBB). They act as estrogens and anti-androgens. Data show an association between PBDEs and premature thelarche, earlier menarche, and earlier pubertal development [ 21, 22]. Furthermore, prenatal exposure to PBDEs is associated with later menarche in girls and earlier development of pubic hair in boys [ 23].

Moreover, exposure during the peri-pubertal period seems to interfere with reproductive development [ 24].

3.5. PCBs

Polychlorinated biphenyl (PCBs) is a dioxin-like compound derived from biphenyl. This chemical is widely used as a dielectric and coolant fluid in electrical apparatuses, in carbonless copy papers, and in heat transfer fluids. Its mechanism of action is rather similar to that of dioxins, but compared to dioxins, there is evidence that exposure during the prenatal period leads to early onset of menarche and to delayed pubertal development [ 25].

3.6. Phthalates

Phthalates are esters of phthalic anhydride. They are used as liquid plasticizers in plastic, flooring, personal care products, medical devices, and tubing. They increase the flexibility, transparency, durability, and longevity of materials. Their most common use is to soften polyvinyl chloride (PVC).

Their endocrine-disrupting mechanism is not fully clarified, but they either act as estrogen receptor agonists and antagonists, or androgen receptor antagonists. Moreover, they can also disrupt androgen synthesis.

Different studies have demonstrated a significant association with premature thelarche and precocious or early puberty [ 26, 27].

Phthalates can be classified as low- or high-molecular-weight phthalates. Depending on the class and the timing of exposure, different outcome have been observed. High-molecular-weight phthalate levels several years before puberty are associated with later pubic hair development and younger age of menarche; in contrast, low-molecular-weight phthalate levels are related to advanced breast or pubic hair development [ 28, 29].

3.7. Pyrethroids

A pyrethroid is an organic compound similar to a natural pyrethrin, which comes from the pyrethrum flower. This chemical is found in most common commercial household insecticides. It has been reported both as a weak androgen and for its association with late onset of puberty in girls [ 30, 31].

4. Puberty and EDCs: A Temporal Window for Breast Cancer

In girls, the first physical sign of puberty is thelarche, with physical breast changes in both the stroma and the epithelium. The amount of fibrous and fatty tissue in the stroma increases (the adult non-lactating breast is more than 80% composed of stroma) [ 32]. The ducts first elongate, proliferate, and form lobular structures composed of the growth ductal tree; with the achievement of sexual maturity, they decrease in size, the proliferative phase reducing [ 33]. Thus, puberty is the time in which the breast tissue undergoes rapid changes regulated by small fluctuations of endogenous hormones [ 34]. These rapid structural and cellular modifications are thought to create a "window of susceptibility" [ 35].

Breast tissue during this period of morphogenesis and re-modeling is particularly sensitive to carcinogenic effects; studies have demonstrated that exposure to radiation, DDT, medications, and disease before and during puberty increases breast cancer risk in adulthood [ 36, 37, 38, 39, 40].

The role of estrogen in breast development and tumorigenesis is confirmed. Prolonged and uninterrupted exposure to endogenous estrogen and administration of exogenous estrogens to postmenopausal women increases the risk of cancer. In contrast, gonadectomy in premenopausal women reduces the risk of breast cancer, while pharmacological agents serve to treat the same condition, blocking estrogen biosynthesis or estrogen receptors.

It is not entirely clear how human exposure to specific EDCs can be linked to breast cancer. The EDCs of interest in this mechanism are those with estrogenic activity: they are called estrogen-mimicking endocrine disruptors (EEDs).

The main effects of EEDs are two-fold: the first is their action on the proliferation of the stromal cells, the second concerns epigenetic mechanisms. EEDs affect stromal cells, interfering with the estrogen signal pathway [ 40, 41].

Moreover, EEDs may modify the breast matrix composition. All these changes macroscopically create a different breast density, which is one of the strongest predictors of premenopausal breast cancer risk [ 42].

Breast cancer is also associated with epigenetic changes such as DNA methylation, histone modifications, and non-coding RNAs (ncRNAs). These mechanisms work together to determine whether the epigenome may expresses itself and how, facilitating gene expression control, X chromosome inactivation, and/or genomic imprinting [ 43].

The most important chemical EEDs and their activities to promote or facilitate breast cancer are analyzed below ().

4.1. Atrazine

Atrazine is an herbicide used to prevent pre- and post-emergence broadleaf weeds in crops (corn and sugarcane) and turf. It can induce aromatase activity, increasing the level of estrogen in the body. Atrazine is not classified as a carcinogen or mutagen because its link with breast cancer is not totally clear. On this issue, studies are contradictory. Some studies on human reaction to atrazine exposure have seemed to detect a weak association between atrazine and breast cancer [ 44].

4.2. BPA

BPA increases the methylation levels of key genes associated with tumor development (e.g., BRCA1, CDKN2A, etc.), altering the epigenome in order to promote proliferation, senescence, and tumor development [ 45].

3D cultures of breast cells have shown that BPA reduces the number of tubules, increases spherical masses, and causes more deformed acini, indicating an ability to induce neoplastic transformation [ 46]. This because BPA exposure increases HOXB9 expression in breast cells, both in vitro and in vivo, through a mechanism that involves increased recruitment of transcription and chromatin modification factors. HOXB9 is a homeobox-containing gene that plays a key role in mammary gland development, and is associated with breast cancer. BPA acts as a competitor for estrogen receptors (ERs), which leads to the activation of this gene through chromatin modifications (histone methylation and acetylation) [ 47].

4.3. DDT/DDE

The estrogenic property of these compounds and their prominent role in mRNA regulation increase epithelial cells' proliferation index and mammary tumor cell proliferation [ 48]. Moreover, a prospective study showed that girls exposed to high levels of DDT and DDE were more likely to develop breast cancer than those who were exposed to a lower dose. On top of that, the younger the age at which a person was exposed to these compounds, the higher the risk of developing breast cancer [ 38]. These data support the hypothesis that the age of exposure must be considered for risk assessment.

4.4. DES

Diethylstilbestrol (DES) is a nonsteroidal estrogen medication that was used in the past to support during pregnancy women with a history of recurrent miscarriage, as hormone therapy for menopausal symptoms and estrogen deficiency, and as a treatment for breast cancer.

The development of breast cancer in women exposed to DES is mediated by the proliferative effects of estrogens, and by a differential expression of ncRNAs. In particular, epigenetically silenced mRNA causes disordered regulation of the p53-mediated apoptotic pathway [ 49].

4.5. Parabens

Parabens are a widely used class of preservatives in cosmetic and pharmaceutical products. They are used for their bactericidal and fungicidal properties. They are commonly found in shampoos, commercial moisturizers, topical/parenteral pharmaceuticals, suntan products, make-up, and toothpaste, and are also used as food preservatives.

They have estrogen-like effects and they may be associated with breast cancer etiology. This is demonstrated by the association between the utilization of paraben-containing body care products in the Western world and the increased incidence of breast cancer [ 50].

4.6. PCBs

Studies have demonstrated that polychlorinated biphenyls accumulate in breast adipose tissue, but so far, an association has not been proven between PCB exposure and breast cancer [ 51]. This is probably because PCBs are mixtures of congeners with varied properties, and because breast cancers of different subtypes are etiologically distinct diseases. The absence of stratified subgroup analysis on individual PCB exposure and patients with specific biological subtypes of breast cancer may have resulted in an underestimation of the correlations between PCBs and breast cancer [ 52]. Moreover, PCBs' mechanisms of action are different. In fact, the natures of the biochemical and toxic effects of PCB congeners are largely determined by their structure, as well as different PCB congeners sometimes exerting conflicting actions. Some PCBs act as estrogen agonists both in vitro and in experimental animal systems; some others have been shown to be anti-estrogenic, while others are enzyme inducers of phenobarbital (PB)-type cytochrome P450 [ 53]. The main mechanism involved in breast cancer seems to be the estrogen agonist mechanism.

4.7. PFOA

Perfluorooctatonic acid (PFOA) serves as a surfactant in the emulsion polymerization of fluoropolymers and as a building block for polymeric materials. It is used for various industrial applications (carpeting, upholstery, floor wax, firefighting foam, sealants etc.). Data show how in CD-1 and C57B1/6 mice, early PFOA exposure alters the mammary gland without changing other pubertal endpoints [ 54]. This development may increase susceptibility to carcinogenesis in future generations.

4.8. Phthalates

One characteristic of phthalates is their lipophilicity, so they accumulate in breast tissue where they cause modification of the mammary gland [ 55]. Epigenetic changes due to exposure to phthalates increase the proliferation of cells in the breast [ 56].

4.9. TCDD

2,3,7,8-Tetracloridibenzo-p-dioxin, or TCDD, is a famous organochlorine mainly known as a contaminant in Agent Orange, an herbicide used in the Vietnam War. The scientific community is aware of its catastrophic effects, including birth defects, pregnancy loss, and increased rates of cancer. In 2012, a retrospective study showed a significantly higher prevalence of breast cancers in women working in a chemical pesticide establishment due to their exposure to high levels of TCDD [ 57].

TCDD has a significant effect on the epigenome in breast and other tissues. It is interesting to note that TCDD induces an epigenetic silencing of key tumor suppressors, which may have a role in breast cancer development [ 58].

4.10. Vinclozolin

Vincozolin is a common dicarboximide fungicide used to control diseases in vineyards and on fruits and vegetables. Exposure to this pesticide in female rats causes the formation of mammary tumors and dysregulates gland development [ 59]. It promotes an epigenetic alteration in the germ line that creates a transgenerational disease state [ 60].

5. Conclusions and Perspectives

Most of these EDCs are present in everyday objects and in various common solutions; it is almost impossible not to be exposed to these chemicals, even if laws were to ban the use of most of them and restrict the others. It is known that environmental factors, including EDCs, are the most important determinant in explaining the puberty secular trend and the timing of menarche. The anticipation of these two endpoints of development can be explained by the action of environment during the pubertal period, and how it may advance and activate the central axis.

According to Parent et al., this paradigm of EDCs could be revised; in fact, environmental factors can affect puberty and reproduction through central and peripheral mechanisms, taking advantage of the critical windows of development [ 61]. During these periods, EDCs can modify genetic or epigenetic pathways.

The epigenetic pathways are interesting because of their long-term action. Studies on animals have demonstrated the effects of a mixture of EDCs on female sexual development over three generations, and that the effects are even bigger in the second and third generations (delayed vaginal opening, decreased percentage of regular estrous cycle, and decreased GnRH interpulse interval) than in the first. The reproductive phenotypes in rats are associated with alterations in both transcriptional and histone posttranslational modifications of hypothalamic genes involved in reproductive competence. Moreover, females of the first generation exposed in utero to these EDCs showed alteration of the thyroid and impaired maternal behavior, which could cause additional epigenetic change in subsequent generations [ 62].

EDCs can also act as obesogens and promote early adiposity rebound, changing metabolic or peripheral signals, and increases in adrenal androgen levels, inducing early pubertal development [ 63].

EDCs, and in particular EEDs, may produce epigenetic changes not only in puberty genes but also in other tissues and cells. In fact, these compounds could act as carcinogens and induce breast cancer. Clearly, EEDs are not the only factors responsible for these phenomena. Their effect is influenced by environmental carcinogens and unhealthy foods such as refined sugar, processed fats, and food additives, which promote molecular damage that increases the risk of breast cancer.

Puberty amplifies this process; young women today live in an obesogenic world and they are exposed to several contemporaneous risk factors, especially girls who live in disadvantaged conditions [ 64].

Moreover, the exposure to these compounds and the development of mammary glands expands the epigenetic changes that EEDs cause in breast tissue, promoting tumorigenesis and increasing the chance of damage that might prove to be significant.

In conclusion, exposure to EDCs during puberty increases the risk of developing breast cancer during the lifetime.

Although these results are still somewhat controversial, it is important for pediatricians to keep in mind the new risks for young girls during puberty, and to provide the right information to their families.

Author Contributions

Conceptualization, L.I.; methodology, L.I. and L.L. (Licia Lugli); resources, L.L. (Laura Lucaccioni), V.T.; writing-original draft preparation, L.L. (Laura Lucaccioni), V.T., L.M.; writing-review and editing, N.B., A.B., B.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Endocrine-Disrupting Chemicals and Their Effects during Female Puberty: A Review of Current Evidence

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Puberty is the process of physical changes between childhood and adulthood during which adolescents reach sexual maturity and become capable of reproduction. It is considered one of the main temporal windows of susceptibility for the influence of the endocrine-disrupting chemicals (EDCs).

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EDCs have proven to have an affect on the timing of puberty. Now the acceleration varies depending on the length of exposure. PCBs also play a role in puberty it can either delay or accelerate puberty in boys. They had some "mixed results" with girls, but EDCs can lead to early timing of their first period.

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INTRODUCTION

Puberty is a fundamental process of maturational changes that mark the transition from sexual infancy to reproductive maturity. It is now recognized that the age of onset of breast development, exemplifying an early stage of puberty in girls, has declined in recent decades, although the age of menarche, an advanced pubertal milestone, has not changed significantly ( 1, 2, 3). The data on pubertal timing in boys is inconsistent, with some reporting an earlier onset and others finding no changes in the age of pubertal development in recent decades ( 4, 5). While genetic factors remain the predominant determinant of pubertal timing, the shift towards an earlier age of puberty in the past century coincided with improvements in public health and nutrition, and more recent changes have been attributed to obesity. It has also been speculated that endocrine disrupting compounds (EDCs) are potential contributors to the observed shifts in pubertal timing ( 1, 2, 5, 6). Here we review publications from 2016 and 2017 that examine EDC associations with pubertal milestones, focusing on possible environmental triggers of puberty in both males and females.

General principles of EDC actions

A number of different classes of exogenous compounds with endocrine active properties interfere with endogenous hormone action ( 7). These Endocrine Disrupting Compounds (EDCs) can bind hormone receptors and affect their synthesis, transport, metabolism, and/or elimination. Among the various types of EDCs are persistent organic compounds that bio-accumulate through the food chain and have half-lives of years to decades. These chemicals, pesticides (dichloro-diphenyl-trichloroethane [DDT], pyrethroids), dioxins, polychlorinated biphenyls (PCBs), and flame retardants (polybrominated diphenyl ethers [PBDEs]), are present ubiquitously across the ecosystem and detectable in humans worldwide (reviewed in ( 5, 7). Other EDCs, such as phthalates and phenols, have shorter biological half-lives and are measurable for only a short duration post-exposure, but may still exert long-term health effects. EDCs are not natural ligands, and therefore do not have the same binding specificity and affinity as endogenous hormones, and may elicit non-receptor mediated effects. They exert antagonistic and agonistic actions on hormonal axes and pathways, and may have divergent effects at low versus high concentrations- consistent with their non-monotonic dose response curves. Both acute and delayed (latent) effects of EDCs have been reported. The prenatal, infancy, and childhood periods are considered critical windows of susceptibility due to ongoing developmental processes that are vulnerable to EDCs. The specific health consequences are dependent on the life stage at the time of exposure, particular compound, the endogenous hormonal milieu, and which health outcomes are being examined.

Phenols and phthalates

Phthalates and phenols include a number of hormonally active chemicals with anti-androgenic and estrogenic activity that are present at relatively high concentrations compared to pesticides and other industrial chemicals.

Bisphenol A (BPA) is the phenol that has drawn the most public interest (reviewed in ( 8)). BPA and other phenols are plasticizers used in both medical products and consumer goods, such as medical tubing, plastic bottles and toys, dental sealants, and linings of metal container. BPA is now banned in the production of infant food and beverage containers such as baby bottles, and has been removed from many consumer products. At lower concentrations, BPA and other phenols have weak estrogenic properties but can also compete with endogenous estrogens for binding, and at higher concentrations, they have been shown to have anti-androgenic properties.

To investigate the associations of BPA exposures with puberty, a Thai study compared urinary BPA concentrations in 41 girls with precocious puberty to 47 age matched controls ( 9). The girls with precocious puberty had higher urinary BPA levels than controls, and among the group with early puberty, those who were obese or overweight had higher values than those of normal weight. One challenge with this study is that the investigators used a relatively advanced cut-off of 8-9 years to define early puberty. In addition, samples were collected at the time of the exam, reflecting contemporaneous exposures rather than exposure assessment prior to the outcome measure- the onset of puberty.

Miao and colleagues also conducted a cross-sectional study to investigate the association between BPA exposure and pubertal development in 655 girls aged 9-18 years in Shanghai, China ( 10). Urinary BPA levels were measured and categorized according to whether they were unmeasurable (<LOD) or measurable (>LOD). If they were measurable, they were grouped as "above" or "below" the median concentrations for the cohort. A physician assessed pubertal development by Tanner staging and collected self-reported age at menarche. After adjusting for potential confounders, girls with higher values of BPA were more likely to have delayed menarche compared to girls with undetectable urinary BPA levels. Girls aged 9-12 years with measurable urinary BPA levels were more likely to have reached pubic hair stage 2; while among girls aged >15 years, those with detectable urinary BPA were less likely to have reached pubic hair stage 5. These authors concluded that contemporaneous BPA exposures were associated with delayed menarche and with earlier onset of P2 but later attainment of P5. Again a limitation of this study was that the exposure assessment occurred concurrently with the outcome assessment, making it difficult to establish causality.

BPA and phthalate levels were compared in 42 Turkish girls with idiopathic central precocious puberty, 42 girls with peripheral precocious puberty, and 50 healthy non-obese age-matched girls as controls ( 11). The authors did not find any differences in mean values of urinary BPA, LH, FSH or estradiol among the three groups. However, they reported that plasma DEHP and MEHP, were higher in the group with central precocious puberty than in the other two groups, suggesting a potential central site of action for these phthalate metabolites.

A longitudinal US puberty cohort study based at three centers (the Bay area in northern California, Cincinnati, Ohio, and Manhattan, NY was established through the Breast Cancer Environmental Research Program (BCERP) to examine the role of EDCs on pubertal development ( 12). 1051 girls had urinary levels of phenols and phthalates measured at age 6-8 years and were followed annually ( 13). Higher 2,5-dichlorophenol was associated with a 7-month earlier onset of menarche, while mono-3-carboxypropyl phthalate (MCPP) was associated with a 4-5 month later age of both thelarche and menarche, that was greater in lean girls. The association of MCPP with delayed pubertal onset and menarche was a new and unanticipated finding. MCPP is a metabolite of several phthalates and exposures is through household products as well as diet. These authors also reported that triclosan, an anti-bacterial and anti-fungal agent in soaps and personal care products, and benzophenone-3, a ketone in cosmetics for UV protection were both associated with earlier thelarche but not menarche ( 13).

Wang and colleagues evaluated the association of urinary BPA with pubertal development in 671 Chinese boys aged 9 to 18 years ( 14). Urinary BPA levels were categorized as <LOD, LOD to the 75 th%ile and >75 th%ile levels. The boys were grouped according to sexual maturity staging as early-, mid-, and late puberty. Of note is that all 13 years olds recruited were already in early puberty and that none of the boys < 11 years of age had progressed to late puberty. Moderate levels of urinary BPA correlated with earlier onset of pubic hair and genital stage 2, and showed a trend towards testicular enlargement. Urinary BPA was associated with later attainment of mature milestones, suggesting an association of moderate levels of urinary BPA with slower pubertal progression. These authors concluded that BPA exposure was associated with earlier age of adrenarche and genital stage 2, but delayed attainment of genital stage 5. Adjusting for BMI did not alter these associations.

Flame retardants/polybrominated diphenyl ethers (PBDEs)

Another class of EDCs of particular interest are the flame retardants, or polybrominated diphenyl ethers (PBDEs) used in carpeting, clothing, and furniture. The Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) was a birth cohort in California established to investigate the health effects of gestational and childhood exposures to agricultural pesticides and other environmental chemicals ( 15). This paper examined the association of flame retardants with pubertal timing in Latino low income mothers in this agricultural community. Four PBDEs were measured in maternal serum during pregnancy or at delivery as a measure of prenatal exposures and childhood exposures were assessed in PBDE measurements at age 9 years in the offspring. The children were seen every 9 months from 9-13 years of age for pubertal staging, and menarche was self-reported. In this cohort of 309 boys and 314 girls, prenatal PBDE exposure was associated with later menarche in girls and earlier pubarche in boys. Associations were stronger for normal weight girls. Childhood PBDE exposure showed a similar trend towards later menarche but it was not statistically significant, and no association was found at all for boys. There was no association between prenatal or childhood PBDE exposure and thelarche or pubarche in girls. These results are similar to those reported for the BCERP cohort, the only other longitudinal cohort study of girls' PBDE exposure ( 12). In the CHAMOCOS cohort, maternal PBDE levels were also associated with higher LH values in the boys at age 12 years ( 16).

A cross sectional case-control study from Italy compared serum PBDEs in children referred for idiopathic central precocious puberty (n=37) and premature thelarche (n=56) to 31 prepubertal control girls ( 17). Serum was collected for PBDE measurement at the time of physical exam. They found that girls with premature thelarche had higher PBDE levels than girls with precocious puberty and controls. This study had the same limitations as the other cross-sectional studies described above, ie they did not assess exposure prior to pubertal onset, and also used a relatively late age of 8 years to define precocious puberty onset. The authors conclude that their findings suggest that PBDE has a peripheral estrogenic action but no central action on the hypothalamic-pituitary-ovarian axis.

PCBs and Dioxins

Eskanazi and colleagues also reported the association of organochlorine chemicals with pubertal timing and reproductive hormones in the CHAMACOS boys, examined at ages 9 to 12.75 years for pubertal staging ( 16). Of the 312 boys who completed the 12 year visit, 234 provided a blood sample for hormone analysis. A 10-fold increase in maternal DDT levels was associated with an 18% decrease in boys' LH and testosterone values, while a 10-fold increase in DDE (a DTT metabolite) was associated only with a decrease in LH. Conversely, higher prenatal PCBs were associated with higher 12 year old FSH values in the boys. These associations between the boys' chemical levels at age 9 years and hormone values at 12 years was mediated by the boys' BMI.

The longitudinal Russian Boys Study cohort examines the associations of mixed childhood exposures with pubertal development ( 18). 473 boys had serum dioxin-like compounds (DLCs) and PCBs measured at 8-9 years of age. Genital staging and testicular volume determination was assessed at annual follow-up visits until age 18 years. The highest quartile of TEQs (a measure of dioxin potency) compared to the lowest was associated with a delay in pubertal onset and maturation by 11.6 months for testicular volume (TV >3 ml for onset; >20 ml for sexual maturity) and 10 months for genital staging (defined as G2 and G5) ( 19). Conversely, non-dioxin-like PCBs co-adjusted for TEQs, were associated with earlier pubertal onset by 8.3 months (TV) and 6.3 months (G2) and earlier sexual maturity by 6-7 months. These compounds were not associated with changes in timing of pubic hair development. In this cohort, higher quartiles of TCDD and PCDD TEQs were also associated with lower sperm concentration, total sperm count, and total motile sperm count (p-trends ≤ 0.05) ( 20), suggesting that childhood exposures to these organochlorines not only altered pubertal timing but were also associated with poorer semen parameters, raising concerns about long term effects of EDCs on reproductive function and fertility.

Dietary exposures

One area of particular interest to parents is whether dietary exposures through dairy, meat or soy intake can alter the timing of puberty. A longitudinal study of 515 Chilean girls recruited at ages 3-4 years investigated the relationship between dairy intake and pubertal changes ( 21). Breast DXA was performed in 290 girls and 324 girls reported age of menarche. Breast volume was highest in girls who drank more sweetened milk-based drinks and lowest in those who ate the most yogurt. Consumption of yogurt and low fat milk was associated with later menarche by 4.6 months. Another group looked at 367 mother-daughter dyads in the UK Avon Longitudinal Study of Parents and Children ( 22). Six phytoestrogens were measured in urine collected during pregnancy. Enterodiol (a lignan metabolite) was associated with lower risk of earlier menarche, defined as <11.5 years of age, while O-DMA (a daidzein metabolite) was associated with an increased risk of earlier menarche, The four other lignans studied, including enterolactone were not associated with age of menarche. This study controlled for timing of sample collection, race, maternal education, age at menarche delivery, BMI, diet and smoking history, infant feeding (maternal breast milk vs soy milk), childhood BMI and vegetarianism. These findings contrasted with the BCERP data that showed an association of enterolactone, a lignan metabolite, both a 4-5 month later age of menarche as well as later thelarche ( 13). This association was more marked in leaner girls and the authors speculate that the conflicting findings may be due to different exposure windows (gestational versus childhood) or sample size.

A prospective study of 456 girls in Bogota, Columbia, investigated the association of red meat intake at ages 5-12 and age at menarche via food-frequency questionnaires ( 23). They found earlier menarche with higher red meat consumption. Those who ate meat more than twice daily had significantly earlier menarche than those who ate it less than four times per week. The investigators also found that those who ate canned tuna/sardine intake more than once per week had later menarche than those who ate them less than once per month. Average age of menarche was 12.4 years in this study. Intake of dairy, poultry, eggs, and other fish were not significantly associated. The meat effect was attenuated when adjusted for BMI.

Pesticides

Another area of considerable interest is whether there is a difference between organic food and conventional food. A study examined 3-phenoxybenzoic acid (3-PBA), a nonspecific metabolite of 18 pyrethroids in 305 9-15 year old Chinese girls with self-reported pubertal staging and menarche status ( 24). These insecticides can be both estrogenic and anti-androgenic, and are used in homes and for agriculture. Exposure occurs via diet as well as through skin. Higher 3-PBA was associated with later pubertal development for breast development, pubic hair, and menarche. The Avon Longitudinal Study examined gestational exposures to weakly estrogenic organochlorines ( 24). Exposure occurs through fatty foods (meat, fish, dairy), as well as via the placenta and maternal breast milk. This nested case-control study measured maternal serum organochlorine concentrations at random times in pregnancy. Among 3682 girls who self-reported age at menarche; 218 girls were 'early' (menarche before 11.5 years), and 230 were "controls" (ages 11.5 years or older). There were no associations between in utero exposure to organochlorines and early menarche. The investigators controlled for race, mother's BMI, mother's age at menarche and at delivery, SES, birth order, child's BMI at age 7, duration of breastfeeding, child's birth weight, and trimester of sample collection.

The associations of insecticides measured as urinary metabolites (3-PBA) with self-reported pubertal staging and self-measured testicular volumes were examined in 463 Chinese boys, ages 9-16 years ( 24). 3-PBA was detected in 98.7% of all urine samples and was inversely correlated with the frequency of organic food intake. After controlling for confounders such as SEC, age, and other birth history factors, higher levels of 3-PBA were associated with increased odds of being at a more advanced stage of puberty and having higher urinary gonadotropins.

Air pollution

Lastly, the BCERP group published two studies on the effects of tobacco exposure and air pollution. Prenatal and contemporary second-hand tobacco exposure was assessed by questionnaire and urinary cotinine measurement at enrollment. Girls with higher prenatal and secondhand smoke exposure have earlier pubarche but not thelarche. Pubarche occurred 10 months earlier in the highest vs the lowest prenatally exposed groups ( 25). To examine the effects of exposure to the polycyclic aromatic hydrocarbons in air pollution, residential proximity to traffic metrics was used as a proxy measure ( 26). Proximity to major roads at age 6-8 years was associated with earlier PH across all ethnic groups, controlling for confounders. Higher exposed girls had pubarche 2-9 months earlier than lower exposed. There was no association with thelarche timing.

Contributor Information

Louise C. Greenspan, Pediatric Endocrinologist, Kaiser Permanente, San Francisco, Clinical Professor of Pediatrics, University of California, San Francisco School of Medicine, (415) 833-3533, fax (415) 833-4177.

Mary M. Lee, Chair and the Stoddard Professor of Pediatrics, Department of Pediatrics, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, 508-857-3199, 508-856-1540.

# data[].attributes.attachment_link.title
Endocrine Disrupters and Pubertal Timing

# data[].attributes.attachment_link.description
Puberty is a fundamental process of maturational changes that mark the transition from sexual infancy to reproductive maturity. It is now recognized that the age of onset of breast development, exemplifying an early stage of puberty in girls, has declined in recent decades, although the age of menarche, an advanced pubertal milestone, has not changed significantly ( 1, 2, 3).

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JaiMarie Washington-Martin: Exposure to EDCs affect the mammary gland development in women and is potentially linked to the onset of precocious puberty.

# data[].attributes.subject
JaiMarie Washington-Martin: Exposure to EDCs affect the mammary gland development in women and is potentially linked to the onset of precocious puberty.

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Affiliation

¹ U.S. Environmental Protection Agency, ORD, National Health and Environmental Effects Research Laboratory, Reproductive Toxicology Division, MD-67, Research Triangle Park, North Carolina 27711, USA. fenton.suzanne@epa.gov

Affiliation

¹ U.S. Environmental Protection Agency, ORD, National Health and Environmental Effects Research Laboratory, Reproductive Toxicology Division, MD-67, Research Triangle Park, North Carolina 27711, USA. fenton.suzanne@epa.gov

Abstract

Breast cancer is the most common non-skin cancer among women in this country. Breast cancer risk is significantly influenced by genetics, but over 70% of the women that are diagnosed have noninherited or sporadic cancer. The risk of breast cancer is thought to be modified by lifestyle and environment. Exposures to certain chemicals and hormone-mimicking or endocrine-disrupting compounds (EDCs) are suspected of contributing to increased breast cancer incidence as well as precocious puberty in the United States. Studies of EDC effects in rodents indicate that multiple toxicants can alter mammary gland development, with or without changing other markers of puberty. EDCs can cause transient and persistent effects on mammary gland development depending on dose, exposure parameters, and whether exposure was during critical periods of gland growth or differentiation. Adverse effects from these abnormal developmental patterns include the presence of carcinogen-sensitive structures in greater numbers or for longer periods in the gland and inhibited functional differentiation leading to malnutrition or increased mortality of their offspring. Developmental toxicants of the mammary gland could lead to an increase in the incidence of mammary tumors if they alter circulating or tissue-localized hormone levels, gland receptor expression patterns, hormone transport, or metabolism that results in altered response to endogenous hormones or growth factors. Environmental disruptors of rodent mammary gland development must be identified for informed decisions in epidemiological studies aimed at identification of environmental factors contributing to breast cancer risk, altered breast development during puberty, or inability to produce sufficient breast milk.

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national center for biotechnology information

# data[].attributes.attachment_link.title
Endocrine-disrupting compounds and mammary gland development: early exposure and later life consequences - PubMed

# data[].attributes.attachment_link.description
Breast cancer is the most common non-skin cancer among women in this country. Breast cancer risk is significantly influenced by genetics, but over 70% of the women that are diagnosed have noninherited or sporadic cancer. The risk of breast cancer is thought to be modified by lifestyle and environmen ...

# data[].attributes.body
In this article they examined how BPA affects puberty. They discovered that high urninary levels of BPA has an association with early onset puberty but could not prove causation.

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Affiliations

¹ Pediatric Clinic, Department of Surgical and Biomedical Sciences, Università degli Studi di Perugia, 06129 Perugia, Italy. alberto.leonardi88@gmail.com.

² Pediatric Clinic, Department of Surgical and Biomedical Sciences, Università degli Studi di Perugia, 06129 Perugia, Italy. marta.cofini@gmail.com.

³ Institute of Pediatrics, Università Cattolica Sacro Cuore, Fondazione Policlinico Universitario A. Gemelli, 00168 Rome, Italy. drigante@gmail.com.

⁴ Pediatric Clinic, Department of Surgical and Biomedical Sciences, Università degli Studi di Perugia, 06129 Perugia, Italy. lauralucchetti@yahoo.it.

⁵ Institute of Pediatrics, Università Cattolica Sacro Cuore, Fondazione Policlinico Universitario A. Gemelli, 00168 Rome, Italy. cipolla.clelia@gmail.com.

⁶ Pediatric Clinic, Department of Surgical and Biomedical Sciences, Università degli Studi di Perugia, 06129 Perugia, Italy. laura.penta@ospedale.perugia.it.

⁷ Pediatric Clinic, Department of Surgical and Biomedical Sciences, Università degli Studi di Perugia, 06129 Perugia, Italy. susanna.esposito@unimi.it.

Free PMC article

Free PMC article

Affiliations

¹ Pediatric Clinic, Department of Surgical and Biomedical Sciences, Università degli Studi di Perugia, 06129 Perugia, Italy. alberto.leonardi88@gmail.com.

² Pediatric Clinic, Department of Surgical and Biomedical Sciences, Università degli Studi di Perugia, 06129 Perugia, Italy. marta.cofini@gmail.com.

³ Institute of Pediatrics, Università Cattolica Sacro Cuore, Fondazione Policlinico Universitario A. Gemelli, 00168 Rome, Italy. drigante@gmail.com.

⁴ Pediatric Clinic, Department of Surgical and Biomedical Sciences, Università degli Studi di Perugia, 06129 Perugia, Italy. lauralucchetti@yahoo.it.

⁵ Institute of Pediatrics, Università Cattolica Sacro Cuore, Fondazione Policlinico Universitario A. Gemelli, 00168 Rome, Italy. cipolla.clelia@gmail.com.

⁶ Pediatric Clinic, Department of Surgical and Biomedical Sciences, Università degli Studi di Perugia, 06129 Perugia, Italy. laura.penta@ospedale.perugia.it.

⁷ Pediatric Clinic, Department of Surgical and Biomedical Sciences, Università degli Studi di Perugia, 06129 Perugia, Italy. susanna.esposito@unimi.it.

Abstract

Many scientific studies have revealed a trend towards an earlier onset of puberty and have disclosed an increasing number of children that display precocious puberty. As an explanation, some authors have considered the global socio-economic improvement across different populations, and other authors have considered the action of endocrine disrupting chemicals (EDCs). Among these, bisphenol A (BPA), an aromatic compound largely used worldwide as a precursor of some plastics and chemical additives, is well known for its molecular oestrogen-like and obesogenic actions. We reviewed the medical literature of the previous 20 years that examined associations between BPA exposure and the age of puberty in humans, considering only those referring to clinical or epidemiological data. Of 19 studies, only 7 showed a correlation between BPA and puberty. In particular, the possible disruptive role of BPA on puberty may be seen in those with central precocious puberty or isolated premature breast development aged 2 months to 4 years old, even if the mechanism is undefined. Some studies also found a close relationship between urinary BPA, body weight, and early puberty, which can be explained by the obesogenic effect of BPA itself. The currently available data do not allow establishment of a clear role for BPA in pubertal development because of the conflicting results among all clinical and epidemiological studies examined. Further research is needed to fully understand the potential role of exposure to EDCs and their adverse endocrine health outcomes.

Keywords: Key-words: bisphenol A; menarche; premature thelarche; pubertal development; puberty.

Conflict of interest statement

The authors declare that they have no competing interests.

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national center for biotechnology information

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The Effect of Bisphenol A on Puberty: A Critical Review of the Medical Literature - PubMed

# data[].attributes.attachment_link.description
Many scientific studies have revealed a trend towards an earlier onset of puberty and have disclosed an increasing number of children that display precocious puberty. As an explanation, some authors have considered the global socio-economic improvement across different populations, and other authors ...

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This journal article explores the effect of fluoride exposure before puberty, finding that fluoride exposure has a high association with late pubertal development in males ages 10-17. There was no association found between fluoride exposure and late pubertal development in females.

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Abstract

Keywords: Fluoride, Puberty, Pubic hair, Genitalia, Menarche

Background

Fluoridation has been used globally for decades to reduce dental caries. Results of earlier studies [ 1, 2] have demonstrated that optimally fluoridated water prevents dental caries without posing serious risks to human health. However, a U.S. National Research Council (NRC) review on fluoride toxicity indicated that high fluoride levels in drinking water (≥4 ppm) are associated with increased risk of enamel fluorosis, bone fractures and joint pain [ 3]. Therefore, the NRC committee recommended that the U.S. Environmental Protection Agency's (EPA) Maximum Contaminant Level Goal for fluoride (4 ppm) should be lowered.

In addition to the effects of fluoride on bone and teeth, growing evidence is showing potential impact of fluoride exposure on the alterations in reproductive hormones, fertility, and possibly timing of sexual maturity [ 3]. However, studies of the association between fluoride exposure and sexual maturity are rare. One animal study reported that fluoride exposure can cause earlier sex maturation in female Mongolian gerbils [ 4]. An ecological study of girls in New York observed an earlier age at menarche in relation to fluoride exposure [ 5], while another ecological investigation of Hungarian girls reported no associations [ 6]. On the other hand, more recent studies have related fluoride exposure to decreased sex hormones in humans [ 7- 9].

The existing epidemiological investigations were limited by the ecological design to estimate childhood exposure to fluoride using drinking water concentrations at population level (i.e. lacked individual fluoride biomarkers) and using single indicator of pubertal development (i.e. menarcheal age). In addition, no analyses have evaluated the association between fluoride exposure and sexual maturity in boys. Therefore, the objective of this cross-sectional study was to address these aforementioned knowledge gaps by examining the association between childhood exposure to fluoride levels (via specific-gravity adjusted urinary measures) and physical markers of pubertal development in 157 boys and 176 girls at age 10-17 years residing in Mexico City. Physical markers of pubertal development in girls included pubic hair, breast maturation and age at menarche, and in boys included pubic hair, genital maturation and testicular volume.

Methods

Study population

In this study, we included participants from the Early Life Exposures in Mexico to ENvironmental Toxicants (ELEMENT) project, which is an on-going birth cohort study of both pregnant women and their offspring in Mexico City. Child participants ( n = 554) were invited to participate in the follow-up study between 2015 and 2017 based on the availability of maternal biological samples and age 10-17 years (thus likely to reach different stages of pubertal transition). Pertinent details of ELEMENT study, such as recruitment, eligibility criteria and collection of maternal information have been described previously [ 10, 11]. During the follow-up visit, child participants provided urine samples, completed anthropometric measures and physician-assessed Tanner stages. Interview-based questionnaires were also completed at the visit. Among these 554 participants, 347 had data on urinary fluoride with measured specific gravity. We further excluded participants who had no measurement of pubertal development, leaving 339 children. Of these 339 participants, 333 had complete information on the covariates of interest including child age and body mass index (BMI) z-score, number of siblings at birth, maternal education and marital status and were included in the final analysis.

Research protocols were approved by the Institutional Review Board at University of Michigan and the University of Indiana, and the Research, Biosafety and Ethics in Research at the Mexico National Institute of Public Health. Prior to enrollment, informed consent from mothers and informed assent from offspring were obtained.

Urinary fluoride

Spot urine samples of about 5 mL were collected in fluoride-free vials, frozen immediately at the research site, and shipped and stored at − 80 °C at University of Michigan School of Public Health. The samples were analyzed at the Indiana University School of Dentistry Oral Health Research Institute. Urinary fluoride levels were assessed using ion-selective electrode-based diffusion assays following standardized analytic methods. Detailed information regarding the protocol, validation and quality control of measuring fluoride in urine has been described elsewhere [ 11- 13]. Specific gravity was used to correct for variations in urine dilution at the time of measurement and was analyzed using a handheld digital refractometer (Atago Co., Ltd., Tokyo, Japan). Specific gravity-adjusted urinary fluoride concentrations (UF sg) were calculated for each participant using the following formula: UF sg = UF child * (1.02-1) / (specific gravity-1). The median level of specific gravity is 1.02 in the study population.

Pubertal indicators

Tanner staging with a range from stage 1 indicating pre-pubertal to stage 5 indicating fully mature was used to assess pubertal development of all participants [ 14]. Tanner staging of breast and pubic hair growth in girls as well as Tanner staging of genitalia and pubic hair growth in boys were assessed by a trained pediatrician using standardized protocols. The assessment of pubertal stages has been validated by comparing with a panel of serum sex hormones in ELEMENT children [ 15]. Menarche was measured via a self-reported questionnaire, where girls at age 10-17 years were asked if and when they had initiated menses. In boys, testicular volume was used as an additional indicator of puberty. Testicular volume (right and left) was determined by comparing with a Prader orchidometer (range 1-25 mL). In the present analysis, the larger volume of the right or left testicles was used. Testicular volume ≥ 20 mL was defined as matured stage [ 14].

Statistical analysis

Univariate and bivariate analyses were conducted. The distribution of specific gravity-adjusted urinary fluoride was right skewed. Given that normality on independent variable is not required for logistic or Cox proportional-hazard regression models, we did not transform the non-normal variable. We compared the differences in key covariates between included and excluded participants due to missing data. We also compared the characteristics of participating children by sex. We performed t-test, Wilcoxon rank-sum test or the analysis of variance (ANOVA) to assess the differences between continuous variables, and adopted Chi-square test to examine the differences between categorical variables. Covariates considered in this analysis as predictors of puberty or potential confounders included child age and BMI z-scores, maternal education [ 16] and marital status [ 17], as well as number of siblings at birth [ 18]. BMI was calculated and then converted to an age- and sex- specific z-score based on the World Health Organization (WHO) standard curves for children aged from 5 to 19 years [ 19]. Lead has also been shown to be associated with pubertal development. In fact, we previously reported that prenatal and early childhood exposure to lead were associated with later pubertal development in girls [ 14]. However, in this study, we did not include peripubertal lead levels at age 10-17 years in the models because we did not observe a significant association between peripubertal lead levels and any markers of pubertal development.

We determined the associations between specific gravity-adjusted urinary fluoride and pubertal stages for the maturation of breast, pubic hair and genitalia using multivariate ordinal regression models. Testicular volume (≥20 mL versus < 20 mL) was examined using logistic regression models. Results for ordinal regression models were reported as the odds of reaching a high stage of puberty (breast, pubic hair or genitalia) versus combined low and middle stages, with a one-interquartile range (IQR) increase in urinary fluoride concentrations. Results for logistic regression models were reported as the odds of having fully matured testicular volume (≥20 mL) versus having juvenile testicular volume (< 20 mL), with a one-IQR increase in urinary fluoride concentrations.

We used survival techniques (time-to-event analysis) to examine the association between fluoride concentration and age at menarche in girls, which adequately account for censored data [ 20]. Hazards ratios (HRs) and 95% confidence intervals (CIs) were estimated using Cox proportional-hazard regression models, which has been widely used to analyze age at menarche. Time to menarche was based on the self-reported age of menarche (years) or right-censored observations using the age at the interview (i.e. for girls who had not initiated menses). First, we included urinary fluoride concentration as a continuous variable in the models. Second, we divided the urinary fluoride into a categorical variable by tertiles. We defined statistical significance as p  < 0.05. All statistical analyses were performed using SAS (version 9.4; SAS Institute Inc., Cary, NC, USA).

Sensitivity analysis

In sensitivity analysis, we examined the adjusted prospective association between prenatal exposure to fluoride during pregnancy and pubertal development in boys and girls. Only a small subset of participants had data on prenatal fluoride, as measured by urinary fluoride in any trimester of pregnancy with the adjustment of creatinine in urine ( n = 90 for boys and n = 111 for girls). The mean of all available creatinine-adjusted urinary fluoride during pregnancy was obtained. In addition, we assessed the adjusted prospective association between childhood exposure to fluoride at age 6-12 years and pubertal development in boys and girls. Similarly, only a small subset of participants had data on childhood exposure fluoride, as measured by urinary fluoride at age 6-12 years with the adjustment of specific gravity in urine ( n = 66 for boys and n = 72 for girls). Detailed information regarding the methods for fluoride analysis has been published elsewhere [ 11].

Results

In this study, only child age and maternal education (borderline significant) of included children were significantly different from those who were excluded (Table ). Children included in the study were younger at the visit (mean 13.9 versus 15.5 years, p < 0.0001), and their mothers received higher education (mean 11.1 versus 10.6 years, p  = 0.05). Most of the participating children's mothers were married (73.6%). Included children had an average BMI z-score at 0.5 and had on average two siblings, and 47.2% were boys. The overall geometric mean and IQR of urinary fluoride (specific gravity adjusted) in participating children were 0.59 mg/L and 0.31 mg/L, respectively.

Table 1

The distributions of key covariates among participating boys and girls are shown in Table . When comparing the characteristics of included participants by sex, we did not find significant differences in any key variables. The concentrations of urinary fluoride did not differ significantly across the categories of these key covariates (Table ). Girls' mothers who received education > 12 years had the lowest level of urinary fluoride (geometric mean: 0.51 mg/L); however, the difference was modest and not statistically significant ( p  = 0.08). Table shows the distribution of physician-assessed pubertal development of boys and girls at age 10-17 years by physical indicators. One hundred and thirty-seven girls (78.3%) had initiated menarche with mean (SD) menarcheal age at 11.4 years (SD = 1.2).

Table 2

Table 3

Table 4

In boys, we found significant negative associations between specific gravity-adjusted urinary fluoride concentrations and pubic hair growth and genital maturation, controlling for child age and BMI z-score, number of siblings at birth, maternal education and marital status (Table ). With a one-IQR increase (0.31 mg/L) in urinary fluoride, the odds of reaching a high stage of pubic hair growth decreased by 29% (Odds ratio (OR): 0.71, 95% confidence interval (CI): 0.51-0.98, p = 0.03); the odds of reaching a high stage of genitalia decreased by 29% (OR: 0.71, 95% CI: 0.53-0.95, p = 0.02) in boys. We did not observe a significant difference in the odds of having matured testicular volume (> = 20 ml) in relation to urinary fluoride levels, although the direction was consistent (OR: 0.90, 95% CI: 0.65-1.27, p  = 0.56).

Table 5

In girls, we found that specific gravity-adjusted urinary fluoride was negatively associated with pubertal stages for pubic hair growth and breast maturation adjusting for the same covariates, although the associations did not reach significance (pubic hair OR: 0.86, p = 0.29; breast OR: 0.85, p  = 0.30) (Table ). Similarly, urinary fluoride was not significantly related to menarcheal age in girls regardless of treating fluoride as a continuous or a categorical variable, adjusted for number of siblings at birth, maternal education and marital status, as well as child BMI z-score. However, the direction of these associations was negative.

Table 6

In sensitivity analysis, we examined the adjusted prospective association of creatinine-adjusted urinary fluoride during pregnancy (See Additional file 1: Table S1 and S2) and specific gravity-adjusted childhood urinary fluoride (age 6-12 years) (See Additional file 1: Table S3 and S4) with physical markers of puberty in a much smaller subset of our participants who had available data. Overall, we found no significant associations with any measures of pubertal development in these boys or girls, although girls with childhood urinary fluoride at age 6-12 years in the 3rd tertile had a later age at menarche compared with girls in the 1st tertile (Hazard ratio (HR): 0.37, 95% CI: 0.16-0.86, p = 0.02) (See Additional file 1: Table S4).

Discussion

This is the first study that examines the relationship between a biomarker of fluoride exposure and multiple physical indicators of pubertal development in children of both sexes. Our findings suggest that fluoride exposure at the levels observed in this study during peripubescence is associated with later pubertal development for pubic hair growth and genital maturation in Mexican boys. We found a negative association between peripubertal fluoride and pubertal development in girls, but the results did not reach significance.

An ecological study of 804 Hungarian girls at age 10-19 years observed no substantial differences in the age at menarche between girls living in the high-fluoride town Kunszentmárton (with life-long exposure to fluoridated drinking water at 1.09 mg/L) and girls living in the low-fluoride town Kiskunmajsa (0.17 mg/L) [ 6]. On the contrary, an animal study of 24 female Mongolian gerbils revealed earlier sex maturation (i.e. earlier vaginal opening and the development of ventral gland) occurred in high fluoride treatment group (3.7 mg/kg/day) versus low fluoride treatment group (0.7 mg/kg/day) [ 4]. An earlier ecological study of 405 girls aged 7-18 years who had been exposed to fluoridated water up to 10 years showed that the average age at menarche was 12 years among girls in fluoridated Newburgh, New York (fluoridated drinking water 1.2 mg/L), versus 12 years 5 months among girls in Kingston, where water was not fluoridated (essentially fluoride-free) [ 5]. The main fluoride exposure sources of the published ecological studies were different from ours: their participants received fluoride through water supply, while our participants were exposed to fluoride through table salt at 200-250 mg/kg salt. In addition to the ecological design, these studies are limited by reliance on self-reported age at menarche, which can be susceptible to recall bias. It has been proposed by Luke that earlier pubertal onset may be a result of reduced release of melatonin from pineal gland in response to fluoride exposure [ 4]. Decreased melatonin has been shown to increase gonadotropins and subsequently stimulate sex hormones and eventually accelerate pubertal development [ 21], but the effect of fluoride on the melatonin production has not been well characterized. A more recent animal study provides some evidence for lower estradiol and progesterone in serum, and damaged ovaries in female rats receiving high fluoride treatment (100 mg/L) [ 22], which may lead to a delayed pubertal development. More studies in this area are warranted.

In males, previous investigations have linked fluoride exposure to decreased sex hormones in humans. A Mexican study revealed that male adults with an average urinary fluoride at 3.2 mg F/g creatinine had lower serum testosterone and inhibin-B levels compared to those with an average urinary fluoride at 1.6 mg F/g creatinine [ 7]. Similarly, the lower concentration of testosterone in serum was observed in male adults who had high versus low urinary fluoride (mean 2.64 versus 0.94 mg/L) in Henan, China [ 9]. An Indian analysis also found that male adult patients with skeletal fluorosis (mean urinary fluoride 3.5 mg/L) had lower concentration of testosterone compared with those who were healthy [ 8]. Although decreased melatonin levels due to fluoride exposure could potentially increase testosterone as described above [ 4], it has been suggested that fluoride may directly impair the structure and function of Leydig cells and disrupt the activities of the hypothalamic-pituitary-thyroid (HPT) axis, and hence the release of testosterone can be reduced [ 23, 24]. In addition, Luke's study showed that the testicular weight in gerbils at 16 weeks was lower in high fluoride treatment group versus low fluoride treatment group [ 4]. It is possible that the decreased levels of testosterone and inhibin-B may in turn postpone puberty, which may explain the negative associations observed in our study.

According to a recent review [ 25], late puberty has been associated with low self-esteem, disruptive behavior disorder and substance use in young adults, depression and anxiety in later adulthood. Late puberty may also be associated with an increased risk of bone fracture and coronary heart disease in adults [ 25- 27].

Unlike our study, the majority of previous studies that measured urinary fluoride concentrations did not correct for variations in urine (e.g. adjust for urinary specific gravity levels). A study of 294 Canadian children aged 3-12 years reported a mean specific gravity-adjusted urinary fluoride concentration at 0.80 mg/L [ 28]. Seventy-eight children from Iran at age 4 years had a mean value of urinary fluoride concentration (specific gravity adjusted) at 0.73 mg/L [ 29]. A Mexican study of 108 children at age 6-12 years residing in rural San Luis Potosi reported a mean specific gravity-adjusted urinary fluoride level at 3.14 mg/L [ 30]. Here we compared the urinary fluoride levels in our cohort (geometric mean: 0.59 mg/L) with the levels reported by other populations, although current studies were quite limited.

The study has some limitations. The sample size of our analysis is small for modeling ordinal outcomes, which may limit the power to detect significant associations between fluoride exposure and pubertal development. The use of spot urine samples (versus 24 h collections) also provides an additional limitation, since fluoride concentration values have been reported to fluctuate at different collection points [ 31]. As noted above, the use of self-reported age at menarche can be subject to recall bias. However, previous studies have shown that the actual menarcheal age was highly correlated with self-reported menarcheal age within 5 years of follow-up among peripubertal girls [ 32], within 7 years [ 33] among women and up to 33 years starting at age 7-9 years [ 34]. This study also has several advantages over the existing human studies including the use of individual biomarkers of fluoride exposure and multiple markers of physician-assessed pubertal development in both boys and girls.

Conclusions

We found a significant association between an increase in peripubertal fluoride exposure at the level observed in our study population and later pubertal development in Mexican boys at age 10-17 years. However, pubertal development in girls was not significantly associated with fluoride exposure, which is inconsistent with previous animal and ecological studies. Further research with a larger sample size and multiple collections of urine samples is needed to confirm our findings.

Additional file

Acknowledgments

We thank the study team and participants from ELEMENT study and thank ABC Hospital for providing facilities for this research. We also thank Christine Buckley and Prithvi Chandrappa for their work in the Indiana fluoride labs.

Abbreviations

Authors' contributions

YL conducted data analyses and drafted the manuscript. KEP and MTR conceived the study and oversaw its coordination. HH, EAMM and NB helped oversee the study and advised the interpretation and drafting of the manuscript. BNS helped oversee the study and advised during the data analysis. AMG and MSG participated in the collection and assembly of data. All authors read and approved the final manuscript.

Notes

Ethics approval and consent to participate

Research protocols were approved by the Institutional Review Board at University of Michigan and the University of Indiana, and the Research, Biosafety and Ethics in Research at the Mexico National Institute of Public Health. Prior to enrollment, informed consent from mothers and informed assent from offspring were obtained.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

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Fluoride exposure and pubertal development in children living in Mexico City

# data[].attributes.attachment_link.description
Keywords: Fluoride, Puberty, Pubic hair, Genitalia, Menarche Fluoridation has been used globally for decades to reduce dental caries. Results of earlier studies [ 1, 2] have demonstrated that optimally fluoridated water prevents dental caries without posing serious risks to human health. However, a U.S.

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V Herron: BPA and Phthalate exposure and female pubertal differences.

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V Herron: BPA and Phthalate exposure and female pubertal differences.

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Proper nutrient intake in adolescence can help with healthy puberty in developing years. Exposure to EDC's have been linked to possibly cause IUGR in children.

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Role nutrition plays in our development, week 5 ass.

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Summary of multiple studies looking at the effects of early-life exposure to EDCs on pubertal onset and tempo for male reproductive development. Concluded there is an effect on timing but the effects on adulthood reproduction factors are yet to be determined.

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Male pubertal development: are endocrine-disrupting compounds shifting the norms?

# data[].attributes.attachment_link.description
Endocrine-disrupting compounds (EDCs) are synthetic or natural compounds that interfere with endogenous endocrine action. The frequent use of chemicals with endocrine active properties in household products and contamination of soil, water, and food sources by persistent chemical pollutants result in ubiquitous exposures. Wildlife observations and animal toxicological studies reveal adverse effects of EDCs on reproductive health.

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Endocrine disruptors have been causing a 1-2 year jumpstart to puberty. A few major ones in this study include phytoestrogens, organohalogens, pesticides, and others. Natural and synthetic endocrine disruptors are discussed as well.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3065309/

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INTRODUCTION

Recent studies have demonstrated a progressive decrease in age of onset of puberty in children around the world. Although the exact reason for this shift is not completely understood, it is generally accepted to be the outcome of a complex interaction between genetic, endocrine and environmental factors. It is also known that owing to the acceleration of industrialization throughout the world, a gradual but significant increase has occurred in the number and amount of environmental pollutants. Some of these environmental pollutants are natural or synthetic chemicals with considerable effects on the endocrine system. The chemicals that have negative effects on the endocrine system are called endocrine disruptors (EDs). EDs exert their effects through different mechanisms: by binding to the relevant hormone receptors; by direct action on cell signaling pathways or on the central nervous system and the neuroendocrine system; by suppression of hormone synthesis; or through their toxic effects on the relevant organs. Several EDs such as phytoestrogens, topical and natural estrogens, pesticides, industrial chemicals and phthalates have been identified as possible agents affecting pubertal development in humans. The potential of EDs to cause precocious puberty has first been noticed in the early 1990s. Afterwards, the effects of EDs on the onset and course of puberty have been demonstrated in numerous animal and human studies and, subsequently, the use of some of these chemicals has even been prohibited. This paper aims to review the present information regarding environmental EDs that may negatively affect pubertal development.
The Effects of Endocrine Disruptors on Puberty
Recent studies have demonstrated that age of onset of puberty has shifted to younger ages by about 1 or 2 years, while no change has been noted in the age of menarche. The triggering mechanism for the earlier onset of puberty is not clearly understood. It is thought to occur as a result of a complex interaction between genetic, hormonal and environmental factors. Recently, ED chemicals have been intensively accused of being potential hazardous environmental factors ( 1, 2, 3, 4, 5, 6).

EDs are environmental chemicals, which may either be natural or synthetic. Some of the major EDs are presented in . EDs accumulate in the environment in the long term and are introduced into the human body through water, air, foodstuffs, or through equipments used in the office and home. Additionally, it has been demonstrated that EDs can be transferred from the mother to the fetus via placenta or to the baby via breast milk ( 1, 2, 3, 4, 5).

Due to their hormone-like characteristics, EDs mostly affect the endocrine system in an agonist- or antagonist-specific manner and can be classified according to their mechanisms of action. They influence puberty through their estrogenic, antiestrogenic, androgenic, antiandrogenic effects or through their direct effects on the gonadotropin-releasing hormone (GnRH). These chemicals may exert their estrogenic effects either directly by binding to estrogen receptors, increasing aromatase activity and increasing estrogen sensitivity or indirectly by their effect on GnRH, leading to an increase in endogenous estrogen production. All of these effects may result in precocious puberty. EDs produce antiestrogenic and androgenic effects through inhibition of aromatase enzyme activity and steroidogenic enzyme production. They display antiandrogenic effects via suppression of testicular steroidogenesis and androgen-receptor blockade. Thus, depending on their mechanism of action, EDs may lead to precocious puberty, to delayed puberty, or to sexual differentiation disorders ( 1, 4, 5, 6, 7, 8, 9, 10, 11). Classification of some of the EDs according to their mechanism of action is presented in .
Natural Endocrine Disruptors
The best known chemicals in this group are phytoestrogens, which are relatively weak compared to endogenous estrogen. They are found in several nutrients that are frequently consumed in daily life (i.e. carrots, garlic, apple, coffee, cherry, parsley, legumes). Phytoestrogens have estrogenic effects when consumed in huge amounts and antiestrogenic effects at low concentrations ( 4, 7, 8).
Synthetic Endocrine Disruptors
Diethylstilbestrol (DES) is the best known ED with strong estrogenic activity. It was first synthesized in 1938 and has since been widely used worldwide for medical zindications including pregnancy toxemia and preterm labor. However, a twofold increase in breast cancer incidence was observed in mothers exposed to DES. It was also shown that the incidence of cervical cancer, ovarian germ cell cancer, cervical or vaginal dysplasia, and vaginal clear-cell adenocarcinoma was increased in female infants born to mothers exposed to DES ( 12). The production or marketing of this chemical is prohibited since 1997.

Many chemicals including pesticides, fungicides, herbicides h ₂o used in agriculture, cleaning substances used in daily life, contents of cosmetic products, dyes, plastic substances and solvents are likely to be EDs. As neutralization or inactivation is difficult and most of these substances often accumulate in fat tissue, they may persist in the body for long periods of time and cause harmful effects ( 4, 5, 6, 7, 8, 9, 10, 11). Time of exposure to EDs is important in terms of their detrimental effects. Male rodents exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in intrauterine life were found to experience problems in masculinization of internal and external genitalia, descent of testicles, androgen production and in spermatogenesis. On the other hand, it was observed that postnatal exposure to TCDD was associated only with impairment of spermatogenesis and somatic and genital growth. The dose and duration of exposure to EDs is also important in terms of potential negative consequences. The negative effects may become more serious as the duration of exposure and dose increase ( 1, 11, 13).

EDs do not necessarily lead to similar effects in all situations. For example, phytoestrogens have estrogenic effects in high doses, while they exert antiestrogenic effects in low doses ( 1, 7).
Studies on Role of EDs in Precocious Puberty
Several animal and human studies have been conducted investigating the role of EDs in precocious puberty. EDs may cause early puberty via their estrogenic or antiandrogenic effects and also by increasing GnRH production ( 1, 4, 5, 6, 7, 8, 9, 10, 11).
Animal Studies
EDs have first been noted in experimental studies and gained significance through evidence from animal experience. Following the accidental spill of high amounts of dichlorodiphenyltrichloroethane (DDT) and dicofol from a chemical company into Lake Apopka in Florida in 1980s, it has been noted that male alligators living in the lake had smaller phalluses, reduced serum testosterone levels and abnormal gonadal structures concurrently with high serum DDT levels ( 14). It was observed that administration of methoxychlor, a pesticide with potential estrogenic effects, in female rats during pregnancy and lactation periods, led to vaginal opening and appearance of puberty at a very early age in their female offspring ( 15). Similarly, accelerated and early vaginal opening was noted in female rats following subcutaneous injections of bisphenol A (BPA), genistein, resvetarol, zearalenone or DES in the prenatal period, findings confirming the estrogenic effects of these chemicals ( 16, 17). Administration of methoxychlor or 17 beta-estradiol after the cessation of breastfeeding also resulted in early vaginal opening in female rats ( 15, 17). In addition, Markey et al ( 18) showed that prenatal exposure to BPA caused early growth and proliferation of breast tissue as well as increased susceptibility to endogenous estrogen in female rats. Methoxychlor, an antiandrogenic and estrogenic agent, and also vinclozolin, an antiandrogenic agent, were shown to lead to delayed puberty in male rats and precocious puberty in female rats ( 19).

Dioxins are environmentally toxic chemicals. TCDD is a type of dioxin, which exerts its estrogenic or antiandrogenic effects by causing alterations in gene expression via binding to aryl hydrocarbon receptors. In utero, exposure of rodents to even low doses of this substance was found to result in reproductive system abnormalities and precocious puberty findings in the females and delayed puberty in the males ( 7, 8, 9, 10, 11, 20, 21, 22). Other chemicals that can produce the same effects by binding to aryl hydrocarbon receptors include industrial products such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ether and the pesticide methoprene ( 1, 7, 8, 9, 10, 11).

Some of the environmental chemicals may impair neuroendocrine functions through their effect on the central nervous system and the hypothalamic-hypophyseal-gonadal (HHG) axis. These include pesticides such as thiram, molinate, metam sodium, chlordimeform, amitraz, triazoles, dichloroacetic acid, atrazine, propazine, simazine, methanol and linuron ( 1). It has been shown in a study on rats that atrazine causes delayed puberty by suppressing luteinizing hormone (LH) and prolactin levels ( 23, 24, 25). In rats prenatally exposed to BPA, an increase in estrogen feedback as well as development of precocious puberty via inhibition of tyrosine hydroxylase activity in rostral preoptic periventricular neurons have been observed ( 26). Howdeshell et al ( 27) also noted precocious puberty in rats prenatally exposed to BPA.

Some of the EDs affect puberty by inhibiting the synthesis of endogenous hormones such as testosterone, 17 beta-estradiol and adrenal steroids via competitive inhibition of P450 steroidogenic enzymes (C17,20-lyase, aromatase) ( 1). Delayed puberty has been reported by exposure to imidazole group fungicides, ketoconazole and fadrozole in the peripubertal period ( 28). Another pesticide, prochloraz, suppresses estrogen and androgen synthesis via inhibition of aromatase and 17,20-lyase ( 29, 30).
Studies in Humans
Studies have shown that several environmental chemical pollutants including DDT/ dichlorodiphenyldichloroethylene (DDE), PCBs, polybrominated biphenyls (PBB),

Following the pollution of the environment by PBB as a result of an industrial accident in the state of Michigan in 2000, Blanck et al ( 31, 32) investigated the serum PBB levels in pregnant women and compared ages of puberty onset and menarche in their daughters who were or were not breast-fed. When the girls with intrauterine exposure to high PBB concentrations (>7 ppm) were compared with the girls who had no exposure (<1 ppm), menarche was observed to occur one year earlier in those who were exposed to high concentrations. Among those exposed to high concentrations, breast-fed girls were observed to have pubarche one year earlier than the girls who were not breast-fed. No significant difference in terms of breast development was reported in this study. Gladen et al ( 33) found a relationship between intrauterine exposure to high doses of these pesticides and early thelarche and early pubarche in girls.

Effects of DDT, with proven estrogenic effect, and its metabolite DDE on pubertal development have been investigated in several studies. After their detrimental effects had been explicated, DDT and its metabolites, formerly used widely as pesticides in agriculture, were banned in many countries. Its use has also been forbidden in Turkey since 1985. Vasiliu et al ( 34) reported that menarche occurred 1 year earlier in girls exposed to high amounts of DDT/DDE in the intrauterine period. Krstevska-Konstantinova et al ( 35) found that precocious puberty was 80 times more frequent and serum DDE levels significantly higher in daughters of immigrants compared to native Belgians. An association between serum DDT/DDE concentrations and early menarche was reported in a study conducted in Chinese textile workers ( 36).

Methoxychlor is an organochlorine widely used as a pesticide in agriculture. It was shown to impair reproductive behavior and functions in male rats due to its estrogenic effect ( 37). There are no human studies on the effects of this pesticide. Methoxychlor, which is a chlorinated hydrocarbon pesticide and has been introduced for use instead of DDT, accumulates in very little quantities in fat tissue. Although it has been reported to produce estrogenic effects similar to DDT and its metabolites in animal studies, there are no studies on the effects of this pesticide on precocious puberty in humans ( 15, 38, 39).

Endosulfan and its derivatives are pesticides widely used in agriculture and are thought to have antiandrogenic and estrogenic effects ( 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). In animal studies, endosulfan was reported to have an estrogenic effect and to cause inhibition of follicle-stimulating hormone (FSH), LH and testosterone production ( 40, 41). In an Indian study, LH was found to be increased and testosterone levels decreased in men exposed to endosulfan ( 41).

In a recent Danish study, as compared to the normal population, significantly earlier thelarche in girls and more frequent genital abnormalities such as hypospadias, micropenis in boys were noted in children of greenhouse owners, and it was suggested that these findings were due to pesticides. However, no pesticide analysis was conducted in this study ( 42). Antiandrogenic effect of vinclozolin has been shown in male animals ( 43). Although the estrogenic effect of this chemical used widely in agriculture is not known for sure, it has been reported that it might have an antiandrogenic effect and produce an estrogen-like effect through stimulation of estrogen receptor a ( 44).

The effects of PCB exposure on puberty have also been investigated by several authors. There are several sub-types of this group of substances that are thought to have estrogenic effects ( 1, 2, 3, 4, 5, 6, 7, 8, 9). While no relationship was detected between intrauterine and/or postnatal exposure to PCB and age of puberty or menarche in some studies, it was reported in others that menarche occurred significantly earlier in girls who were exposed to PCB 52, 70, 101, +90 and 187 sub-groups ( 45, 46).

Chlorine is used as a whitener in many solutions used in daily life. Dioxins are formed as by-products of incomplete combustion of chlorinated waste products. Contact of plastic products with hot surfaces (serving hot liquids or food in plastic glasses or plates, microwave use) may also lead to production of dioxins. Exposure of humans to huge amounts of dioxin is only possible in industrial accidents. However, exposure to very small amounts may occur due to the contact of several daily used substances such as plastic glasses, plates, toys, cleaning substances or paper whitened by chlorine with hot surfaces. Dioxins can be taken by animals through ingestion of contaminated soil, vegetables and water, and due to long-term adipose tissue accumulation, they may be transferred to humans from animal meat or milk ( 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). There are two human studies investigating this toxic substance, which has been found to be associated with estrogenic effect and precocious puberty in animal studies. In a Belgian study, Den Hond et al ( 47) observed a reduction in testicular volume in boys exposed to a substance with dioxin-like activity, but failed to note a change in pubertal development. In this same study, the authors reported a delay in the breast development in girls, but no change in age of menarche or pubarche. In a study on Italian girls, Warner et al ( 48) found no change in age of menarche in girls exposed to TCDD postnatally before the age of 5 years. Mothers of these children were not exposed to this substance during pregnancy or before.

Colon et al ( 49) observed significantly higher levels of phthalate esters in 31 Puerto Rican girls with early thelarche compared to controls. Phthalates are used as plastic softeners and as preservatives in some cosmetic products and they may be found in plastic toys, hair sprays, deodorants, shampoo, nail polish and perfumes, and may easily enter the human body ( 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). The use of these substances, which possess estrogenic and antiandrogenic properties, has been prohibited in many European countries.

Lead, a heavy metal and one of the major environmental pollutants, has also been found to affect puberty. It was observed that menarche and pubarche were delayed in girls with high serum lead levels ( 50, 51).

In addition to its teratogenic and cancerogenic features, BPA, which is found in huge amounts in polycarbonate plastics (i.e. baby feeding bottles), has also been suggested to have estrogenic effects causing precocious puberty. Although BPA has been investigated thoroughly in many animal studies, human studies are lacking. It has been suggested in some studies that exposure to BPA at early ages is associated with an increased incidence of breast cancer ( 52).

# data[].attributes.attachment_link.title
Effects of Environmental Endocrine Disruptors on Pubertal Development

# data[].attributes.attachment_link.description
The onset and course of puberty are under the control of the neuroendocrine system. Factors affecting the timing and regulation of the functions of this system may alter the onset and course of puberty. Several environmental endocrine disruptors (EDs) ...

# data[].attributes.body
Exposure to EDCs such as phthalates or phenols both in-utero and during early development is associated with altered pubertal timing in girls, but little evidence suggests boys are affected in the same way despite exposure.

# data[].attributes.body
This study analyzed phenols and phthalates, the timing of menarche, and the association between pre-pubescent and pubescent. They found a later menarche was associated with an increase of di(2-ethylhexl) phthalate biomarkers but a higher concentration of 2,5 dichlorophenol and benzophenone-3 were associated with a later menarche.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5883544/

# data[].attributes.attachment_link.readable_text
Abstract

Keywords: Menarche, Puberty, Phenols, Phthalates, Endocrine disrupting chemicals

Background

Determinants of pubertal timing are of public health concern because of the association between the age of onset and adult disease susceptibility. Among females, early menarche has been associated with an increased risk of type 2 diabetes, cardiovascular disease, and reproductive cancers, including breast [ 1- 3]. The average age of menarche has decreased from 16-17 years at the end of the nineteenth century, to less than 13 years across Europe and the United States, likely due to improvements in nutrition and general health [ 4, 5]. While the age of menarche was thought to have stabilized in the past 50 years, more recent studies have suggested that the age of menarche has continued to decline in South America, the United States, and many countries in Europe [ 6- 9]. The secular trend towards earlier menarche has been observed across race/ethnicity groups, despite differences in timing [ 7, 10- 12]. Within the United States, the average age of menarche is earlier among Hispanic and black females relative to white females, adjusting for BMI, as well as social and economic indicators [ 7, 13]. Across Europe and South America, the fastest decline has been among impoverished girls, in spite of earlier menarche being previously more common among affluent families [ 6, 8, 9, 14, 15].

The two primary hypotheses for this shift towards earlier menarche are the growing childhood obesity epidemic and increasing exposure to endocrine disruptor chemicals (EDCs) [ 16- 18]. EDCs can influence the endocrine system through a multitude of mechanisms, including competitive binding to hormone receptors to modulation of hormone synthesis and metabolism [ 19]. There is building evidence that EDCs can affect proper function across a number of different tissues, including breast tissue and female reproductive organs [ 19], as well as influencing the neuroendocrine control of reproduction. Results of both in vivo studies and a few longitudinal human cohorts suggest some of these compounds may be obesogenic, with early life exposure associated with childhood body size [ 20- 23]. Phenols and phthalates are two classes of EDCs of particular concern due to their use in an extensive array of products, including plastics, building materials, personal care products, insecticides, and herbicides [ 24]. In this study, we analyzed the association between pre-pubescent and pubescent biomarkers of exposure to select phenols and phthalates and the timing of menarche within a longitudinal cohort of Chilean girls born in 2002-2003. Two cross-sectional studies and four longitudinal studies have analyzed the influence of childhood EDC exposure on the age of menarche [ 25- 30]. The results of these studies have been largely discordant, potentially reflecting regional and socioeconomic influences on exposure profile, differences in the age of biomarker measurement, and possible effect modification by race/ethnicity. For this study, we quantified a broad panel of 26 phenol and phthalate urinary biomarker concentrations prior to the onset of breast development (Tanner 1; B1) and at Tanner 4 (B4). This study provides unique insight into the influence of these compounds on pubertal timing in Latina girls.

Methods

Study population

Our study population was a random subset of 200 girls part of the longitudinal Growth and Obesity Cohort Study (GOCS) with urine samples collected at B1 (ages 6.7 to 9.6 years; median age: 7.9 years) and B4 (ages 9.4 to 13.1 years; median age: 11.2 years). The GOCS children were born in 2002-2003, and are representative of the low to middle-income families in Santiago, Chile. A description of the cohort has been provided elsewhere [ 31]. Starting in 2009, breast development was assessed by two trained dietitians (kappa with pediatric endocrinologist = 0.85) by visual inspection using Tanner's rating scale approximately every 6 months [ 32]. Palpation was additionally used to distinguish breast Tanner 1 from Tanner 2. Sex and age-adjusted BMI Z-score at each visit were calculated based on the Centers for Disease Control and Prevention (CDC) growth charts. Girls were categorized as having excess weight (overweight or obese) if their BMI Z-score was equal or above the 85th percentile. Informed consent was obtained from all parents or guardians of children before the start of data collection.

Biomarker measurements

Fasting spot urine samples were collected between 10 AM and 12 PM in polypropylene sterile cups, and were immediately vortexed and aliquoted. Concentrations of 26 phenol and phthalate biomarkers were quantified in urines collected at breast Tanner 1 (B1) and Tanner 4 (B4) from 200 girls (400 samples). The analytical measurements were performed at the CDC National Center for Environmental Health Laboratory using previously described analytical methods [ 33, 34]. Concentrations below the limit of detection (LOD) were given an imputed value equal to LOD/sqrt(2) [ 35]. Biomarker concentrations (ng/ml) were corrected for specific gravity. Dilution adjustment was performed using the formula P c = P[(1.015-1)/(SG-1)], where P c is the specific gravity-corrected biomarker concentration, P is the observed biomarker concentration, SG is the specific gravity of the urine sample, and 1.015 is the median SG of the study population [ 36, 37]. To calculate the summation of di(2-ethylhexyl) phthalate (DEHP) metabolites (⅀DEHP), concentrations of MEHP, MEOHP, MEHHP, and MECPP were converted to nmol/L before being added together. The analysis of blinded specimens by the CDC laboratory was determined not to constitute engagement in human subjects' research.

Age of menarche

Prior to the onset of B4, girls were asked to report the date of their first menstrual bleeding at each 6-month visit. After achieving B4, girls were contacted by study dieticians every 3 months to survey whether the girl had reached menarche. During this phone interview, a questionnaire was used to differentiate menarche from other potential causes of vaginal bleeding, such as vaginal infection, urinary infection or trauma. Longitudinal follow-up of participants enabled the confirmation of menarche onset.

Statistical methods

Multivariable accelerated failure time (AFT) models were used to assess the influence of individual phenol and phthalate biomarker concentrations on time to menarche, assuming a Weibull distribution. To account for possible confounding by body size, models were adjusted for BMI Z-score at EDC measurement [ 16- 18, 38, 39]. Models were additionally adjusted for maternal education as an indicator of socioeconomic status, which is related to both exposure profile and age of menarche [ 6, 8, 9, 14, 15, 40]. Sensitivity analyses were conducted further adjusting for the mother's age of menarche (recall; ≤11, 12, 13,≥14 years), which may capture confounding by transgenerational exposures correlated with socioeconomic status. These analyses were restricted to the subset of the families that reported mother's age of menarche ( N = 181), assuming recall data was missing completely at random. A cluster statement was used to account for within-subject correlation between B1 and B4 biomarker measurements. Accordingly, inference was based on robust standard errors estimated using the Huber sandwich estimator. For incident cases, survival time was the age at menarche, estimated based on time between the self-reported date of first menses and date of birth. Survival time for right censored individuals was the age at last clinic visit, based on the time between date of last visit and date of birth. Time-varying associations between biomarker concentration (log(ng/ml)) and the age of menarche were investigated by adding Tanner stage as well as an interaction between biomarker concentrations and Tanner stage to our models. Association estimates from the AFT models were reported as the relative change in hazard of menarche (hazard ratio; HR) associated with a log(ng/ml) increase in biomarker concentration, which generally corresponded to a change from the lowest concentration quartile to the highest quartile. If the interaction between Tanner stage and concentration on timing of menarche was significant (Wald test; p < 0.05), the reported associations were stratified by Tanner stage. Similarly, a Wald test was used to assess effect modification by BMI Z-score. If the interaction term significantly improved model fit (p < 0.05), the influence of the biomarker on the timing of menarche was reported separately for overweight/obese girls and normal weight girls. To identify potential non-monotonic dose-response relationships, we additionally modeled tertiles of biomarker concentration, with tertile cut-points stratified by Tanner stage. Significant trends across categories were evaluated by modeling the log(median) concentration within tertiles as a continuous variable. To visualize the change in the timing of menarche, we computed median age at menarche for tertiles of urinary concentrations using the baseline survivor function of multivariable adjusted AFT models. All statistical analysis was performed in R Version 3.3.1 and figures were generated using ggplot2 [ 41].

Results

The influence of EDC exposure across childhood and adolescence on the age of menarche was assessed among 200 Chilean girls born in 2002-2003. The median age at urine collection prior to the onset of breast development (B1) was 7.9 years and 11.2 years at Tanner 4 (Table ). Proportion of samples with measurements above the limit of detection, as well as the geometric mean (95% CI) biomarker concentrations at B1 and B4 are reported in Additional file 1: Tables S1a and S1b. Analysis was restricted to the subset of 21 biomarkers with detectable concentrations at Tanner 1 and Tanner 4 in at least 75% of the girls. These 21 biomarkers included: 2,4-dichlorophenol, 2,5-dichlorophenol, benzophenone-3, BPA, MBP, MBzP, MCNP, MCOP, MCPP, MECPP, MEHHP, MEHP, MEOHP, MEP, methyl paraben, MHBP, MHiBP, MiBP, MMP, propyl paraben, triclosan; excluded were: MNP, B-PB, BPF, BPS, and E-PB (see Abbreviation List; Additional file 1: Table S1a and S1b). For reference, geometric mean (95% CI) phenol and phthalate creatine-adjusted urinary biomarker concentrations among U.S. females of a similar age range from the 2011-2012 National Health and Nutrition Examination Survey are reported in Additional file 1: Table S2. In both the U.S. and Chilean populations, most biomarker concentrations decreased with age (Additional file 1: Table S1a and S1b; Additional file 1: Table S2). The intra-individual correlation between phenol and phthalate biomarkers at B1 and B4 was moderate to low (Additional file 1: Table S3). The mean age of menarche was 12.0 years; the median age was similarly 12.0 years (95% CI: 11.9-12.2 years). At the time of analysis, 18 girls had not reached menarche or had dropped out of the cohort. Of those that achieved menarche during the course of the study, 10.5% ( n  = 21) reached menarche prior to the first detection of B4. The occurrence of menarche prior to B4 did not significantly modify the linear association between any biomarkers and the age of menarche in adjusted models. Therefore, B4 biomarker measurements for these 21 girls were included in all subsequent models. Biomarker concentrations at B1 were not significantly correlated with BMI Z-score. Similarly, most B4 biomarker concentrations were not correlated with B4 BMI Z-score, with the exception of monoethyl phthalate (MEP) and 2,5-dichlorophenol, which had moderate positive associations with BMI Z-score (ρ = 0.21 and ρ = 0.22, respectively). Additional study population characteristics are outlined in Table .

Table 1

We did not detect a significant linear impact of average concentration across Tanner stages on the age of menarche for any biomarker (Table ). However, the association between urinary biomarker concentrations and the timing of menarche was significantly modified by the Tanner stage at biomarker measurement for 2,5-dichlorophenol, benzophenone-3, monomethyl phthalate (MMP), and all of the metabolites of DEHP after adjusting for BMI Z-score and maternal education (Table ). Further adjustment for mother's age of menarche (≤11, 12, 13, ≥14 years) did not change the shape or strength of the associations between EDC biomarker levels and daughter's age of menarche (Additional file 1: Tables S4 and S5). For ease of interpretation, and to identify potential non-linear influences, we evaluated the association between tertiles of each biomarker and the timing of menarche in adjusted models (Table ; Fig. ; T1=lowest tertile, T2=middle tertile, T3=highest tertile). Among the subset of biomarkers for which we did not detect significant effect modification by Tanner stage at measurement, we found a non-monotonic change in the timing of menarche between tertiles of monobenzyl phthalate (MBzP) across puberty (Table ; Fig. ). While there was a later age of menarche among the second tertile of MBzP relative to the first (HR: 0.70; 95% CI: 0.54, 0.93), the highest tertile was not significantly different from the lowest (Table ; Fig. ).

Table 2

Table 3

Increased B1 concentrations of 2,5-dichlorophenol and benzophenone-3 were associated with decreased time to menarche (Table ). In contrast, higher B1 concentrations of the DEHP biomarkers mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), and mono(2-ethylhexyl) phthalate (MEHP) were associated with later menarche (Table ). Girls in the highest tertile of B1 2,5-dichlorophenol concentration had an earlier age of menarche compared to those in the lowest tertile (Table ; HR: 1.58; 95% CI: 1.09, 2.29), corresponding to 4.09 months earlier median age of menarche (Fig. ). The influence of benzophenone-3 and the DEHP metabolites on the relative time to menarche was less linear (Table ; Fig. ). The median age of menarche was 4.10 months earlier among girls in the second tertile of B1 benzophenone-3 concentration compared to the lowest tertile (HR: 1.58; 95% CI: 1.12, 2.22), but the change in timing was not significant between the lowest and highest tertiles (Table ). For all of the secondary oxidative DEHP biomarkers quantified (MEHHP, MECPP, and MEOHP), the median age of menarche was 6.57 to 7.37 months later among girls in the middle tertile relative to those in the lowest tertile of B1 concentration (Table ). For MEHHP, girls in the highest tertile at B1 also had a significantly later age of menarche, but the median delay was not as great as that among girls in the second tertile (Table ; Fig. ).

Table 4

Table 5

A log(ng/ml) increase in B4 concentrations of MMP was associated with a faster time to menarche (Table ; HR: 1.30; 95% CI: 1.10, 1.53). The influence of B4 concentrations of MMP on the age of menarche was relatively linear. Among girls in the highest B4 concentration tertile, the median age of menarche was 3.15 months earlier than girls in the lowest MMP tertile (Table ; HR: 1.42; 95%: 1.02, 1.99).

For MEP and triclosan, the influence on the age of menarche was significantly modified by age and sex-adjusted BMI Z-score (Table ). The average effect of triclosan across various body sizes was negligible, whereas there was a shift towards earlier menarche among girls with higher concentrations of MEP (Table ). Stratifying by BMI category, a log(ng/ml) increase in triclosan concentration was associated with earlier age of menarche (Table ; HR: 1.16; 95% CI: 1.01, 1.34) among girls that were either overweight or obese (≥85th percentile BMI Z-score). Similarly, a log(ng/ml) increase in MEP among overweight or obese girls was associated with earlier menarche (HR: 1.24; 95%CI: 1.05, 1.47), with the median age of menarche 5.03 months earlier in the highest compared to the lowest tertile (Fig. ).There was no significant association with either triclosan or MEP among girls that were normal weight.

Discussion

In this study, we report changes in the timing of menarche associated with earlier childhood and adolescent biomarker urinary concentrations of several phthalates and phenols. For most of these biomarkers, the strength of the association was dependent on the timing of biomarker measurement, suggesting specific periods of susceptible hormone-driven development. High childhood (Tanner 1) urinary concentrations of DEHP metabolites were associated with later menarche. In contrast, increased childhood levels of 2,5-dichlorophenol and benzophenone-3 were associated with early menarche. Increased adolescent (Tanner 4) concentrations of MMP were similarly associated with earlier menarche. This sensitivity to exposure window may partially explain some discordance between prior studies of childhood EDC exposure and the age of menarche, which evaluated biomarker concentrations at various ages [ 25- 28].

Among this cohort of Latina girls, increased urinary concentrations of DEHP biomarkers during childhood were associated with later menarcheal age. High pre-pubertal concentrations of these biomarkers have been associated with decreased concentrations of adolescent androgens, suggesting DEHP may inhibit adrenal androgen synthesis in humans [ 29]. This anti-androgenic influence of DEHP is further supported by the later age of pubarche among girls with relatively high concentrations of DEHP metabolites [ 42, 43]. Another mechanism by which childhood DEHP exposure may influence pubertal timing is through its influence on concentrations of IGF-1 and thyroid hormones [ 44- 47]. DEHP may also act by modifying the production of luteinizing hormone and follicle-stimulating hormone, reducing estrogen formation, or through the activation of peroxisome proliferation-activated receptors [ 48]. Similar to our findings, one longitudinal study of German adolescents recently reported that higher urinary concentrations of DEHP metabolites from ages 8-10 years were associated with decreased odds of having reached menarche at subsequent visits [ 30]. Other studies have not reported the same association between urinary DEHP biomarker concentrations and age of menarche. A cross-sectional study of U.S. girls 12-16 years reported the summation of phthalate urinary biomarker concentrations was not associated with the onset of menarche [ 27]. A smaller cross-sectional study of Hispanic girls in Mexico City also did not report a significant influence of these exposures [ 28]. In contrast, greater concentrations of DEHP biomarkers between 7 and 15 years were associated with increased odds of earlier menarche within a cohort of Chinese girls [ 26]. Our study results suggest that the influence of the biomarkers was modified by pubertal stage at the time of exposure assessment, and suggested the correlation in DEHP biomarker concentrations across puberty was weak. Therefore, these disparate findings may partially reflect differences in exposure window.

In contrast to the influence of the DEHP biomarkers, increased concentrations of 2,5-dichlorophenol, benzophenone-3, and MMP were associated with earlier menarche. 2,5-dichlorophenol is the major biomarker of para-dichlorobenzene, a chemical used in moth repellants, room deodorants, and fumigants. Two prior studies of U.S. girls reported a similar association between 2,5-dichlorophenol and age of menarche, which is consistent with the estrogenic activity 2,5-dichlorophenol has demonstrated in vitro and in vivo [ 25, 27, 49]. The sunscreen agent benzophenone-3 has also been shown to have estrogenic characteristics, including exerting uterotrophic effects in rats, simulating proliferation of breast cancer cell lines, as well as binding and activating estrogen receptors [ 50, 51]. While these compounds may have a similar mechanism of action, prior studies did not report a significant association between benzophenone-3 and menarcheal age [ 25, 27]. In our study, only pre-pubertal concentrations of 2,5-dichlorophenol and benzophenone-3 were associated with earlier menarche. Conversely, MMP concentrations close to the onset of menarche were associated with earlier timing. Two studies of Chinese girls reported conflicting associations between MMP concentrations and pubertal timing [ 52, 53]. The parent compound of MMP, dimethyl phthalate (DMP), can be found in insect repellants and plastics. While no estrogenic effect of DMP has been demonstrated in vivo, concentrations of MMP among children 5-7 years have been inversely associated with concentrations of IGF-1, with no significant impact on thyroid function [ 54]. The potential influence on the regulation of growth and development suggests the need for further investigation into the impact of MMP, or its precursor DMP, on adolescent development.

BMI significantly modified the influence of both MEP and triclosan on age of menarche. An increase in either biomarker was only associated with earlier menarche among girls that were either overweight or obese. Among U.S. children, neither association was significantly modified by BMI; however, the association with MEP was in a similar direction among all girls [ 25, 27]. It is possible that these differences in effect modification by BMI are due to race/ethnicity disparities in adolescent body composition [ 55]. Compared to white U.S. girls, Asian and Hispanic adolescents have greater trunk fat placement independent of total body fat [ 55]. Furthermore, Hispanic girls tend to have higher percent body fat than white girls, adjusting for body size [ 56]. It is possible the action of MEP and triclosan may be modified by elements of metabolic profile shaped by fat distribution, such as modulations in insulin resistance or leptin production [ 18, 57]. Alternatively, the observed statistical interaction with BMI may reflect an influence of MEP and triclosan on both adiposity and menarcheal age. Prenatal exposure to MEP has been associated with decreased childhood BMI Z-score [ 23], but this association has been inconsistent across populations [ 22]. In one cross-sectional study, triclosan exposure was similarly associated with a decrease in BMI and waist circumference among children [ 58], but this correlation was not observed in a second cross-sectional study [ 38]. Given the impact of childhood adiposity on pubertal timing [ 59- 63], the potential influence of EDCs on adiposity raises important consideration of the direction of causation. Specifically, EDC exposure could have a direct effect on menarcheal age, or influence adiposity to impact pubertal timing, or influence pubertal timing to impact adiposity. These theories are not mutually exclusive, and should be addressed by follow-up studies.

One limitation to this study is that we cannot account for the influence of earlier EDC exposure on pubertal timing that may have partially confounded the observed associations due to consistent home environment. For example, there is indication that exposure to certain EDCs in utero, such as DEHP, may influence both childhood adiposity and pubertal timing [ 21- 23, 28]. A second limitation is that while this study did measure urinary biomarkers at two developmental time points, concentrations in a single spot urine may not be representative of average exposure. Nonetheless, prior longitudinal assessments of EDC concentrations suggest that spot measurements can estimate relative EDC exposures over the course of several months to a year due to recurrent exposures [ 64- 67]. A third important consideration is that these girls were primarily from low socioeconomic status families, which may have an exposure profile that is not representative of all children and adolescents. However, given the faster decline in menarcheal age among impoverished girls [ 6, 8, 9, 14, 15], the determinants of pubertal timing within this subset of the population is of particular public health concern. Finally, this study did not account for potential synergistic or antagonistic impacts of EDC co-exposures on pubertal timing. In addition to the studied biomarkers, exposure to other types of unmeasured EDCs, such as persistent organic pollutants, may have modified our observed associations with pubertal development. These interactions may contribute to the lack of reproducibility in the associations between EDC biomarkers and the onset of menarche across populations. Given the concurrence of EDCs in consumer products and the home environment [ 68], it is difficult to disentangle the independent influence of any single chemical on menarcheal age. However, we hope these findings spur additional research into modifiable sources of EDC exposure during childhood and adolescence.

Conclusions

Exposure to certain phenols and phthalates during specific stages of pubertal development was associated with menarcheal age in Chilean girls. This study highlights the necessity of capturing critical windows of exposure during periods of rapid physiological change. Additionally, the unique associations observed in this cohort emphasize the need to integrate investigations from across the world to identify population-specific sensitivities, which can guide global health recommendations. As data collection in this longitudinal cohort continues, we plan to explore the impact of EDC exposure on additional facets of pubertal development, such as the onset of regular cycling. These shifts in pubertal timing may have both immediate psychological implications for these girls, and may indicate changes in the predisposition to adverse health in adulthood. Girls with early puberty have been shown to suffer from higher rates of adolescent depression, anxiety, and delinquent behavior [ 69- 72]. Early onset of menarche has also been associated with increased risk of type-2 diabetes, cardiovascular disease, and cancer risk. Future studies will be necessary to directly link the variation in pubertal timing associated with early life EDC exposure to adult health, as well as the potential direct effect of childhood/adolescent EDC exposure on adult health independent of age of menarche.

Additional file

Acknowledgements

We acknowledge Manori Silva, Ella Samandar, Jim Preau, Prabha Dwivedi, Xiaoliu Zhou, and Tao Jia who performed the measurements of phthalates and phenols biomarkers.

Abbreviations

Authors' contributions

AMB, CC, AP, and KBM designed the research plan; CC, VM, and AP acquired the data; AMC and XY measured the urinary biomarkers; AMB, CC, AP, and KBM interpreted the data; AMB performed the statistical analysis and wrote the paper; AMB, AMC, CC, VM, AP, and KBM provided critical revision of the manuscript for important intellectual content; AMB and KBM had primary responsibility for final content. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

Notes

Ethics approval and consent to participate

The study protocol was approved by the Ethics Committee of the Institute of Nutrition and Food Technology, University of Chile. The study was also reviewed and approved by the IRB of the University of California, Los Angeles. Informed consent was obtained from all parents or guardians of children before the start of data collection.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Footnotes
Electronic supplementary material
The online version of this article (10.1186/s12940-018-0376-z) contains supplementary material, which is available to authorized users.

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References

# data[].attributes.attachment_link.title
Childhood and adolescent phenol and phthalate exposure and the age of menarche in Latina girls

# data[].attributes.attachment_link.description
Keywords: Menarche, Puberty, Phenols, Phthalates, Endocrine disrupting chemicals Determinants of pubertal timing are of public health concern because of the association between the age of onset and adult disease susceptibility. Among females, early menarche has been associated with an increased risk of type 2 diabetes, cardiovascular disease, and reproductive cancers, including breast [ 1- 3].

# data[].attributes.body
In this article it goes over how phytoestrogen effects the sexual development, the research is coming mostly from animal studies but the few human studies results have agreed. Some of these effects include altered puberty timing, impaired ovarian function, and hypothalamus/pituitary dysfunction.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433562/

# data[].attributes.attachment_link.readable_text
Abstract

Phytoestrogen is an estrogenic compound that occurs naturally in plants. The most common sources of phytoestrogen are soybean products, which contain high levels of isoflavones. This compound, which has structural similarity with estrogen, can act as an estrogen receptor agonist or antagonist. Animal studies provide evidence of the significant effects of phytoestrogen on sexual development, including altered pubertal timing, impaired estrous cycling and ovarian function, and altered hypothalamus and pituitary functions. Although human studies examining the effects of phytoestrogen on sexual development are extremely limited, the results of some studies agree with those of the animal studies. In this paper, we review the possible mechanism of phytoestrogen action and the evidence showing the effects of phytoestrogen on sexual development in animal and human studies.

Keywords: Phytoestrogens, Puberty, Sexual development

Introduction

Phytoestrogen is a plant-derived compound that mimics estrogen 1). Phytoestrogens can be classified as isoflavones, coumestans, and lignans (). Isoflavones, which are the most extensively studied phytoestrogens, are found predominately in soybeans and soy product-containing foods 2). The major isoflavones are genistein and daidzein, and their conjugates account for greater than 65% of the total isoflavones 3).

Phytoestrogen has significant binding affinity to estrogen receptor (ER), although much weaker than that of estradiol. Experimental animal studies have demonstrated several adverse effect of isoflavones on the reproductive system, such as premature pubertal onset 4, 5), reduced fertility 6, 7), altered estrous cyclicity 5, 8), and disrupted pituitary responsiveness to gonadotropin releasing hormone (GnRH) 9). Therefore, concerns over the possible adverse effects of phytoestrogens on reproductive health have been raised in humans as well.

Studies on the hormonal effects of isoflavones are especially important in Koreans because soy products are a major component of the Korean diet; in fact, the consumption of an isoflavone-containing diet such as soy-based infant formula and soy-fortified foods and beverages has recently increased. In this paper, we review the possible mode of actions of phytoestrogen and summarize the results of animal and human studies showing the effects of phytoestrogens on sexual development, especially in childhood.

Mode of phytoestrogen action and target tissues

Estrogens act through 2 subtypes of their receptors found in target tissues: ERα and ERβ. Both ERs are widely expressed in various mammalian tissues; however, there are some differences in their distribution. ERα is most abundant in the uterus, and smaller quantities are detected in the ovaries, Leydig cells of the testes, female mammary glands, bones, and hypothalamus 10, 11). ERβ expression is highest in ovarian granulose cells and the gastrointestinal tract, whereas it is moderate to low in spermatids, the prostate, epididymis, female mammary glands, hypothalamus, and pituitary gland 11, 12).

The structure of isoflavones is closely similar to that of estrogens 13). The presence of the 2 phenolic rings in isoflavones enables these compounds to bind to ERs (). Isoflavones exert estrogenic effects on a number of target organs that possess ERα and/or ERβ. Although the binding affinities to ERα and ERβ of isoflavones is much lower than those of 17β-estradiol, they can compete with 17β-estradiol for binding to the ERα and ERβ 1). The binding affinity of genistein for ERβ is 87%, while that for ERα is 4%, while that of daidzein for ERβ and ERα is 0.5% and 0.1%, respectively 14). The effect of phytoestrogen has been demonstrated to be either agonistic or antagonistic depending on its own concentrations 15) or the estrogen concentrations of the environment 16). At low concentrations, genistein appears to act as an agonist, stimulating the proliferation of estrogen-dependent breast cancer cells, whereas high concentrations inhibit estradiol-stimulated cell growth 17). Hwang et al. 16) showed that isoflavone metabolites act as ER agonists in a low-estrogen environment (postmenopausal level of estradiol) but act as ER antagonists in a high-estrogen environment (premenopausal level of estradiol).

Phytoestrogen exposure and sexual development in animals

A number of animal studies have shown the adverse effects of phytoestrogens on reproductive organs (). Exposure to genistein affects mammary gland development and differentiation in rodents. Chronic genistein exposure from gestation day (GD) 7 to postnatal day (PND) 50 has been shown to result in ductal/alveolar hyperplasia of the mammary gland in pups 18). Neonatal female mice treated with genistein showed a differential effect on mammary gland growth according to genistein dosage: low dose-treated mice exhibited advanced maturation with increased ductal elongation, whereas high dose-treated mice showed reduced lobular alveolar development 19).

Table 1

In the aspect of pubertal onset, the effects of phytoestrogens are quite different according to exposure time, dosage, and route. Signs of pubertal onset in rats/mice are vaginal opening in females and preputial separation in males. One study demonstrated that perinatal exposure to genistein did not affect the age of pubertal onset in male and female rats 20). However, other studies found that dietary exposure to high doses of isoflavone from GD 1 to PND 21 induced the acceleration of vaginal opening in female offspring but did not affect preputial separation in male offspring 21, 22). Short-term exposure to high doses of isoflavone in the neonatal period has also been shown to expedite vaginal opening in female rats 5, 8, 23). In fact, we recently found that exposure to high doses of genistein in the prepubertal period (from PND 21 until puberty) induces early vaginal opening in female rats 4).

The adverse effects of genistein exposure on the developing ovaries have been shown in rodent studies. At birth, mice have large oocyte nests, and during the first week of life, these oocyte nests dissociate into individual oocytes surrounded by granulose cells 24). This ovarian differentiation process requires a postpartum decrease in estrogen and progesterone 24). Neonatal treatment with estrogen compounds such as 17β-estradiol and genistein disrupts this process, leaving the oocytes together in nests and leading to multi-oocyte follicles (MOFs) 7, 25, 26). Since MOF-derived oocytes have been observed to have reduced fertility rates during in vitro fertilization 27), an increased number of MOFs is one possible explanation for infertility in genistein-treated rodents 28).

Numerous studies have shown that exposure of rodents to genistein in the neonatal period caused abnormal estrous cyclicity 5, 7, 8) and altered ovulation 6, 25). These phenomena are mainly explained by the effect of genistein on the hypothalamic-pituitary-gonadal (HPG) axis, upon which normal estrous cyclicity is dependent 6) and from which a mid-cycle surge of luteinizing hormone (LH) for ovulation is derived 29). The response of the HPG axis in the neonatal period to genistein exposure seems to be dose-dependent. One study demonstrated that exposure to low doses of genistein in the neonatal period increased GnRH-induced LH release, whereas a high dose of genistein decreased GnRH-induced LH release 9). Similarly, mice treated neonatally with low doses of genistein showed increased LH production and enhanced ovulation 6, 25), whereas rats exposed to high doses of genistein in the neonatal period demonstrated decreased GnRH activation and kisspeptin fiber density in the hypothalamus 5, 30). These data suggest that exposure to genistein in the neonatal period disrupts HPG function in a dose-dependent manner.

Relatively few studies have demonstrated the effects of phytoestrogen on male sexual development in rodents. Delclos et al. 18) showed decreased ventral prostate weight and delayed spermatogenesis in male rats after genistein exposure from conception to puberty. However, exposure for short duration (GD 1 to the prepubertal period) did not affect pubertal timing, gonadal weight/histology, or fertility rate in male rodents 7, 20- 22).

Phytoestrogen exposure and sexual development in children

Human phytoestrogen exposure is highly dependent on the consumption of soy-based foods. The traditional Asian diet includes greater amounts of soy products such as tofu, soy sauce, and tempeh than the typical Western diet; therefore, the Asian population has higher concentrations of isoflavone in the urine and plasma than the Western population 31- 33). Because phytoestrogens cross the placenta, fetuses are exposed to them through maternal dietary consumption 34). After birth, infants are exposed to phytoestrogens though breastfeeding; however, the most extensive exposure to phytoestrogens occurs via soy-based formula feeding 35).

A few studies have investigated the relationship between phytoestrogen exposure and sexual development in children, and an association was mainly reported in girls (). Neonatal consumption of soy-based formula was independently associated with premature thelarche in Puerto Rican girls 36). Zung et al. 37) also reported that premature thelarche was more prevalent among soy formula-fed infants. In addition, the estrogenic activity of soy-based formula in infants involves vaginal cell maturation 38). Vaginal wall cells show maximal maturation index at birth due to maternal estrogen and then rapidly lose the estrogen effect by the age of 1 month. This low maturation index is maintained in breast milk- or cow-based formula-fed infants; in contrast, the vaginal maturation index rises again in infants fed soy-based formula by the age of 6 months, indicating the estrogen activity of soy-based formula.

Table 2

There are conflicting results in regard with pubertal timing. A telephone survey of young adults who had been fed soy formula during the infancy reported no significant changes in pubertal maturation 39). Two studies reported that phytoestrogen exposure was associated with delayed puberty in girls. A cross-sectional study of 192 healthy 9-year-old girls found that delayed breast development was observed in girls with high concentrations of urinary daidzein and genistein 40). A prospective cohort study reported that high isoflavone intake was associated with delayed breast development in German girls 41). On the other hand, recent studies support that phytoestrogen exposure may induce earlier pubertal maturation. A prospective longitudinal study that enrolled pregnant women in the United Kingdom reported that early soy-fed infants had earlier menarche compared with girls fed breast milk or other types of formula 42). We recently demonstrated that high serum isoflavone levels are associated with central precocious puberty in Korean girls 43).

These conflicting results reflect the complex influence of phytoestrogens on the HPG axis. The effects of isoflavones on puberty seems be dependent on several factors, such as the amount of isoflavones consumed, timing of isoflavone exposure, and endogenous estrogen status of individuals. As discussed earlier, genistein exposure alters GnRH activation and pituitary responsiveness; specifically, a low dose of genistein increased GnRH-induced LH release, whereas a high dose of genistein decreased GnRH-induced LH release 9). In the peripubertal period when endogenous estradiol levels are low, phytoestrogen exposure could induce increased sensitivity of the pituitary gland to GnRH challenge 9), whereas exposure after pubertal onset could interfere with gonadotropin release as demonstrated in premenopausal women 44).

For boys, one epidemiologic study found that maternal vegetarian diets containing large amounts of isoflavones are associated with hypospadias in male offspring 45). However, most studies failed to show a significant association between phytoestrogen exposure and pubertal onset 39, 41).

Summary

Several animal studies have demonstrated significant effects of phytoestrogen on sexual development, including altered pubertal timing, impaired estrous cyclicity and ovarian function, and altered function of the HPG axis. Human studies are also examining the possible effects of isoflavone exposure on sexual development in childhood. Especially during infancy, which is a critical period for growth and development, soy formula feeding may exert estrogenic effects on multiple organs including the mammary glands, gonads, and brain. In human studies, it is difficult to absolutely control other factors influencing clinical outcomes, such as diet, genetic factors, or other environmental disruptors. Further prospective human studies are needed that control these factors as much as possible to reveal the potential health effects of soy-based formulas or other sources of phytoestrogens on sexual development in children.

Acknowledgment

This work was supported by a grant 09182KFDA600 from the Korea Food & Drug Administration in 2010.

References

3. Barnes S, Kirk M, Coward L. Isoflavones and their conjugates in soy foods: extraction conditions and analysis by HPLC-mass spectrometry. J Agric Food Chem. 1994;42:2466-2474. [ Google Scholar]

4. Lee W, Lee SH, Ahn RS, Park MJ. Effect of genistein on the sexual maturation in immature female rats. Korean J Pediatr. 2009;52:111-118. [ Google Scholar]

29. Freeman ME. Neuroendocrine control of the ovarian cycle of the rat. In: Knobil E, Neil JD, editors. The physiology of reproduction. New York: Raven Press; 1994. pp. 613-658. [ Google Scholar]

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Effects of phytoestrogen on sexual development

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Phytoestrogen is an estrogenic compound that occurs naturally in plants. The most common sources of phytoestrogen are soybean products, which contain high levels of isoflavones. This compound, which has structural similarity with estrogen, can act as an estrogen receptor agonist or antagonist.

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Lizbeth Cisneros: Exposure to EDCs as well as PCB can effect the life cycle of an animal or person, and are especially potent when exposure occurs during fetal and early postnatal development.

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Lizbeth Cisneros: Exposure to EDCs as well as PCB can effect the life cycle of an animal or person, and are especially potent when exposure occurs during fetal and early postnatal development.

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https://link.springer.com/article/10.1007/s11154-007-9048-y

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Reviews in Endocrine and Metabolic Disorders

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Endocrine disrupting chemicals (EDCs) are natural or synthetic compounds that interfere with the normal function of an organism's endocrine system. Many EDCs are resistant to biodegradation, due to their structural stability, and persist in the environment. The focus of this review is on natural and artificial EDCs that act through estrogenic mechanisms to affect reproductive neuroendocrine systems. This endocrine axis comprises the hypothalamic gonadotropin-releasing hormone (GnRH), pituitary gonadotropins, and gonadal steroid hormones, including estrogens. Although it is not surprising that EDCs that mimic or antagonize estrogen receptors may exert actions upon reproductive targets, the mechanisms for these effects are complex and involve all three levels of the hypothalamic-pituitary-gonadal (HPG) system. Nevertheless, considerable evidence links exposure to estrogenic environmental EDCs with neuroendocrine reproductive deficits in wildlife and in humans. The effects of an EDC are variable across the life cycle of an animal, and are particularly potent when exposure occurs during fetal and early postnatal development. As a consequence, abnormal sexual differentiation, disrupted reproductive function, or inappropriate sexual behavior may be detected later in life. This review will cover the effects of two representative classes of estrogenic EDCs, phytoestrogens and polychlorinated biphenyls (PCBs), on neuroendocrine reproductive function, from molecules to behavior, across the vertebrate life cycle. Finally, we identify the gaps of knowledge in this field and suggest future directions for study.

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Estrogenic environmental endocrine-disrupting chemical effects on reproductive neuroendocrine function and dysfunction across the life cycle - Reviews in Endocrine and Metabolic Disorders

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Endocrine disrupting chemicals (EDCs) are natural or synthetic compounds that interfere with the normal function of an organism's endocrine system. Many EDCs are resistant to biodegradation, due to their structural stability, and persist in the environment.

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Shelby Wilson: Summary of Endocrine Disruptors and Timing of Puberty

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Shelby Wilson: Summary of Endocrine Disruptors and Timing of Puberty

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The observed endocrine disruptors include Pthalates & Phenols, Flame Retardants/PBDEs, PCBs & Dioxins, Pesticides, Air Pollution, and dietary exposures such as dairy and soy. These compiled studies are able to show a strong relationship between the disruptors and pubertal timing, however, it is unable to determine if the relationship is causal.

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INTRODUCTION

Puberty is a fundamental process of maturational changes that mark the transition from sexual infancy to reproductive maturity. It is now recognized that the age of onset of breast development, exemplifying an early stage of puberty in girls, has declined in recent decades, although the age of menarche, an advanced pubertal milestone, has not changed significantly ( 1, 2, 3). The data on pubertal timing in boys is inconsistent, with some reporting an earlier onset and others finding no changes in the age of pubertal development in recent decades ( 4, 5). While genetic factors remain the predominant determinant of pubertal timing, the shift towards an earlier age of puberty in the past century coincided with improvements in public health and nutrition, and more recent changes have been attributed to obesity. It has also been speculated that endocrine disrupting compounds (EDCs) are potential contributors to the observed shifts in pubertal timing ( 1, 2, 5, 6). Here we review publications from 2016 and 2017 that examine EDC associations with pubertal milestones, focusing on possible environmental triggers of puberty in both males and females.

General principles of EDC actions

A number of different classes of exogenous compounds with endocrine active properties interfere with endogenous hormone action ( 7). These Endocrine Disrupting Compounds (EDCs) can bind hormone receptors and affect their synthesis, transport, metabolism, and/or elimination. Among the various types of EDCs are persistent organic compounds that bio-accumulate through the food chain and have half-lives of years to decades. These chemicals, pesticides (dichloro-diphenyl-trichloroethane [DDT], pyrethroids), dioxins, polychlorinated biphenyls (PCBs), and flame retardants (polybrominated diphenyl ethers [PBDEs]), are present ubiquitously across the ecosystem and detectable in humans worldwide (reviewed in ( 5, 7). Other EDCs, such as phthalates and phenols, have shorter biological half-lives and are measurable for only a short duration post-exposure, but may still exert long-term health effects. EDCs are not natural ligands, and therefore do not have the same binding specificity and affinity as endogenous hormones, and may elicit non-receptor mediated effects. They exert antagonistic and agonistic actions on hormonal axes and pathways, and may have divergent effects at low versus high concentrations- consistent with their non-monotonic dose response curves. Both acute and delayed (latent) effects of EDCs have been reported. The prenatal, infancy, and childhood periods are considered critical windows of susceptibility due to ongoing developmental processes that are vulnerable to EDCs. The specific health consequences are dependent on the life stage at the time of exposure, particular compound, the endogenous hormonal milieu, and which health outcomes are being examined.

Phenols and phthalates

Phthalates and phenols include a number of hormonally active chemicals with anti-androgenic and estrogenic activity that are present at relatively high concentrations compared to pesticides and other industrial chemicals.

Bisphenol A (BPA) is the phenol that has drawn the most public interest (reviewed in ( 8)). BPA and other phenols are plasticizers used in both medical products and consumer goods, such as medical tubing, plastic bottles and toys, dental sealants, and linings of metal container. BPA is now banned in the production of infant food and beverage containers such as baby bottles, and has been removed from many consumer products. At lower concentrations, BPA and other phenols have weak estrogenic properties but can also compete with endogenous estrogens for binding, and at higher concentrations, they have been shown to have anti-androgenic properties.

To investigate the associations of BPA exposures with puberty, a Thai study compared urinary BPA concentrations in 41 girls with precocious puberty to 47 age matched controls ( 9). The girls with precocious puberty had higher urinary BPA levels than controls, and among the group with early puberty, those who were obese or overweight had higher values than those of normal weight. One challenge with this study is that the investigators used a relatively advanced cut-off of 8-9 years to define early puberty. In addition, samples were collected at the time of the exam, reflecting contemporaneous exposures rather than exposure assessment prior to the outcome measure- the onset of puberty.

Miao and colleagues also conducted a cross-sectional study to investigate the association between BPA exposure and pubertal development in 655 girls aged 9-18 years in Shanghai, China ( 10). Urinary BPA levels were measured and categorized according to whether they were unmeasurable (<LOD) or measurable (>LOD). If they were measurable, they were grouped as "above" or "below" the median concentrations for the cohort. A physician assessed pubertal development by Tanner staging and collected self-reported age at menarche. After adjusting for potential confounders, girls with higher values of BPA were more likely to have delayed menarche compared to girls with undetectable urinary BPA levels. Girls aged 9-12 years with measurable urinary BPA levels were more likely to have reached pubic hair stage 2; while among girls aged >15 years, those with detectable urinary BPA were less likely to have reached pubic hair stage 5. These authors concluded that contemporaneous BPA exposures were associated with delayed menarche and with earlier onset of P2 but later attainment of P5. Again a limitation of this study was that the exposure assessment occurred concurrently with the outcome assessment, making it difficult to establish causality.

BPA and phthalate levels were compared in 42 Turkish girls with idiopathic central precocious puberty, 42 girls with peripheral precocious puberty, and 50 healthy non-obese age-matched girls as controls ( 11). The authors did not find any differences in mean values of urinary BPA, LH, FSH or estradiol among the three groups. However, they reported that plasma DEHP and MEHP, were higher in the group with central precocious puberty than in the other two groups, suggesting a potential central site of action for these phthalate metabolites.

A longitudinal US puberty cohort study based at three centers (the Bay area in northern California, Cincinnati, Ohio, and Manhattan, NY was established through the Breast Cancer Environmental Research Program (BCERP) to examine the role of EDCs on pubertal development ( 12). 1051 girls had urinary levels of phenols and phthalates measured at age 6-8 years and were followed annually ( 13). Higher 2,5-dichlorophenol was associated with a 7-month earlier onset of menarche, while mono-3-carboxypropyl phthalate (MCPP) was associated with a 4-5 month later age of both thelarche and menarche, that was greater in lean girls. The association of MCPP with delayed pubertal onset and menarche was a new and unanticipated finding. MCPP is a metabolite of several phthalates and exposures is through household products as well as diet. These authors also reported that triclosan, an anti-bacterial and anti-fungal agent in soaps and personal care products, and benzophenone-3, a ketone in cosmetics for UV protection were both associated with earlier thelarche but not menarche ( 13).

Wang and colleagues evaluated the association of urinary BPA with pubertal development in 671 Chinese boys aged 9 to 18 years ( 14). Urinary BPA levels were categorized as <LOD, LOD to the 75 th%ile and >75 th%ile levels. The boys were grouped according to sexual maturity staging as early-, mid-, and late puberty. Of note is that all 13 years olds recruited were already in early puberty and that none of the boys < 11 years of age had progressed to late puberty. Moderate levels of urinary BPA correlated with earlier onset of pubic hair and genital stage 2, and showed a trend towards testicular enlargement. Urinary BPA was associated with later attainment of mature milestones, suggesting an association of moderate levels of urinary BPA with slower pubertal progression. These authors concluded that BPA exposure was associated with earlier age of adrenarche and genital stage 2, but delayed attainment of genital stage 5. Adjusting for BMI did not alter these associations.

Flame retardants/polybrominated diphenyl ethers (PBDEs)

Another class of EDCs of particular interest are the flame retardants, or polybrominated diphenyl ethers (PBDEs) used in carpeting, clothing, and furniture. The Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) was a birth cohort in California established to investigate the health effects of gestational and childhood exposures to agricultural pesticides and other environmental chemicals ( 15). This paper examined the association of flame retardants with pubertal timing in Latino low income mothers in this agricultural community. Four PBDEs were measured in maternal serum during pregnancy or at delivery as a measure of prenatal exposures and childhood exposures were assessed in PBDE measurements at age 9 years in the offspring. The children were seen every 9 months from 9-13 years of age for pubertal staging, and menarche was self-reported. In this cohort of 309 boys and 314 girls, prenatal PBDE exposure was associated with later menarche in girls and earlier pubarche in boys. Associations were stronger for normal weight girls. Childhood PBDE exposure showed a similar trend towards later menarche but it was not statistically significant, and no association was found at all for boys. There was no association between prenatal or childhood PBDE exposure and thelarche or pubarche in girls. These results are similar to those reported for the BCERP cohort, the only other longitudinal cohort study of girls' PBDE exposure ( 12). In the CHAMOCOS cohort, maternal PBDE levels were also associated with higher LH values in the boys at age 12 years ( 16).

A cross sectional case-control study from Italy compared serum PBDEs in children referred for idiopathic central precocious puberty (n=37) and premature thelarche (n=56) to 31 prepubertal control girls ( 17). Serum was collected for PBDE measurement at the time of physical exam. They found that girls with premature thelarche had higher PBDE levels than girls with precocious puberty and controls. This study had the same limitations as the other cross-sectional studies described above, ie they did not assess exposure prior to pubertal onset, and also used a relatively late age of 8 years to define precocious puberty onset. The authors conclude that their findings suggest that PBDE has a peripheral estrogenic action but no central action on the hypothalamic-pituitary-ovarian axis.

PCBs and Dioxins

Eskanazi and colleagues also reported the association of organochlorine chemicals with pubertal timing and reproductive hormones in the CHAMACOS boys, examined at ages 9 to 12.75 years for pubertal staging ( 16). Of the 312 boys who completed the 12 year visit, 234 provided a blood sample for hormone analysis. A 10-fold increase in maternal DDT levels was associated with an 18% decrease in boys' LH and testosterone values, while a 10-fold increase in DDE (a DTT metabolite) was associated only with a decrease in LH. Conversely, higher prenatal PCBs were associated with higher 12 year old FSH values in the boys. These associations between the boys' chemical levels at age 9 years and hormone values at 12 years was mediated by the boys' BMI.

The longitudinal Russian Boys Study cohort examines the associations of mixed childhood exposures with pubertal development ( 18). 473 boys had serum dioxin-like compounds (DLCs) and PCBs measured at 8-9 years of age. Genital staging and testicular volume determination was assessed at annual follow-up visits until age 18 years. The highest quartile of TEQs (a measure of dioxin potency) compared to the lowest was associated with a delay in pubertal onset and maturation by 11.6 months for testicular volume (TV >3 ml for onset; >20 ml for sexual maturity) and 10 months for genital staging (defined as G2 and G5) ( 19). Conversely, non-dioxin-like PCBs co-adjusted for TEQs, were associated with earlier pubertal onset by 8.3 months (TV) and 6.3 months (G2) and earlier sexual maturity by 6-7 months. These compounds were not associated with changes in timing of pubic hair development. In this cohort, higher quartiles of TCDD and PCDD TEQs were also associated with lower sperm concentration, total sperm count, and total motile sperm count (p-trends ≤ 0.05) ( 20), suggesting that childhood exposures to these organochlorines not only altered pubertal timing but were also associated with poorer semen parameters, raising concerns about long term effects of EDCs on reproductive function and fertility.

Dietary exposures

One area of particular interest to parents is whether dietary exposures through dairy, meat or soy intake can alter the timing of puberty. A longitudinal study of 515 Chilean girls recruited at ages 3-4 years investigated the relationship between dairy intake and pubertal changes ( 21). Breast DXA was performed in 290 girls and 324 girls reported age of menarche. Breast volume was highest in girls who drank more sweetened milk-based drinks and lowest in those who ate the most yogurt. Consumption of yogurt and low fat milk was associated with later menarche by 4.6 months. Another group looked at 367 mother-daughter dyads in the UK Avon Longitudinal Study of Parents and Children ( 22). Six phytoestrogens were measured in urine collected during pregnancy. Enterodiol (a lignan metabolite) was associated with lower risk of earlier menarche, defined as <11.5 years of age, while O-DMA (a daidzein metabolite) was associated with an increased risk of earlier menarche, The four other lignans studied, including enterolactone were not associated with age of menarche. This study controlled for timing of sample collection, race, maternal education, age at menarche delivery, BMI, diet and smoking history, infant feeding (maternal breast milk vs soy milk), childhood BMI and vegetarianism. These findings contrasted with the BCERP data that showed an association of enterolactone, a lignan metabolite, both a 4-5 month later age of menarche as well as later thelarche ( 13). This association was more marked in leaner girls and the authors speculate that the conflicting findings may be due to different exposure windows (gestational versus childhood) or sample size.

A prospective study of 456 girls in Bogota, Columbia, investigated the association of red meat intake at ages 5-12 and age at menarche via food-frequency questionnaires ( 23). They found earlier menarche with higher red meat consumption. Those who ate meat more than twice daily had significantly earlier menarche than those who ate it less than four times per week. The investigators also found that those who ate canned tuna/sardine intake more than once per week had later menarche than those who ate them less than once per month. Average age of menarche was 12.4 years in this study. Intake of dairy, poultry, eggs, and other fish were not significantly associated. The meat effect was attenuated when adjusted for BMI.

Pesticides

Another area of considerable interest is whether there is a difference between organic food and conventional food. A study examined 3-phenoxybenzoic acid (3-PBA), a nonspecific metabolite of 18 pyrethroids in 305 9-15 year old Chinese girls with self-reported pubertal staging and menarche status ( 24). These insecticides can be both estrogenic and anti-androgenic, and are used in homes and for agriculture. Exposure occurs via diet as well as through skin. Higher 3-PBA was associated with later pubertal development for breast development, pubic hair, and menarche. The Avon Longitudinal Study examined gestational exposures to weakly estrogenic organochlorines ( 24). Exposure occurs through fatty foods (meat, fish, dairy), as well as via the placenta and maternal breast milk. This nested case-control study measured maternal serum organochlorine concentrations at random times in pregnancy. Among 3682 girls who self-reported age at menarche; 218 girls were 'early' (menarche before 11.5 years), and 230 were "controls" (ages 11.5 years or older). There were no associations between in utero exposure to organochlorines and early menarche. The investigators controlled for race, mother's BMI, mother's age at menarche and at delivery, SES, birth order, child's BMI at age 7, duration of breastfeeding, child's birth weight, and trimester of sample collection.

The associations of insecticides measured as urinary metabolites (3-PBA) with self-reported pubertal staging and self-measured testicular volumes were examined in 463 Chinese boys, ages 9-16 years ( 24). 3-PBA was detected in 98.7% of all urine samples and was inversely correlated with the frequency of organic food intake. After controlling for confounders such as SEC, age, and other birth history factors, higher levels of 3-PBA were associated with increased odds of being at a more advanced stage of puberty and having higher urinary gonadotropins.

Air pollution

Lastly, the BCERP group published two studies on the effects of tobacco exposure and air pollution. Prenatal and contemporary second-hand tobacco exposure was assessed by questionnaire and urinary cotinine measurement at enrollment. Girls with higher prenatal and secondhand smoke exposure have earlier pubarche but not thelarche. Pubarche occurred 10 months earlier in the highest vs the lowest prenatally exposed groups ( 25). To examine the effects of exposure to the polycyclic aromatic hydrocarbons in air pollution, residential proximity to traffic metrics was used as a proxy measure ( 26). Proximity to major roads at age 6-8 years was associated with earlier PH across all ethnic groups, controlling for confounders. Higher exposed girls had pubarche 2-9 months earlier than lower exposed. There was no association with thelarche timing.

Contributor Information

Louise C. Greenspan, Pediatric Endocrinologist, Kaiser Permanente, San Francisco, Clinical Professor of Pediatrics, University of California, San Francisco School of Medicine, (415) 833-3533, fax (415) 833-4177.

Mary M. Lee, Chair and the Stoddard Professor of Pediatrics, Department of Pediatrics, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, 508-857-3199, 508-856-1540.

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Endocrine Disrupters and Pubertal Timing

# data[].attributes.attachment_link.description
Puberty is a fundamental process of maturational changes that mark the transition from sexual infancy to reproductive maturity. It is now recognized that the age of onset of breast development, exemplifying an early stage of puberty in girls, has declined in recent decades, although the age of menarche, an advanced pubertal milestone, has not changed significantly ( 1, 2, 3).

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Taya Warren: Endocrine Disrupters and Pubertal Timing. The effects of ECDs on ones timing of puberty

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Taya Warren: Endocrine Disrupters and Pubertal Timing. The effects of ECDs on ones timing of puberty

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6009831/

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INTRODUCTION

Puberty is a fundamental process of maturational changes that mark the transition from sexual infancy to reproductive maturity. It is now recognized that the age of onset of breast development, exemplifying an early stage of puberty in girls, has declined in recent decades, although the age of menarche, an advanced pubertal milestone, has not changed significantly ( 1, 2, 3). The data on pubertal timing in boys is inconsistent, with some reporting an earlier onset and others finding no changes in the age of pubertal development in recent decades ( 4, 5). While genetic factors remain the predominant determinant of pubertal timing, the shift towards an earlier age of puberty in the past century coincided with improvements in public health and nutrition, and more recent changes have been attributed to obesity. It has also been speculated that endocrine disrupting compounds (EDCs) are potential contributors to the observed shifts in pubertal timing ( 1, 2, 5, 6). Here we review publications from 2016 and 2017 that examine EDC associations with pubertal milestones, focusing on possible environmental triggers of puberty in both males and females.

General principles of EDC actions

A number of different classes of exogenous compounds with endocrine active properties interfere with endogenous hormone action ( 7). These Endocrine Disrupting Compounds (EDCs) can bind hormone receptors and affect their synthesis, transport, metabolism, and/or elimination. Among the various types of EDCs are persistent organic compounds that bio-accumulate through the food chain and have half-lives of years to decades. These chemicals, pesticides (dichloro-diphenyl-trichloroethane [DDT], pyrethroids), dioxins, polychlorinated biphenyls (PCBs), and flame retardants (polybrominated diphenyl ethers [PBDEs]), are present ubiquitously across the ecosystem and detectable in humans worldwide (reviewed in ( 5, 7). Other EDCs, such as phthalates and phenols, have shorter biological half-lives and are measurable for only a short duration post-exposure, but may still exert long-term health effects. EDCs are not natural ligands, and therefore do not have the same binding specificity and affinity as endogenous hormones, and may elicit non-receptor mediated effects. They exert antagonistic and agonistic actions on hormonal axes and pathways, and may have divergent effects at low versus high concentrations- consistent with their non-monotonic dose response curves. Both acute and delayed (latent) effects of EDCs have been reported. The prenatal, infancy, and childhood periods are considered critical windows of susceptibility due to ongoing developmental processes that are vulnerable to EDCs. The specific health consequences are dependent on the life stage at the time of exposure, particular compound, the endogenous hormonal milieu, and which health outcomes are being examined.

Phenols and phthalates

Phthalates and phenols include a number of hormonally active chemicals with anti-androgenic and estrogenic activity that are present at relatively high concentrations compared to pesticides and other industrial chemicals.

Bisphenol A (BPA) is the phenol that has drawn the most public interest (reviewed in ( 8)). BPA and other phenols are plasticizers used in both medical products and consumer goods, such as medical tubing, plastic bottles and toys, dental sealants, and linings of metal container. BPA is now banned in the production of infant food and beverage containers such as baby bottles, and has been removed from many consumer products. At lower concentrations, BPA and other phenols have weak estrogenic properties but can also compete with endogenous estrogens for binding, and at higher concentrations, they have been shown to have anti-androgenic properties.

To investigate the associations of BPA exposures with puberty, a Thai study compared urinary BPA concentrations in 41 girls with precocious puberty to 47 age matched controls ( 9). The girls with precocious puberty had higher urinary BPA levels than controls, and among the group with early puberty, those who were obese or overweight had higher values than those of normal weight. One challenge with this study is that the investigators used a relatively advanced cut-off of 8-9 years to define early puberty. In addition, samples were collected at the time of the exam, reflecting contemporaneous exposures rather than exposure assessment prior to the outcome measure- the onset of puberty.

Miao and colleagues also conducted a cross-sectional study to investigate the association between BPA exposure and pubertal development in 655 girls aged 9-18 years in Shanghai, China ( 10). Urinary BPA levels were measured and categorized according to whether they were unmeasurable (<LOD) or measurable (>LOD). If they were measurable, they were grouped as "above" or "below" the median concentrations for the cohort. A physician assessed pubertal development by Tanner staging and collected self-reported age at menarche. After adjusting for potential confounders, girls with higher values of BPA were more likely to have delayed menarche compared to girls with undetectable urinary BPA levels. Girls aged 9-12 years with measurable urinary BPA levels were more likely to have reached pubic hair stage 2; while among girls aged >15 years, those with detectable urinary BPA were less likely to have reached pubic hair stage 5. These authors concluded that contemporaneous BPA exposures were associated with delayed menarche and with earlier onset of P2 but later attainment of P5. Again a limitation of this study was that the exposure assessment occurred concurrently with the outcome assessment, making it difficult to establish causality.

BPA and phthalate levels were compared in 42 Turkish girls with idiopathic central precocious puberty, 42 girls with peripheral precocious puberty, and 50 healthy non-obese age-matched girls as controls ( 11). The authors did not find any differences in mean values of urinary BPA, LH, FSH or estradiol among the three groups. However, they reported that plasma DEHP and MEHP, were higher in the group with central precocious puberty than in the other two groups, suggesting a potential central site of action for these phthalate metabolites.

A longitudinal US puberty cohort study based at three centers (the Bay area in northern California, Cincinnati, Ohio, and Manhattan, NY was established through the Breast Cancer Environmental Research Program (BCERP) to examine the role of EDCs on pubertal development ( 12). 1051 girls had urinary levels of phenols and phthalates measured at age 6-8 years and were followed annually ( 13). Higher 2,5-dichlorophenol was associated with a 7-month earlier onset of menarche, while mono-3-carboxypropyl phthalate (MCPP) was associated with a 4-5 month later age of both thelarche and menarche, that was greater in lean girls. The association of MCPP with delayed pubertal onset and menarche was a new and unanticipated finding. MCPP is a metabolite of several phthalates and exposures is through household products as well as diet. These authors also reported that triclosan, an anti-bacterial and anti-fungal agent in soaps and personal care products, and benzophenone-3, a ketone in cosmetics for UV protection were both associated with earlier thelarche but not menarche ( 13).

Wang and colleagues evaluated the association of urinary BPA with pubertal development in 671 Chinese boys aged 9 to 18 years ( 14). Urinary BPA levels were categorized as <LOD, LOD to the 75 th%ile and >75 th%ile levels. The boys were grouped according to sexual maturity staging as early-, mid-, and late puberty. Of note is that all 13 years olds recruited were already in early puberty and that none of the boys < 11 years of age had progressed to late puberty. Moderate levels of urinary BPA correlated with earlier onset of pubic hair and genital stage 2, and showed a trend towards testicular enlargement. Urinary BPA was associated with later attainment of mature milestones, suggesting an association of moderate levels of urinary BPA with slower pubertal progression. These authors concluded that BPA exposure was associated with earlier age of adrenarche and genital stage 2, but delayed attainment of genital stage 5. Adjusting for BMI did not alter these associations.

Flame retardants/polybrominated diphenyl ethers (PBDEs)

Another class of EDCs of particular interest are the flame retardants, or polybrominated diphenyl ethers (PBDEs) used in carpeting, clothing, and furniture. The Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) was a birth cohort in California established to investigate the health effects of gestational and childhood exposures to agricultural pesticides and other environmental chemicals ( 15). This paper examined the association of flame retardants with pubertal timing in Latino low income mothers in this agricultural community. Four PBDEs were measured in maternal serum during pregnancy or at delivery as a measure of prenatal exposures and childhood exposures were assessed in PBDE measurements at age 9 years in the offspring. The children were seen every 9 months from 9-13 years of age for pubertal staging, and menarche was self-reported. In this cohort of 309 boys and 314 girls, prenatal PBDE exposure was associated with later menarche in girls and earlier pubarche in boys. Associations were stronger for normal weight girls. Childhood PBDE exposure showed a similar trend towards later menarche but it was not statistically significant, and no association was found at all for boys. There was no association between prenatal or childhood PBDE exposure and thelarche or pubarche in girls. These results are similar to those reported for the BCERP cohort, the only other longitudinal cohort study of girls' PBDE exposure ( 12). In the CHAMOCOS cohort, maternal PBDE levels were also associated with higher LH values in the boys at age 12 years ( 16).

A cross sectional case-control study from Italy compared serum PBDEs in children referred for idiopathic central precocious puberty (n=37) and premature thelarche (n=56) to 31 prepubertal control girls ( 17). Serum was collected for PBDE measurement at the time of physical exam. They found that girls with premature thelarche had higher PBDE levels than girls with precocious puberty and controls. This study had the same limitations as the other cross-sectional studies described above, ie they did not assess exposure prior to pubertal onset, and also used a relatively late age of 8 years to define precocious puberty onset. The authors conclude that their findings suggest that PBDE has a peripheral estrogenic action but no central action on the hypothalamic-pituitary-ovarian axis.

PCBs and Dioxins

Eskanazi and colleagues also reported the association of organochlorine chemicals with pubertal timing and reproductive hormones in the CHAMACOS boys, examined at ages 9 to 12.75 years for pubertal staging ( 16). Of the 312 boys who completed the 12 year visit, 234 provided a blood sample for hormone analysis. A 10-fold increase in maternal DDT levels was associated with an 18% decrease in boys' LH and testosterone values, while a 10-fold increase in DDE (a DTT metabolite) was associated only with a decrease in LH. Conversely, higher prenatal PCBs were associated with higher 12 year old FSH values in the boys. These associations between the boys' chemical levels at age 9 years and hormone values at 12 years was mediated by the boys' BMI.

The longitudinal Russian Boys Study cohort examines the associations of mixed childhood exposures with pubertal development ( 18). 473 boys had serum dioxin-like compounds (DLCs) and PCBs measured at 8-9 years of age. Genital staging and testicular volume determination was assessed at annual follow-up visits until age 18 years. The highest quartile of TEQs (a measure of dioxin potency) compared to the lowest was associated with a delay in pubertal onset and maturation by 11.6 months for testicular volume (TV >3 ml for onset; >20 ml for sexual maturity) and 10 months for genital staging (defined as G2 and G5) ( 19). Conversely, non-dioxin-like PCBs co-adjusted for TEQs, were associated with earlier pubertal onset by 8.3 months (TV) and 6.3 months (G2) and earlier sexual maturity by 6-7 months. These compounds were not associated with changes in timing of pubic hair development. In this cohort, higher quartiles of TCDD and PCDD TEQs were also associated with lower sperm concentration, total sperm count, and total motile sperm count (p-trends ≤ 0.05) ( 20), suggesting that childhood exposures to these organochlorines not only altered pubertal timing but were also associated with poorer semen parameters, raising concerns about long term effects of EDCs on reproductive function and fertility.

Dietary exposures

One area of particular interest to parents is whether dietary exposures through dairy, meat or soy intake can alter the timing of puberty. A longitudinal study of 515 Chilean girls recruited at ages 3-4 years investigated the relationship between dairy intake and pubertal changes ( 21). Breast DXA was performed in 290 girls and 324 girls reported age of menarche. Breast volume was highest in girls who drank more sweetened milk-based drinks and lowest in those who ate the most yogurt. Consumption of yogurt and low fat milk was associated with later menarche by 4.6 months. Another group looked at 367 mother-daughter dyads in the UK Avon Longitudinal Study of Parents and Children ( 22). Six phytoestrogens were measured in urine collected during pregnancy. Enterodiol (a lignan metabolite) was associated with lower risk of earlier menarche, defined as <11.5 years of age, while O-DMA (a daidzein metabolite) was associated with an increased risk of earlier menarche, The four other lignans studied, including enterolactone were not associated with age of menarche. This study controlled for timing of sample collection, race, maternal education, age at menarche delivery, BMI, diet and smoking history, infant feeding (maternal breast milk vs soy milk), childhood BMI and vegetarianism. These findings contrasted with the BCERP data that showed an association of enterolactone, a lignan metabolite, both a 4-5 month later age of menarche as well as later thelarche ( 13). This association was more marked in leaner girls and the authors speculate that the conflicting findings may be due to different exposure windows (gestational versus childhood) or sample size.

A prospective study of 456 girls in Bogota, Columbia, investigated the association of red meat intake at ages 5-12 and age at menarche via food-frequency questionnaires ( 23). They found earlier menarche with higher red meat consumption. Those who ate meat more than twice daily had significantly earlier menarche than those who ate it less than four times per week. The investigators also found that those who ate canned tuna/sardine intake more than once per week had later menarche than those who ate them less than once per month. Average age of menarche was 12.4 years in this study. Intake of dairy, poultry, eggs, and other fish were not significantly associated. The meat effect was attenuated when adjusted for BMI.

Pesticides

Another area of considerable interest is whether there is a difference between organic food and conventional food. A study examined 3-phenoxybenzoic acid (3-PBA), a nonspecific metabolite of 18 pyrethroids in 305 9-15 year old Chinese girls with self-reported pubertal staging and menarche status ( 24). These insecticides can be both estrogenic and anti-androgenic, and are used in homes and for agriculture. Exposure occurs via diet as well as through skin. Higher 3-PBA was associated with later pubertal development for breast development, pubic hair, and menarche. The Avon Longitudinal Study examined gestational exposures to weakly estrogenic organochlorines ( 24). Exposure occurs through fatty foods (meat, fish, dairy), as well as via the placenta and maternal breast milk. This nested case-control study measured maternal serum organochlorine concentrations at random times in pregnancy. Among 3682 girls who self-reported age at menarche; 218 girls were 'early' (menarche before 11.5 years), and 230 were "controls" (ages 11.5 years or older). There were no associations between in utero exposure to organochlorines and early menarche. The investigators controlled for race, mother's BMI, mother's age at menarche and at delivery, SES, birth order, child's BMI at age 7, duration of breastfeeding, child's birth weight, and trimester of sample collection.

The associations of insecticides measured as urinary metabolites (3-PBA) with self-reported pubertal staging and self-measured testicular volumes were examined in 463 Chinese boys, ages 9-16 years ( 24). 3-PBA was detected in 98.7% of all urine samples and was inversely correlated with the frequency of organic food intake. After controlling for confounders such as SEC, age, and other birth history factors, higher levels of 3-PBA were associated with increased odds of being at a more advanced stage of puberty and having higher urinary gonadotropins.

Air pollution

Lastly, the BCERP group published two studies on the effects of tobacco exposure and air pollution. Prenatal and contemporary second-hand tobacco exposure was assessed by questionnaire and urinary cotinine measurement at enrollment. Girls with higher prenatal and secondhand smoke exposure have earlier pubarche but not thelarche. Pubarche occurred 10 months earlier in the highest vs the lowest prenatally exposed groups ( 25). To examine the effects of exposure to the polycyclic aromatic hydrocarbons in air pollution, residential proximity to traffic metrics was used as a proxy measure ( 26). Proximity to major roads at age 6-8 years was associated with earlier PH across all ethnic groups, controlling for confounders. Higher exposed girls had pubarche 2-9 months earlier than lower exposed. There was no association with thelarche timing.

Contributor Information

Louise C. Greenspan, Pediatric Endocrinologist, Kaiser Permanente, San Francisco, Clinical Professor of Pediatrics, University of California, San Francisco School of Medicine, (415) 833-3533, fax (415) 833-4177.

Mary M. Lee, Chair and the Stoddard Professor of Pediatrics, Department of Pediatrics, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, 508-857-3199, 508-856-1540.

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Endocrine Disrupters and Pubertal Timing

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Puberty is a fundamental process of maturational changes that mark the transition from sexual infancy to reproductive maturity. It is now recognized that the age of onset of breast development, exemplifying an early stage of puberty in girls, has declined in recent decades, although the age of menarche, an advanced pubertal milestone, has not changed significantly ( 1, 2, 3).

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Summer Esquivel: Endocrine Disrupting Chemicals and early puberty in girls-- phthalates, BPA, pesticides, and flame retardant may affect timing of puberty, results show only minor affects and are inconsistent. 

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Summer Esquivel: Endocrine Disrupting Chemicals and early puberty in girls-- phthalates, BPA, pesticides, and flame retardant may affect timing of puberty, results show only minor affects and are inconsistent. 

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8226958/

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Pierluigi Marzuillo, Academic Editor

Abstract

In recent decades, pubertal onset in girls has been considered to occur at an earlier age than previously. Exposure to endocrine-disrupting chemicals (EDCs) has been associated with alterations in pubertal timing, with several reports suggesting that EDCs may have a role in the secular trend in pubertal maturation, at least in girls. However, relevant studies give inconsistent results. On the other hand, the majority of girls with idiopathic precocious or early puberty present the growth pattern of constitutional advancement of growth (CAG), i.e., growth acceleration soon after birth. Herein, we show that the growth pattern of CAG is unrelated to exposure to endocrine-disrupting chemicals and is the major determinant of precocious or early puberty. Presented data suggest that EDCs, at most, have a minor effect on the timing of pubertal onset in girls.

Keywords: puberty, endocrine-disrupting chemicals, girls, precocious puberty, early puberty, constitutional advancement of growth

1. Introduction

Pubertal timing is multifactorial involving a predominant effect of genetic and epigenetic factors and to a lesser-but still significant-extent, environmental factors. Although genetic factors are considered to explain 50-80% of the onset of puberty [ 1], genes that have been found to play a role in pubertal onset have a minor role in the onset of puberty of the population. Gene mutations, e.g., ESR1, KISS1, KISSR1, MKRN3, are rarely identified as the cause of disordered pubertal timing; thus, these genes do not seem to determine the timing of puberty nor menarche in the female population [ 2]. Recently, it was shown that the onset of puberty is regulated by epigenetic mechanisms, Kiss1 expression is negatively regulated by two polycomb group proteins ( Cbx7 and Eed) [ 3].

Environmental factors are major determinants of the onset of puberty and the age at menarche. Nutrition of the mother or/and of the infant [ 4], chronic diseases and chronic somatic stress, like strenuous exercise or psychological stress, e.g., violence exposure [ 5], or adoption of a girl from an underprivileged environment, exert a major influence on the timing of the pubertal events. Although environmental factors may result in epigenetic modifications in an organism, the epigenetic effects of the environment on the hypothalamic regulation of puberty are still to be discovered.

In girls, when pubertal onset occurs before the age of 8 years, it is considered precocious and when it occurs after 8 years but before 9 years of age, it is considered early. Precocious puberty (PP) when it is gonadotropin dependent is called central or true PP and when it is gonadotropin independent is called peripheral PP. The causes of central precocious puberty may be organic, e.g., due to tumors of the central nervous system (CNS) or most commonly idiopathic, i.e., no etiological factor is identified with appropriate imaging of the CNS. Obviously, it is important to differentiate between organic or idiopathic precocious puberty (IPP) because the former may have dire health consequences. On the other hand, early puberty lies on the extreme of normal variation of timing of pubertal onset [ 6].

In recent decades, there have been reports from several countries detailing that the onset of puberty in girls occurs at a younger age than previously [ 7, 8]. At the same time, endocrine-disrupting chemicals (EDC) have been suggested as affecting the age of pubertal onset, especially in girls. Hence, researchers were led to hypothesize that increasing exposure to EDC had a role in the secular trend for earlier sexual maturation. Moreover, it was suggested that early puberty manifesting in immigrants from developing countries was the result of previous exposure to organochloride pesticides [ 9].

Constitutional advancement of growth (CAG) is the growth pattern of early growth acceleration, which is present in the majority of girls with idiopathic precocious puberty, and in girls with early puberty [ 6, 10]. Herein, we show that CAG is unrelated to EDC exposure and we maintain that EDCs have, at most, a minor role on female pubertal timing.

2. Endocrine-Disrupting Chemicals

Endocrine-disrupting chemicals are compounds that can interfere with the activity of endocrine systems. EDCs action is exerted by imitating or blocking hormone signaling through the relevant hormonal receptor. EDCs may also modulate the synthesis, metabolism, and binding of natural hormones.

EDCs are usually used by industry, as plastics (bisphenol A (BPA)), plasticizers (phthalates), solvents/lubricants (polybrominated biphenyls (PBBs), polychlorinated biphenyls (PCBs), dioxins), pesticides (chlorpyrifos, dichlorodiphenyltrichloroethane (DDT), methoxychlor), fungicides (vinclozolin) and also as flame retardant additives in manufactured materials and pharmaceutical agents, e.g., diethylstilbestrol (DES), a non-steroidal synthetic estrogen [ 11].

EDCs may also be made by nature, e.g., phytoestrogens, which interfere with endogenous endocrine function, are produced by plants and act primarily through estrogen receptors [ 12].

The abundance of EDCs and their ability to interfere with the endocrine system combined with the secular trend for earlier onset of puberty has led many researchers to associate EDCs with early puberty, especially since some EDCs have estrogenic activity.

3. Association between Exposure to Endocrine-Disrupting Chemicals and Timing of Puberty

Commonly used and studied EDCs are phthalates, bisphenol, pesticides and flame retardants.

Phthalates are mainly used as plasticizers, i.e., substances added to plastics to increase their flexibility, transparency, durability, and longevity. They are used primarily to soften polyvinyl chloride (PVC). Human exposure to phthalates [ 13] is extremely prevalent, and it may occur through multiple routes: i.e., oral (via phthalate-contaminated food, water and other liquids and in children through mouthing of toys and teethers), dermal (via cosmetics and other personal care products), inhaled (from breathing phthalate particles in the air) ( https://www.cdc.gov/biomonitoring/Phthalates_FactSheet.html accessed 5 June 2021). Phthalates may have anti-androgenic activity [ 14], and also possess some estrogenic activity [ 15]. In Puerto Rico, high phthalate levels have been linked to premature thelarche [ 16], a normal variant of female premature sexual development. In a study of Danish schoolgirls, high phthalate excretion in urine was associated with delayed pubarche, but not thelarche, which suggests anti-androgenic actions of phthalate [ 17]. Similar results were obtained in a study of US girls [ 18]. Some studies reporting early/precocious puberty associated with phthalate exposure [ 19, 20], whereas in a US study on girls with central precocious puberty, such an association was not found [ 21]. In contrast to the previous reports, in a Chinese study performed in boys, elevated levels of phthalates were associated with constitutional delay of growth and puberty [ 22]. Furthermore, a recent Korean study showed that phthalate metabolites in girls with central precocious puberty were significantly lower than the prepubertal control girls [ 23]. Therefore, more studies are warranted to explore the effect of phthalate exposure on pubertal timing.

BPA (bisphenol A) is found in plastics (e.g., bottles, Tupperware, etc.), and in epoxy resins coating the inside of beverage and food cans, and humans are exposed mainly through food contamination from plastic and can packaging. BPA is the most commonly found estrogen-like endocrine disruptor in the environment. In experimental animals, it has been shown that BPA advances puberty [ 24], but on the other hand, it also has been shown it has no effect on pubertal timing [ 25]. Similar to the experimental animals, results of BPA on human puberty are inconsistent. In a US study of girls, Wolff et al. reported that BPA had no influence on breast development [ 26], however in studies performed in Turkey and in Thailand, idiopathic central precocious puberty was associated with higher levels of BPA than in control girls [ 27, 28]. Watkins et al. studied the in-utero and peripubertal exposure to phthalates and BPA in relation to sexual maturation and did not find any association between BPA and sexual maturation, although in utero phthalate exposure impacted on earlier timing of sexual maturation [ 29].

Pesticides are classified into various classes, e.g., insecticides, herbicides and fungicides. Pesticides enter the human body through water, air, food, and also, they can pass from mother to fetus via the placenta and to the infant through mother's milk. Most of these substances are lipophilic and accumulate in adipose tissue, where they remain for long periods of time. The most widely studied pesticide compounds are dichlorodiphenyltrichloroethane (DDT) and its metabolite dichlorodiethyldichloroethane (DDE). Although the agricultural use of DDT has been banned worldwide, in some developing countries, it is still used against mosquitos. In a study performed in Denmark, mothers who worked in greenhouses in the first trimester of pregnancy were prenatally categorized as exposed or unexposed to pesticides. Female offspring of exposed mothers had decreased age of breast development at 8.9 years, compared with 10.4 years in the unexposed, and 10.0 years in a Danish reference population [ 30]. Other researchers reported decreased menarcheal age to girls exposed in utero to DDE [ 31]. However, the significance of the association disappeared when weight at menarche was controlled for. Adopted or immigrant girls in Belgium, who presented central precocious puberty (CPP), had increased plasma levels of pesticides (DDE), thus CPP could be attributed to pesticide exposure [ 9]. However, other studies did not find an association between DDE levels and the timing of menarche [ 32]. Additionally, in inner-city girls, DDE, Pb, and dietary intakes of phytoestrogens were not significantly associated with breast stage [ 33]. In a study examining prenatal DDT exposure in relation to anthropometric and pubertal measures in adolescent males, no associations between prenatal exposure to any of the DDT compounds and any pubertal measures were noted [ 34].

Flame-retardant chemicals are added to manufactured materials (plastics, textiles, surface finishes and coatings) intended to prevent or slow the further development of ignition with their physical and chemical properties. Among them, organohalogen compounds such as polybrominated diphenyl ethers (PBDEs) are lipophilic persistent endocrine disruptors exhibiting estrogenic as well as androgenic properties. It has been proposed that PBDEs might alter pubertal timing resulting in later menarche in girls [ 35, 36] but earlier pubarche in boys [ 36]. Curiously, girls with idiopathic central precocious puberty, particularly those with higher body mass index (BMI) have been found with higher serum concentrations of PBDEs [ 37].

Thus, the inconsistency of the results of the various studies examining the association of endocrine disruptor chemicals with the onset of puberty [ 38] makes it imperative that more studies on the subject are performed.

4. Constitutional Advancement of Growth

Constitutional advancement of growth (CAG) is a growth pattern characterized by early growth acceleration [ 10]. Children with CAG are born with an average length but present growth acceleration soon after birth, reaching a zenith centile in the first 2 to 4 years of life. Then, the child grows along this centile until the onset of puberty, which is usually early. The growth pattern of CAG is the mirror image of the well-established growth pattern of constitutional delay of growth (CDG), which is characterized by growth deceleration in the first 2 to 3 years of life, although birth length is usually average. As a result of the growth deceleration, the child's height may fall to a nadir centile, which is at or below the third centile, depending on parental height. Then, growth resumes at a normal rate, and the child grows along this centile until the onset of puberty, which is usually delayed.

For a child to be considered as presenting the growth pattern of CAG, his/her Height Standard Deviation Score (SDS) should be ≥1.5 than Target Height (TH) SDS and other conditions that lead to early growth acceleration, such as genetic tall stature, overfeeding, and intrauterine growth restraint have to be excluded. In the case of a typical girl, the growth pattern of CAG is depicted in .

Red bullets depict actual height, orange squares depict bone age. Target Height (TH) derived from the heights of the mother (yellow dot) and father (blue dot) is shown on the right-hand side of the figure and as a blue line within the graph.

5. Constitutional Advancement of Growth and Early Puberty

Based on the observation that girls with precocious puberty (i.e., with breast development before 8 years of age) are tall for age, and also taller for TH, even at the onset of pubertal development, we examined the growth of 47 girls with IPP from birth until diagnosis [ 39]. The vast majority, i.e., 79%, of the girls with IPP manifested the growth pattern of CAG. Thus, we suggested that this pattern may be used as an additional clue in favor of idiopathic precocious puberty in the differential diagnosis of precocious puberty [ 10], a claim that was supported by the fact that all girls with CAG presented no CNS abnormalities in brain magnetic resonance imaging (MRI) [ 39]. We also observed a similar growth pattern in girls with early puberty, i.e., with breast development at an age between 8 and 9 years [ 40]. The greater the difference HSDS-THSDS is above +1.5 SD the greater the possibility for the girl to present precocious than early puberty.

6. Is There an Association between EDC Exposure and CAG?

Since CAG is associated with early growth acceleration, if there was an association between EDCs and CAG it would be related to fetal exposure during pregnancy or early postnatal exposure.

Several studies have examined the association of fetal exposure to EDCs, especially phthalates and BPA, and fetal growth. These compounds, besides hormonal perturbations, may cause oxidative stress and epigenetic modifications that might have a deleterious effect on fetal growth [ 41, 42, 43]. Most relevant studies were cross-sectional, measurements of length and weight were performed at delivery [ 44] and the results were inconsistent [ 45, 46, 47]. In a longitudinal study that examined the relationship between average exposure measures and fetal growth [ 48], researchers observed inverse associations between head and abdominal circumferences, femur length, and estimated fetal weight and Di (2-ethylhexyl) phthalate (DEHP) metabolites. However, no consistent associations were observed for other phthalate metabolites or for BPA. Taken together, these data indicate that it is unlikely EDC exposure in utero has a substantial adverse effect on fetal growth. In our studies on CAG girls, there was no difference in birth weight or length compared to control girls.

What about the effect of postnatal exposure on growth? In a study in which chlordecone (an organochlorine insecticide with estrogenic properties) was measured at cord blood and in breast milk at the age of 3 months, postnatal exposure in girls was associated with lower height at 3, 8 and 18 months [ 49].

Taking into account that girls with CAG present growth acceleration soon after birth, it is unlikely that growth acceleration is induced by estradiol. In neonatal life and early infancy increased estrogen levels are experienced by all normal girls. Estradiol levels peak around the second month after birth and reach levels characteristic of early- to mid-puberty, hence the period of the first 6 months of life is termed as mini-puberty. Despite increased estradiol levels, mini-puberty is not associated with growth acceleration contrary to puberty occurring during childhood.

How could the early growth acceleration characteristic of CAG be explained? Clinical evidence suggests that after the age of 2 to 3 months growth hormone (GH) is necessary for normal growth [ 50]. However, the majority of small for gestational age (SGA) infants present catch-up growth in length since early postnatal life placing them at or above the 3d percentile for length by 6 months of age [ 51]. Catch-up growth in these children is mostly attributed to overnutrition, thereby increasing insulin levels, a hormone with growth-promoting properties. However, from the 3d day of life SGA neonates present functional hypersomatotropism, i.e., increased GH and insulin growth factor 1 (IGF-1) levels relative to appropriate for gestational age neonates, suggesting that the somatotropic axis is fully operational since the first days of life [ 52, 53]. In line with a functional GH/IGF-1 axis from the first days of life is the observation that, in healthy full-term neonates, the postnatal growth velocity is positively related to a spurt in immediate postnatal life IGF-1 levels [ 54]. It is noteworthy that the increased IGF-1 levels may persist in later years [ 55].

SGA children presenting catch-up growth are more prone to insulin resistance and development of metabolic syndrome [ 56]. Recently a study on Sprague-Dawley rats examined whether a post-receptor crosstalk of GH and insulin signaling might affect insulin resistance in catch-up growth SGA animals [ 57]. The authors demonstrated that catch-up growth SGA rats exhibit increased insulin resistance associated with an impaired IRS-1-PI3K-AKT signaling pathway, which resulted from GH signaling-induced upregulation of SOCS3 expression. Thus, these data suggest a link between increased GH levels and insulin resistance in catch-up growth.

Constitutional advancement of growth presents similarities to catch-up growth only that the CAG children are, auxologically, appropriate for gestational age. Accordingly, they are susceptible to developing obesity during childhood, therefore we suggested that the growth pattern of CAG may be a predictor, not only of early puberty, but of childhood obesity as well [ 10]. Moreover, it has been reported that earlier menarche was associated with greater height, adiposity, and significantly increased serum IGF-1 at 8 years of age, even after adjustment for height and BMI [ 58]. Thus, these data allow us to speculate that the growth pattern of CAG is induced by the early activation of the GH/IGF-1 axis.

7. Concluding Remarks

The secular trend of pubertal onset in girls as reflected by the age at menarche has started since the early 20th century with the improvement of the socioeconomic and hygienic conditions. This trend, however, has levelled off in many countries of the developed world [ 59] and it seems that it has reached the age range of menarche occurrence in the paleolithic female [ 60], suggesting that human females evolved to enter puberty at a young age.

There is a growing body of evidence that some EDCs alter the metabolic set-points and increase the risk of obesity [ 61], and that EDC exposure during fetal life is associated with breast cancer [ 62]. It is of interest that the increase in obesity prevalence coincides with increases in endocrine-disrupting chemical production and exposures [ 63]. The rapid increase in the prevalence of obesity in the last three decades suggests that environmental factors are major players in the obesity epidemic. The association between EDCs and early onset of puberty not only is inconsistent but is complicated by the fact that, at least in girls, pubertal events may be advanced by obesity [ 64].

Undoubtedly, there is a great need for the EDC effects on the human body systems to be studied thoroughly. However, from the data presented in this review, it is clear that the major determinant of early puberty, at least in girls, is the presentation of the growth pattern of constitutional advancement of growth, which is unrelated to EDC exposure. Therefore, if there is a role of EDCs on female pubertal timing it seems, at the most, to be a minor one.

Abbreviations

BPA: bisphenol A, CAG: constitutional advancement of growth, CDG: constitutional delay of growth, CNS: central nervous system, CPP: central precocious puberty, DDE: dichlorodiethyldichloroethane, DDT: dichlorodiphenyltrichloroethane, DEHP: di (2-ethylhexyl)phthalate, DES: diethylstilbestrol, EDCs: endocrine-disrupting chemicals, IPP: idiopathic precocious puberty, PBBs: polybrominated biphenyls, PCBs: polychlorinated biphenyls, PP: precocious puberty, SGA: small for gestational age, TH: target height.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

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Endocrine-Disrupting Chemicals and Early Puberty in Girls

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Pierluigi Marzuillo, Academic Editor In recent decades, pubertal onset in girls has been considered to occur at an earlier age than previously. Exposure to endocrine-disrupting chemicals (EDCs) has been associated with alterations in pubertal timing, with several reports suggesting that EDCs may have a role in the secular trend in pubertal maturation, at least in girls.

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Gabi Wagoner: Endocrine Disrupters and Pubertal Timing 

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Gabi Wagoner: Endocrine Disrupters and Pubertal Timing 

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In this article, they talk about the effect of EDCs with puberty is compound specific, sexually dimorphic, and varies with the exposure window. With low levels with a mix of environment compounds may disguise the effects of the individual compounds. In the end they talk about that they have found that the EDCs have been later in puberty and not early on. They said the effects were dependent on the expose window and the specific study of a compound, and the specific pubertal milestone

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INTRODUCTION

Puberty is a fundamental process of maturational changes that mark the transition from sexual infancy to reproductive maturity. It is now recognized that the age of onset of breast development, exemplifying an early stage of puberty in girls, has declined in recent decades, although the age of menarche, an advanced pubertal milestone, has not changed significantly ( 1, 2, 3). The data on pubertal timing in boys is inconsistent, with some reporting an earlier onset and others finding no changes in the age of pubertal development in recent decades ( 4, 5). While genetic factors remain the predominant determinant of pubertal timing, the shift towards an earlier age of puberty in the past century coincided with improvements in public health and nutrition, and more recent changes have been attributed to obesity. It has also been speculated that endocrine disrupting compounds (EDCs) are potential contributors to the observed shifts in pubertal timing ( 1, 2, 5, 6). Here we review publications from 2016 and 2017 that examine EDC associations with pubertal milestones, focusing on possible environmental triggers of puberty in both males and females.

General principles of EDC actions

A number of different classes of exogenous compounds with endocrine active properties interfere with endogenous hormone action ( 7). These Endocrine Disrupting Compounds (EDCs) can bind hormone receptors and affect their synthesis, transport, metabolism, and/or elimination. Among the various types of EDCs are persistent organic compounds that bio-accumulate through the food chain and have half-lives of years to decades. These chemicals, pesticides (dichloro-diphenyl-trichloroethane [DDT], pyrethroids), dioxins, polychlorinated biphenyls (PCBs), and flame retardants (polybrominated diphenyl ethers [PBDEs]), are present ubiquitously across the ecosystem and detectable in humans worldwide (reviewed in ( 5, 7). Other EDCs, such as phthalates and phenols, have shorter biological half-lives and are measurable for only a short duration post-exposure, but may still exert long-term health effects. EDCs are not natural ligands, and therefore do not have the same binding specificity and affinity as endogenous hormones, and may elicit non-receptor mediated effects. They exert antagonistic and agonistic actions on hormonal axes and pathways, and may have divergent effects at low versus high concentrations- consistent with their non-monotonic dose response curves. Both acute and delayed (latent) effects of EDCs have been reported. The prenatal, infancy, and childhood periods are considered critical windows of susceptibility due to ongoing developmental processes that are vulnerable to EDCs. The specific health consequences are dependent on the life stage at the time of exposure, particular compound, the endogenous hormonal milieu, and which health outcomes are being examined.

Phenols and phthalates

Phthalates and phenols include a number of hormonally active chemicals with anti-androgenic and estrogenic activity that are present at relatively high concentrations compared to pesticides and other industrial chemicals.

Bisphenol A (BPA) is the phenol that has drawn the most public interest (reviewed in ( 8)). BPA and other phenols are plasticizers used in both medical products and consumer goods, such as medical tubing, plastic bottles and toys, dental sealants, and linings of metal container. BPA is now banned in the production of infant food and beverage containers such as baby bottles, and has been removed from many consumer products. At lower concentrations, BPA and other phenols have weak estrogenic properties but can also compete with endogenous estrogens for binding, and at higher concentrations, they have been shown to have anti-androgenic properties.

To investigate the associations of BPA exposures with puberty, a Thai study compared urinary BPA concentrations in 41 girls with precocious puberty to 47 age matched controls ( 9). The girls with precocious puberty had higher urinary BPA levels than controls, and among the group with early puberty, those who were obese or overweight had higher values than those of normal weight. One challenge with this study is that the investigators used a relatively advanced cut-off of 8-9 years to define early puberty. In addition, samples were collected at the time of the exam, reflecting contemporaneous exposures rather than exposure assessment prior to the outcome measure- the onset of puberty.

Miao and colleagues also conducted a cross-sectional study to investigate the association between BPA exposure and pubertal development in 655 girls aged 9-18 years in Shanghai, China ( 10). Urinary BPA levels were measured and categorized according to whether they were unmeasurable (<LOD) or measurable (>LOD). If they were measurable, they were grouped as "above" or "below" the median concentrations for the cohort. A physician assessed pubertal development by Tanner staging and collected self-reported age at menarche. After adjusting for potential confounders, girls with higher values of BPA were more likely to have delayed menarche compared to girls with undetectable urinary BPA levels. Girls aged 9-12 years with measurable urinary BPA levels were more likely to have reached pubic hair stage 2; while among girls aged >15 years, those with detectable urinary BPA were less likely to have reached pubic hair stage 5. These authors concluded that contemporaneous BPA exposures were associated with delayed menarche and with earlier onset of P2 but later attainment of P5. Again a limitation of this study was that the exposure assessment occurred concurrently with the outcome assessment, making it difficult to establish causality.

BPA and phthalate levels were compared in 42 Turkish girls with idiopathic central precocious puberty, 42 girls with peripheral precocious puberty, and 50 healthy non-obese age-matched girls as controls ( 11). The authors did not find any differences in mean values of urinary BPA, LH, FSH or estradiol among the three groups. However, they reported that plasma DEHP and MEHP, were higher in the group with central precocious puberty than in the other two groups, suggesting a potential central site of action for these phthalate metabolites.

A longitudinal US puberty cohort study based at three centers (the Bay area in northern California, Cincinnati, Ohio, and Manhattan, NY was established through the Breast Cancer Environmental Research Program (BCERP) to examine the role of EDCs on pubertal development ( 12). 1051 girls had urinary levels of phenols and phthalates measured at age 6-8 years and were followed annually ( 13). Higher 2,5-dichlorophenol was associated with a 7-month earlier onset of menarche, while mono-3-carboxypropyl phthalate (MCPP) was associated with a 4-5 month later age of both thelarche and menarche, that was greater in lean girls. The association of MCPP with delayed pubertal onset and menarche was a new and unanticipated finding. MCPP is a metabolite of several phthalates and exposures is through household products as well as diet. These authors also reported that triclosan, an anti-bacterial and anti-fungal agent in soaps and personal care products, and benzophenone-3, a ketone in cosmetics for UV protection were both associated with earlier thelarche but not menarche ( 13).

Wang and colleagues evaluated the association of urinary BPA with pubertal development in 671 Chinese boys aged 9 to 18 years ( 14). Urinary BPA levels were categorized as <LOD, LOD to the 75 th%ile and >75 th%ile levels. The boys were grouped according to sexual maturity staging as early-, mid-, and late puberty. Of note is that all 13 years olds recruited were already in early puberty and that none of the boys < 11 years of age had progressed to late puberty. Moderate levels of urinary BPA correlated with earlier onset of pubic hair and genital stage 2, and showed a trend towards testicular enlargement. Urinary BPA was associated with later attainment of mature milestones, suggesting an association of moderate levels of urinary BPA with slower pubertal progression. These authors concluded that BPA exposure was associated with earlier age of adrenarche and genital stage 2, but delayed attainment of genital stage 5. Adjusting for BMI did not alter these associations.

Flame retardants/polybrominated diphenyl ethers (PBDEs)

Another class of EDCs of particular interest are the flame retardants, or polybrominated diphenyl ethers (PBDEs) used in carpeting, clothing, and furniture. The Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) was a birth cohort in California established to investigate the health effects of gestational and childhood exposures to agricultural pesticides and other environmental chemicals ( 15). This paper examined the association of flame retardants with pubertal timing in Latino low income mothers in this agricultural community. Four PBDEs were measured in maternal serum during pregnancy or at delivery as a measure of prenatal exposures and childhood exposures were assessed in PBDE measurements at age 9 years in the offspring. The children were seen every 9 months from 9-13 years of age for pubertal staging, and menarche was self-reported. In this cohort of 309 boys and 314 girls, prenatal PBDE exposure was associated with later menarche in girls and earlier pubarche in boys. Associations were stronger for normal weight girls. Childhood PBDE exposure showed a similar trend towards later menarche but it was not statistically significant, and no association was found at all for boys. There was no association between prenatal or childhood PBDE exposure and thelarche or pubarche in girls. These results are similar to those reported for the BCERP cohort, the only other longitudinal cohort study of girls' PBDE exposure ( 12). In the CHAMOCOS cohort, maternal PBDE levels were also associated with higher LH values in the boys at age 12 years ( 16).

A cross sectional case-control study from Italy compared serum PBDEs in children referred for idiopathic central precocious puberty (n=37) and premature thelarche (n=56) to 31 prepubertal control girls ( 17). Serum was collected for PBDE measurement at the time of physical exam. They found that girls with premature thelarche had higher PBDE levels than girls with precocious puberty and controls. This study had the same limitations as the other cross-sectional studies described above, ie they did not assess exposure prior to pubertal onset, and also used a relatively late age of 8 years to define precocious puberty onset. The authors conclude that their findings suggest that PBDE has a peripheral estrogenic action but no central action on the hypothalamic-pituitary-ovarian axis.

PCBs and Dioxins

Eskanazi and colleagues also reported the association of organochlorine chemicals with pubertal timing and reproductive hormones in the CHAMACOS boys, examined at ages 9 to 12.75 years for pubertal staging ( 16). Of the 312 boys who completed the 12 year visit, 234 provided a blood sample for hormone analysis. A 10-fold increase in maternal DDT levels was associated with an 18% decrease in boys' LH and testosterone values, while a 10-fold increase in DDE (a DTT metabolite) was associated only with a decrease in LH. Conversely, higher prenatal PCBs were associated with higher 12 year old FSH values in the boys. These associations between the boys' chemical levels at age 9 years and hormone values at 12 years was mediated by the boys' BMI.

The longitudinal Russian Boys Study cohort examines the associations of mixed childhood exposures with pubertal development ( 18). 473 boys had serum dioxin-like compounds (DLCs) and PCBs measured at 8-9 years of age. Genital staging and testicular volume determination was assessed at annual follow-up visits until age 18 years. The highest quartile of TEQs (a measure of dioxin potency) compared to the lowest was associated with a delay in pubertal onset and maturation by 11.6 months for testicular volume (TV >3 ml for onset; >20 ml for sexual maturity) and 10 months for genital staging (defined as G2 and G5) ( 19). Conversely, non-dioxin-like PCBs co-adjusted for TEQs, were associated with earlier pubertal onset by 8.3 months (TV) and 6.3 months (G2) and earlier sexual maturity by 6-7 months. These compounds were not associated with changes in timing of pubic hair development. In this cohort, higher quartiles of TCDD and PCDD TEQs were also associated with lower sperm concentration, total sperm count, and total motile sperm count (p-trends ≤ 0.05) ( 20), suggesting that childhood exposures to these organochlorines not only altered pubertal timing but were also associated with poorer semen parameters, raising concerns about long term effects of EDCs on reproductive function and fertility.

Dietary exposures

One area of particular interest to parents is whether dietary exposures through dairy, meat or soy intake can alter the timing of puberty. A longitudinal study of 515 Chilean girls recruited at ages 3-4 years investigated the relationship between dairy intake and pubertal changes ( 21). Breast DXA was performed in 290 girls and 324 girls reported age of menarche. Breast volume was highest in girls who drank more sweetened milk-based drinks and lowest in those who ate the most yogurt. Consumption of yogurt and low fat milk was associated with later menarche by 4.6 months. Another group looked at 367 mother-daughter dyads in the UK Avon Longitudinal Study of Parents and Children ( 22). Six phytoestrogens were measured in urine collected during pregnancy. Enterodiol (a lignan metabolite) was associated with lower risk of earlier menarche, defined as <11.5 years of age, while O-DMA (a daidzein metabolite) was associated with an increased risk of earlier menarche, The four other lignans studied, including enterolactone were not associated with age of menarche. This study controlled for timing of sample collection, race, maternal education, age at menarche delivery, BMI, diet and smoking history, infant feeding (maternal breast milk vs soy milk), childhood BMI and vegetarianism. These findings contrasted with the BCERP data that showed an association of enterolactone, a lignan metabolite, both a 4-5 month later age of menarche as well as later thelarche ( 13). This association was more marked in leaner girls and the authors speculate that the conflicting findings may be due to different exposure windows (gestational versus childhood) or sample size.

A prospective study of 456 girls in Bogota, Columbia, investigated the association of red meat intake at ages 5-12 and age at menarche via food-frequency questionnaires ( 23). They found earlier menarche with higher red meat consumption. Those who ate meat more than twice daily had significantly earlier menarche than those who ate it less than four times per week. The investigators also found that those who ate canned tuna/sardine intake more than once per week had later menarche than those who ate them less than once per month. Average age of menarche was 12.4 years in this study. Intake of dairy, poultry, eggs, and other fish were not significantly associated. The meat effect was attenuated when adjusted for BMI.

Pesticides

Another area of considerable interest is whether there is a difference between organic food and conventional food. A study examined 3-phenoxybenzoic acid (3-PBA), a nonspecific metabolite of 18 pyrethroids in 305 9-15 year old Chinese girls with self-reported pubertal staging and menarche status ( 24). These insecticides can be both estrogenic and anti-androgenic, and are used in homes and for agriculture. Exposure occurs via diet as well as through skin. Higher 3-PBA was associated with later pubertal development for breast development, pubic hair, and menarche. The Avon Longitudinal Study examined gestational exposures to weakly estrogenic organochlorines ( 24). Exposure occurs through fatty foods (meat, fish, dairy), as well as via the placenta and maternal breast milk. This nested case-control study measured maternal serum organochlorine concentrations at random times in pregnancy. Among 3682 girls who self-reported age at menarche; 218 girls were 'early' (menarche before 11.5 years), and 230 were "controls" (ages 11.5 years or older). There were no associations between in utero exposure to organochlorines and early menarche. The investigators controlled for race, mother's BMI, mother's age at menarche and at delivery, SES, birth order, child's BMI at age 7, duration of breastfeeding, child's birth weight, and trimester of sample collection.

The associations of insecticides measured as urinary metabolites (3-PBA) with self-reported pubertal staging and self-measured testicular volumes were examined in 463 Chinese boys, ages 9-16 years ( 24). 3-PBA was detected in 98.7% of all urine samples and was inversely correlated with the frequency of organic food intake. After controlling for confounders such as SEC, age, and other birth history factors, higher levels of 3-PBA were associated with increased odds of being at a more advanced stage of puberty and having higher urinary gonadotropins.

Air pollution

Lastly, the BCERP group published two studies on the effects of tobacco exposure and air pollution. Prenatal and contemporary second-hand tobacco exposure was assessed by questionnaire and urinary cotinine measurement at enrollment. Girls with higher prenatal and secondhand smoke exposure have earlier pubarche but not thelarche. Pubarche occurred 10 months earlier in the highest vs the lowest prenatally exposed groups ( 25). To examine the effects of exposure to the polycyclic aromatic hydrocarbons in air pollution, residential proximity to traffic metrics was used as a proxy measure ( 26). Proximity to major roads at age 6-8 years was associated with earlier PH across all ethnic groups, controlling for confounders. Higher exposed girls had pubarche 2-9 months earlier than lower exposed. There was no association with thelarche timing.

Contributor Information

Louise C. Greenspan, Pediatric Endocrinologist, Kaiser Permanente, San Francisco, Clinical Professor of Pediatrics, University of California, San Francisco School of Medicine, (415) 833-3533, fax (415) 833-4177.

Mary M. Lee, Chair and the Stoddard Professor of Pediatrics, Department of Pediatrics, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, 508-857-3199, 508-856-1540.

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Endocrine Disrupters and Pubertal Timing

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Puberty is a fundamental process of maturational changes that mark the transition from sexual infancy to reproductive maturity. It is now recognized that the age of onset of breast development, exemplifying an early stage of puberty in girls, has declined in recent decades, although the age of menarche, an advanced pubertal milestone, has not changed significantly ( 1, 2, 3).

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Sage Baker: "Fetal malnourishment as well as fetal exposure to endocrine disrupters such as DES and BPA were shown to possibly result in low birth weight, early puberty, ovulatory disorders, obesity in adulthood and metabolic syndrome" https://www.sciencedirect.com/science/article/pii/S0303720710001231?via%3Dihub 

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Sage Baker: "Fetal malnourishment as well as fetal exposure to endocrine disrupters such as DES and BPA were shown to possibly result in low birth weight, early puberty, ovulatory disorders, obesity in adulthood and metabolic syndrome" https://www.sciencedirect.com/science/article/pii/S0303720710001231?via%3Dihub 

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Abstract

The involvement of environmental factors such as endocrine disrupting chemicals (EDCs) in the timing of onset of puberty is suggested by recent changes in age at onset of puberty and pattern of distribution that are variable among countries, as well as new forms of sexual precocity after migration. However, the evidence of association between early or late pubertal timing and exposure to EDCs is weak in humans, possibly due to heterogeneity of effects likely involving mixtures and incapacity to assess fetal or neonatal exposure retrospectively. The neuroendocrine system which is crucial for physiological onset of puberty is targeted by EDCs. These compounds also act directly in the gonads and peripheral sex-steroid sensitive tissues. Feedbacks add to the complexity of regulation so that changes in pubertal timing caused by EDCs can involve both central and peripheral mechanisms. In experimental conditions, several neuroendocrine endpoints are affected by EDCs though only few studies including from our laboratory aimed at EDC involvement in the pathophysiology of early sexual maturation. Recent observations support the concept that EDC cause disturbed energy balance and account for the obesity epidemic. Several aspects are linking this system and the reproductive axis: coexisting neuroendocrine and peripheral effects, dependency on fetal/neonatal programming and the many factors cross-linking the two systems, for instance leptin, adiponectin, Agouti Related Peptide (AgRP). This opens perspectives for future research and, hopefully, measures preventing the disturbances of homeostasis caused by EDCs.

View full text

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Neuroendocrine disruption of pubertal timing and interactions between homeostasis of reproduction and energy balance

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The involvement of environmental factors such as endocrine disrupting chemicals (EDCs) in the timing of onset of puberty is suggested by recent changes in age at onset of puberty and pattern of distribution that are variable among countries, as well as new forms of sexual precocity after migration.

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Holly Udris: phthalates, parabens and phenols found in personal care products tied to earlier pubertal timing in girls 

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Holly Udris: phthalates, parabens and phenols found in personal care products tied to earlier pubertal timing in girls 

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https://pubmed.ncbi.nlm.nih.gov/30517665/

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. 2019 Jan 1;34(1):109-117.

doi: 10.1093/humrep/dey337.

Affiliations

¹ Center for Environmental Research and Children's Health (CERCH), School of Public Health, University of California, Berkeley, 1995 University Avenue, Berkeley, CA, USA.

² Department of Pediatrics, University of California, San Francisco, 550 16th Street, San Francisco, CA, USA.

³ Department of Pediatrics, Kaiser Permanente, 2238 Geary Blvd., San Francisco, CA, USA.

⁴ Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Hwy, Atlanta, GA, USA.

Free PMC article

Kim G Harley et al. Hum Reprod.

Free PMC article

. 2019 Jan 1;34(1):109-117.

doi: 10.1093/humrep/dey337.

Affiliations

¹ Center for Environmental Research and Children's Health (CERCH), School of Public Health, University of California, Berkeley, 1995 University Avenue, Berkeley, CA, USA.

² Department of Pediatrics, University of California, San Francisco, 550 16th Street, San Francisco, CA, USA.

³ Department of Pediatrics, Kaiser Permanente, 2238 Geary Blvd., San Francisco, CA, USA.

⁴ Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Hwy, Atlanta, GA, USA.

Abstract

Study question: Are in-utero or peripubertal exposures to phthalates, parabens and other phenols found in personal care products associated with timing of pubertal onset in boys and girls?

Summary answer: We found some associations of altered pubertal timing in girls, but little evidence in boys.

What is known already: Certain chemicals in personal care and consumer products, including low molecular weight phthalates, parabens and phenols, or their precursors, are associated with altered pubertal timing in animal studies.

Study design, size, duration: Data were from the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) longitudinal cohort study which followed 338 children in the Salinas Valley, California, from before birth to adolescence.

Participants/materials, setting, methods: Pregnant women were enrolled in 1999-2000. Mothers were mostly Latina, living below the federal poverty threshold and without a high school diploma. We measured concentrations of three phthalate metabolites (monoethyl phthalate [MEP], mono-n-butyl phthalate and mono-isobutyl phthalate), methyl and propyl paraben and four other phenols (triclosan, benzophenone-3 and 2,4- and 2,5-dichlorophenol) in urine collected from mothers during pregnancy and from children at age 9. Pubertal timing was assessed among 179 girls and 159 boys every 9 months between ages 9 and 13 using clinical Tanner staging. Accelerated failure time models were used to obtain mean shifts of pubertal timing associated with concentrations of prenatal and peripubertal biomarkers.

Main results and the role of chance: In girls, we observed earlier onset of pubic hair development with prenatal urinary MEP concentrations and earlier menarche with prenatal triclosan and 2,4-dichlorophenol concentrations. Regarding peripubertal biomarkers, we observed: earlier breast development, pubic hair development and menarche with methyl paraben; earlier menarche with propyl paraben; and later pubic hair development with 2,5-dichlorophenol. In boys, we observed no associations with prenatal urinary biomarker concentrations and only one association with peripubertal concentrations: earlier genital development with propyl paraben.

Limitations, reasons for caution: These chemicals are quickly metabolized and one to two urinary measurements per developmental point may not accurately reflect usual exposure. Associations of peripubertal measurements with parabens may reflect reverse causality: children going through puberty early may be more likely to use personal care products. The study population was limited to Latino children of low socioeconomic status living in a farmworker community and may not be widely generalizable.

Wider implications of the findings: This study contributes to a growing literature that suggests that exposure to certain endocrine disrupting chemicals may impact timing of puberty in children.

Study funding/competing interest(s): This study was funded by the National Institute of Environmental Health Sciences and the US Environmental Protection Agency. The authors declare no conflicts of interest.

Trial registration number: N/A.

Figures

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prenatal exposure delayed effects / chemically induced*

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prenatal exposure delayed effects / physiopathology

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national center for biotechnology information

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Association of phthalates, parabens and phenols found in personal care products with pubertal timing in girls and boys - PubMed

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2019 Jan 1;34(1):109-117. doi: 10.1093/humrep/dey337. 1 Center for Environmental Research and Children's Health (CERCH), School of Public Health, University of California, Berkeley, 1995 University Avenue, Berkeley, CA, USA. 2 Department of Pediatrics, University of California, San Francisco, 550 16th Street, San Francisco, CA, USA.

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Danielle Daguison: The Effects of Environmental Exposure on Development

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Danielle Daguison: The Effects of Environmental Exposure on Development

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Danielle Daguison: Effects of Environmental EDCs on varying stages of childhood development, including puberty. Using human and animal studies, it is suggested that PCBs, PBDEs, pesticides, phthalates, and BPA, in addition to numerous other EDCs, can affect thyroid signaling and other developmental issues.

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Abstract

Exposure to exogenous chemicals can impact endocrine function at multiple sites and through numerous specific modes of action, which may have far-reaching impacts on human health and development. Widespread human exposure to numerous known or suspected endocrine disrupting chemicals (EDCs) has been documented in the US and worldwide, as have trends for increased rates of endocrine-related diseases and disorders among children. While human epidemiology studies of exposure to EDCs and children's health remain extremely limited, there is a growing body of evidence showing that exposure to a number of chemicals commonly found in consumer goods, personal care products, food, drinking water, and other sources may adversely impact child development through altered endocrine function. This narrative review provides a brief introduction to several common EDCs (with a specific focus on persistent organic pollutants, phthalates, bisphenol A, and contemporary use pesticides, which only represents a small number of all known or suspected EDCs), an overview of the state of the human evidence for adverse impacts of EDCs on child development (fetal growth, early reproductive tract development, pubertal development, neurodevelopment, and obesity), guidance for health care providers based on current knowledge, and recommendations for future research.

1. INTRODUCTION

The development and use of synthetic chemicals has grown exponentially since the 1940s, and there are now greater than 84,000 different chemicals in commerce. While many of these chemicals have allowed for modern conveniences (e.g. plastics) or were developed to address safety (e.g. flame retardants) and other societal needs (e.g. pesticides) to improve our quality of life, recent advances in the fields of exposure science and analytical chemistry have documented widespread exposure to hundreds of these chemicals among men, women and children throughout the world. Depending on the chemical, exposure can occur through food, drinking water, air, soil and house dust, or through direct contact with various household materials or consumer products. Exposures among children are especially of concern since they have contact with potentially contaminated soil and dust; they have frequent hand-to-mouth or object-to-mouth activity; they eat, drink and breathe more per body weight compared to adults; and they are undergoing many rapid changes that are susceptible to even minute doses of environmental stressors. Fetal and infant exposures are also of concern since many chemicals have been shown to pass through the placenta and/or can be passed from mother to infant through breast milk.

Numerous high production volume (HPV) chemicals - those produced or imported in the US in quantities of 1 million pounds or more per year (which currently number approximately 3,000) - are known or suspected endocrine disrupting chemicals (EDCs). EDCs can impact endocrine system through a multitude of specific mechanisms which can target different levels of the hypothalamic-pituitary-gonad/thyroid/adrenal axes, ranging from effects on hormone receptors to effects on hormone synthesis, secretion or metabolism; therefore, they can have far-reaching health implications throughout the life course. Transgenerational effects are also possible, as is now being observed in offspring of women who had been exposed to diethylstilbestrol (DES) in utero decades ago. The realization that exposure to many environmental EDCs is now ubiquitous, coupled with proven or suggested trends for increased rates of certain endocrine-related diseases and disorders among children, has resulted in growing concern regarding potential links between the two among scientists, governments, physicians, and patients. Several scientific bodies, including the Endocrine Society, now support that EDCs can impact human health. However, human studies investigating possible adverse health effects of EDCs, especially among children, are surprisingly deficient for most EDCs.

This review provides a brief introduction to several common EDCs, an overview of the state of the human evidence for adverse impacts of EDCs on child development, guidance for health care providers based on current knowledge, and recommendations for future research. As an overview, it does not provide indepth information on any of the individual exposures or outcomes introduced, and discusses only a small portion of all known or suspected EDCs. Seminal studies, select examples, and previous review articles focusing on various subtopics are referenced here, and the interested reader is directed toward those for additional detail.

2. Endocrine Disrupting Chemicals

2.1. Persistent Organic Pollutants

Persistent organic pollutants (POPs) are lipophilic chemicals with long half-lives that bioaccumulate up the food chain, and include polychlorinated biphenyls (PCBs), organochlorine pesticides (such as DDT, chlordane, and hexachlorobenzene), polybrominated diphenyl ethers (PBDEs) and other brominated flame retardants (BFRs), among others. PCBs are a class of chemicals that were widely used as transformer and hydraulic fluids, and as additives in paints, oils and building materials. DDT and other organochlorine pesticides were widely used after World War II and were regarded at the time as safe and effective alternatives to arsenic-based pesticides. In response to reports of high concentrations in the environment due to their persistence, effects on wildlife, and the potential to harm human health, PCBs and some of the more heavily used organochlorine pesticides were banned in industrialized nations in the 1970s. The use of DDT still occurs in some developing countries as an effective and affordable method to combat malaria and other vector-borne illnesses. PBDEs and other flame retardants are found in furniture, carpet backing, electronics, and many other consumer products. The use of PBDEs has recently been banned or is currently being phased out in the US and other countries. Due to their environmental ubiquity and persistence, human exposure to POPs can continue for decades after their use ceases. Continued exposures to PCBs and DDT in countries that have banned their use occur primarily through the diet (dairy, meat and higher trophic level fish), though PCB exposure from building materials used in the mid-20 th century is also possible. PBDE exposures can also occur through the same dietary sources, though exposure to some congeners may occur to a greater extent through contact with house dust after they have leached from products in the home.

POPs have been associated with a wide range adverse health effects in studies of adults, including male and female reproductive problems, thyroid effects, obesity, diabetes, and endocrine-related cancers. Most of this evidence comes from research on PCBs and organochlorine pesticides, though a growing number of studies are now investigating health effects related to PBDE exposures.

2.2. Phthalates

Phthalates are a diverse class of widely used industrial chemicals. They are used as plasticizers to make plastics more flexible, and are also used as solubilizing or stabilizing agents. Phthalates can be found in an extensive array of products. Low molecular weight phthalates are found in personal care products, certain dietary supplements and medications, and other consumer goods. High molecular weight phthalates are found in flexible PVC commonly found in consumer products, food packaging, home furnishings and other building materials. Medical equipment can also contain phthalates, and elevated exposures have been documented among NICU infants. Due to this widespread use urinary phthalate metabolites can be measured in virtually everyone. Several phthalates are anti-androgenic, and in rodents have demonstrated significant adverse impacts on male reproductive development and numerous other endpoints at high doses. In human studies of adults phthalates have been related to decreases in sex steroid and thyroid hormone levels, poor sperm quality, endometriosis, insulin resistance, obesity, and possibly breast cancer.

2.3. Bisphenol A (BPA)

Bisphenol A is a high production chemical that is used in the manufacture of polycarbonate plastics, epoxy resins, and thermal paper. Polycarbonate is a clear, rigid plastic that has been used for water bottles, and other items, while epoxy resins are found in the lining of many canned foods and in certain construction materials. As with phthalates, medical equipment may also contain BPA, as NICU infants have been shown to have elevated exposure levels. For most people exposure to BPA occurs primarily through the diet, and measurable levels of BPA can be found in most people. BPA has been known to be weakly estrogenic for 75 years, and recent animal studies have reported a diverse variety of developmental problems following early life exposure, such as altered reproductive organ development and neurobehavioral effects. Human studies of BPA remain limited, though in adults there have been suggestive relationships with male and female reproductive endpoints, altered thyroid hormones and liver function, cardiovascular disease, and diabetes.

2.4. Contemporary use pesticides

Due to the long-lasting environmental issues related to the persistent organochlorine pesticides described earlier, nearly all pesticides used today are non-persistent. While they are designed to break down in a matter of hours or days, it has been shown that many non-persistent pesticides can remain for years after an application in homes and other indoor environments where they are protected from moisture, sunlight, and other degradation mechanisms. Exposure is multi-media, multi-pathway and multi-route, and can depend on the chemical, scenario, and population. Pesticides are known neurotoxins, especially in acute high-dose situations. Many commonly used pesticides are also known or suspected EDCs, though studies of potential endocrine effects have greatly lagged behind research focused on their designed mode of action (e.g. cholinesterase inhibition). Among all the types of pesticides (insecticides, herbicides, fungicides, rodenticides, etc.) the most human research has been conducted for insecticides. The most common classes of insecticides are organophosphates, carbamates, and pyrethroids. Of these the organophosphates, some of which have been banned or restricted for residential but not agricultural use in the US, have been the most studied class.

2.6. Other chemicals

There are many other types of known or suspected EDCs for which widespread human exposure has been documented. There is a large body of evidence showing that a number of heavy metals, such as lead, cadmium and mercury, may impact endocrine function in addition to their other modes of toxicity. There is also a growing list of emerging EDCs of concern which includes, among others, parabens, triclosan, perchlorate, alternative brominated and chlorinated flame retardants, and fluorinated organic compounds such as perfluorooctane octanoate (PFOA) and perfluorooctane sulfonate (PFOS). A handful of studies suggest that many of these emerging compounds may be associated with endocrine-related endpoints in animals and humans. However, the number of human studies that have been conducted for any one chemical or class of chemicals is sparse. Thus, the remainder of this article will focus on developmental endpoints associated with those EDCs introduced in previous paragraphs.

3. Developmental Endpoints Related to EDCs

3.1. Fetal growth and gestation length

Low birth weight (LBW) and preterm birth are the leading cause of infant mortality and precursors to future morbidity, and both have increased significantly in the US since 1990. Numerous studies have assessed relationships between chemical exposures and birth weight which have been extensively reviewed. Studies among EDCs are more limited compared to those of agents related to ambient air pollution, such as particulate matter, carbon monoxide, ozone, nitrogen dioxide, and sulfur dioxide. There is some inconsistent evidence for reduced birth weight in association with exposure to POPs, organophosphate insecticides, and triazine herbicides. A meta-analysis of nearly 8,000 women from 12 European birth cohorts dating back to 1990 reported significant declines in birth weight in relation to markers of low-level exposure to PCBs, but not DDT. More recently, evidence for reduced birth weight in relation to phthalates, BPA, and PBDE has been reported, though several studies have also reported no associations. Since study designs and results have varied across studies, for most EDCs it is currently difficult to conclude whether a relationship exists between exposure and birth weight.

Few studies have investigated the potential relationship between EDC exposure and preterm birth, and most have focused on PCBs and organochlorine pesticides. Since the time these exhaustive literature reviews were published, a small nested case-control study taking place in Mexico City (n=60) reported higher concentrations of phthalates and BPA in third trimester urine of women who delivered preterm compared to women who delivered at term. For phthalates, this was consistent with two studies reporting inverse relationships between phthalates and gestation length. On the other hand, two studies have reported null or even positive associations between phthalates and gestation length. Future studies are needed on the relationship between EDCs and preterm birth, and should account for the heterogenous etiologies of preterm birth to enhance study precision.

3.2. Male reproductive tract development

There is evidence that birth anomalies of the male reproductive tract (cryptorchidism, hypospadias) may have increased in some countries in recent decades. A leading hypothesis for a collection of linked conditions in human males exposed to EDCs in utero is termed "testicular dysgenesis syndrome (TDS)". TDS represents a number of reproductive disorders of varying severity that are associated with disturbed gonadal development, including cryptorchidism, hypospadias, and smaller reproductive organs. Later in life, the effects of TDS are hypothesized to manifest as a reduction in semen quality and infertility, and an increased risk for testicular cancer. The evidence for widespread endocrine disruption among males is thus further fueled by reports of significant secular declines in semen quality and testosterone, as well as increased rates of testicular cancer, in men over the past 50 years or so. Several studies have assessed relationships between EDCs, particularly POPs, and male genital birth defects with inconsistent findings which have been reviewed previously. More recently, a large case-control study (n=471 cases and 490 controls) of maternal occupation reported significantly increased risk of hypospadias associated with exposure to hairspray and phthalates, some of which are anti-androgenic. Another recent case-control study reported increased risk of hypospadias and cryptorchidism in relation to parental occupational exposure to pesticides, though the number of cases in the study was relatively small (14 hypospadia, 18 cryptorchidism). A relationship between PBDE measured in breast milk, but not placenta, and risk of cryptorchidism was also reported in a Danish case-control study of 95 cryptorchid boys.

More subtle measures of reproductive tract development have been studied in relation to EDCs in recent years. A landmark US study reported reduced anogenital distance (a marker for insufficient fetal androgenization), penis size, and incomplete testicular descent in 106 2-24 month old boys in relation to several urinary phthalate metabolites measured in third trimester maternal urine. A newer Japanese study that measured phthalate metabolites in 111 pregnant women also reported inverse associations with anogenital distance in male infants. Finally, a Danish study reported significant decreases in serum measures of free testosterone and Leydig cell function in 3 month-old boys in relation to phthalates in maternal breast milk. Thus, there is evidence that phthalates and possibly other EDCs disrupt early male reproductive development, but additional studies are needed.

3.3. Pubertal development

A secular decline in the age at onset of puberty and an increased frequency of precocious puberty has been observed over the past couple of decades in the US and other countries. These trends are considered to be a significant public health concern since precocious puberty is a risk factor for endocrine-related diseases in adulthood. The trends cannot be fully explained by known predictors (e.g. living conditions, BMI), and exposure to EDCs have been hypothesized to be a significant contributor. Several studies have reported an association between exposure to POPs and precocious puberty or earlier menarche in girls, and delayed puberty in boys; however, there have also been a number of studies that have not observed these relationships.

There has been a lack of research on non-persistent EDCs and pubertal development. Several phthalates were found to be elevated in serum of girls with premature thelarche compared to controls in a small study in Puerto Rico. However, the study has been criticized on several aspects of the study design, including the potential for sample contamination when measuring phthalates in serum. A more recent US study of pubertal development among 1,151 girls aged 6 to 8 years at enrollment reported subtle but statistically suggestive positive associations between low molecular weight urinary phthalate metabolites and breast and pubic hair development. The authors also reported a weak inverse relationship between high molecular weight phthalate and pubic hair stage, but no associations between BPA and pubertal development. Among boys, a small study conducted in Turkey reported that plasma concentrations of high molecular weight phthalates were significantly higher in 11 to 15 year olds with pubertal gynecomastia compared to a control group. Additional studies on EDC exposure and pubertal development are greatly needed due to the current lack of human data.

3.4. Neurodevelopment

Neurodevelopmental disorders are prevalent in the US and worldwide, with evidence that rates of certain disorders, such as attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorders (ASD) have been increasing in recent years. While it is likely that these trends are attributable to numerous factors, including changes in diagnostic practices, there is growing evidence that exposure to EDCs may play a significant role. Sex steroid hormones are vital to central nervous system development. Thus, developing neuroendocrine systems may be particularly sensitive to EDC exposure. Longitudinal and/or cross-sectional human studies have related exposure to EDCs, both in utero and during early childhood, and neurodevelopmental disorders such as decreased IQ, poorer memory, ASD, ADHD, and other behavioral problems. These data are most robust for PCBs and pesticides. However, there is recent evidence that certain phthalates may be associated with each of these endpoints as well as reduced masculine play in boys, that gestational BPA exposure may be associated with poorer executive function and behavior in 3 year-old girls, and that gestational exposure to PBDEs could be related to lower scores on tests of mental development in the first few years of life.

Thyroid hormones also play an essential role in neurodevelopment in addition to many other functions related to growth and metabolism. Rates of thyroid dysfunction, including congenital hypothyroidism, have been on the rise over the past several decades. There is a growing list of EDCs that have been found capable of disrupting thyroid function. Both human and animal studies suggest that PCBs, PBDEs, pesticides, phthalates, and BPA, in addition to numerous other EDCs, may disrupt thyroid signaling through diverse mechanisms. More research is needed to establish dose-response relationships between EDCs and adverse neurodevelopment, as well as to determine the most sensitive stages and precise biological mechanisms involved.

3.5. Obesity

The prevalence of overweight and obesity is rapidly increasing in the US and worldwide. In addition to diet, physical activity and genetics, environmental "obesogens" may play a role in these trends. Obesogens are defined as chemicals that inappropriately alter lipid homeostasis to promote adipogenesis and lipid accumulation, and experimental evidence showing that numerous chemicals may impart these effects is growing. Human studies of environmental exposures in relation to obesity among children are lacking. They are primarily limited to studies of POPs and results have been inconsistent. Cord blood PCB and DDE concentrations were associated with increased BMI or change in BMI from ages 1 to 3 years in a Belgian prospective study. In a cross-sectional study of adolescents, three serum PCB congeners (138, 153, 180) were associated with decreased BMI, but a fourth (118) was positively associated with BMI, among 14 and 15 year olds. Several studies have reported null results as well. For nonpersistent EDCs, research has been limited to two cross sectional studies that have reported suggestive positive associations between certain phthalates and BMI in girls. More research is needed to more adequately assess whether EDC exposure is associated with childhood obesity and related conditions (metabolic syndrome, diabetes, and future cardiovascular disease).

4. Clinical Perspectives

Based on the available data it is currently impossible to determine individual-level risk and whether there are "safe" levels of exposure to EDCs. Although the effect estimates or expected changes in developmental markers reported in individual studies may seem subtle, because exposure is ubiquitous a seemingly small shift in the population distribution for these measures in relation to exposure is of great public health concern. Given the range of potential serious developmental effects described above, efforts to reduce EDC exposure as a precaution among pregnant women and children are warranted. The first level of concern is for those highly exposed, and physicians should obtain occupational and environmental exposure history information. Occupational exposure history for the parents may shed light on potentially important in utero exposures. Current "take home" exposures may also occur for some children, where the parent's clothing or other articles contaminated from the workplace can lead to exposures in the home or car. Patient or parent accounts of product overuse, hobbies, home-based businesses, or contaminated locations where children spend time are some examples of potentially useful environmental history information.

Because exposure to the chemicals described here occurs among virtually everyone as they go about their normal activities, all patients could be advised to take certain steps in an attempt to reduce exposure. They can purchase consumer goods or personal care products labeled phthalate-free or BPA- free, which are becoming more commonly available. However, this trend has been primarily due to marketing purposes and products containing these chemicals are generally not required to be labeled as such. For BPA and high molecular weight phthalates, replacing foods in the diet that involve plastic food packaging with "fresh" alternatives may reduce exposure by over 50%. Among fresh foods, replacing a diet of conventional produce with organic produce may significantly reduce exposure to organophosphate pesticides for children who are exposed primarily through residues in food. For legacy POPs like PCBs and DDT that are no longer used in most countries, as well as for more contemporary POPs such as PBDEs, consuming a diet lower in animal fats may reduce exposure. Individuals who live near polluted waters should also adhere to local fishing advisories. Since homes, offices, daycares and cars still have many products that contain PBDEs, and other flame retardants are being used in new products, careful product selection and cleaning practices to reduce indoor dust exposure (e.g., cleaning carpets and dusty surfaces regularly, using a vacuum cleaner with a HEPA filter) may help reduce exposure.

Healthcare providers are encouraged to practice primary prevention by learning about EDC exposure sources and potential health impacts. Several resources are available for health professionals and patients, including handouts designed by university-based Pediatric Environmental Health Specialty Units (PEHSU)( http://www.aoec.org/PEHSU/facts.html), a pediatric environmental health toolkit from the Physicians for Social Responsibility ( http://www.psr.org/resources/pediatric-toolkit.html), the "Green Book" on Pediatric Environmental Health from the American Academy of Pediatrics ( http://ebooks.aap.org/product/pediatric-environmental-health-3rd-edition), and guidance on collecting pediatric exposure history from the National Environmental Education Foundation ( http://www.neefusa.org/health/PEHI/index.htm). Hospitals may help prevent exposures among sensitive populations through their purchasing practices, such as seeking out PVC-free products for use in the NICU. Finally, clinicians are encouraged to consult with physicians or other professionals trained in environmental and occupational health as needed to address specific exposures or potential environmentally related health conditions. A resource for clinician referrals the PEHSU network of pediatric environmental health providers ( http://www.aoec.org/pehsu.htm).

5. Regulatory Perspectives

While the steps listed here may help reduce exposure to some extent, exposure to most EDCs is multi-source, multi-pathway, and multi-route. Individual exposure scenarios depend on many factors, many of which are not modifiable through personal choices and activities. Thus, the most effective way to reduce risk is at the regulatory level. The regulatory framework for controlling chemical risks in the US is considered outdated and ineffective. An extremely small proportion of chemicals in current use have been thoroughly tested for endocrine disrupting potential, and there has been little motivation for chemical producers to assess this during product development and registration. In 1996, the US EPA enacted legislation requiring special considerations be given to child susceptibility in the risk assessment of pesticides but not other industrial or consumer chemicals. The 1996 legislation also mandated that pesticides and certain drinking water contaminants be screened for endocrine activity which resulted in implementation of the EPA Endocrine Disrupting Screening Program. In 2009 EPA finally ordered producers to conduct the first set of endocrine screening tests for a truncated list of chemicals, but at the time this article was written data from these new tests were still not available. In addition to data on health risks, data on exposure sources and pathways are also needed to inform the most effective and cost-efficient regulations. For example, several phthalates have been banned from use in children's toys in the US. However, the most sensitive exposure period may be to the developing fetus, which would likely not be reduced with legislation only geared toward children's toys. Finally, there must be caution when replacing chemicals deemed high-risk with new chemicals for which we know less about the potential toxicity.

6. Conclusions and Future Research Needs

There is a growing body of evidence showing that exposure to a number of chemicals may adversely impact child development through altered endocrine function. However, many of the potential exposure-response relationships described here have not been adequately explored. For those that have been investigated in multiple studies, results have been inconsistent across studies. There are many possible explanations for variability in study findings, including differences in sample size, study design, study populations, life stage, data analysis approaches, and/or strategies for attaining data on exposure, endpoint, and important covariates. Many studies on EDCs to date among both children and adults are cross-sectional; there is a great need for well-designed longitudinal studies that measure both exposure and developmental endpoints at multiple potentially sensitive time points. For non-persistent EDCs it is especially important for studies to consider intra-individual variability in exposure levels over time. Future research aimed at explaining sexually dimorphic impacts of EDCs on development is also needed, as are studies capable of exploring health effects resulting from exposure to multiple EDCs simultaneously to more closely reflect the reality of being exposed to chemical mixtures. Research that combines molecular epidemiology and toxicology approaches should be conducted to establish causality and to elucidate specific biological mechanisms of EDCs in humans, individual susceptibility factors, and the stages of development most sensitive to exposure. Finally, more research is needed in the area of exposure science to enable more precise effect estimates in epidemiology studies used for risk assessment, as well as to provide solid data on important exposure sources and pathways for risk management. The US National Children's Study ( http://www.nationalchildrensstudy.gov) and Centers for Children's Environmental Health & Disease Prevention Research ( http://www.niehs.nih.gov/research/supported/centers/prevention/) are important sources of cutting-edge research for developing new information on these topics.

Acknowledgments

Work supported by grants R01ES018872, P42ES017198, P20ES018171, and P30ES017885 from the National Institute of Environmental Health Sciences (NIEHS), and RD83480001 from the US Environmental Protection Agency (USEPA). The content is solely the responsibility of the author and does not necessarily represent the official views of the NIEHS or USEPA.

Footnotes

Conflict of Interest: None

References

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Exposure to Environmental Endocrine Disruptors and Child Development

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Exposure to exogenous chemicals can impact endocrine function at multiple sites and through numerous specific modes of action, which may have far-reaching impacts on human health and development. Widespread human exposure to numerous known or suspected endocrine disrupting chemicals (EDCs) has been documented in the US and worldwide, as have trends for increased rates of endocrine-related diseases and disorders among children.

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Maddie Click: Age-dependent vulnerability of the ovary to AhR-mediated TCDD action before puberty: Evidence from mouse models.

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Maddie Click: Age-dependent vulnerability of the ovary to AhR-mediated TCDD action before puberty: Evidence from mouse models.

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Highlights

*

Infantile and juvenile mice deleted for Ahr or exposed to FICZ or TCDD were used.

*

Intrinsic AhR signaling mediated ovarian estradiol synthesis in juvenile mice only.

*

FICZ and TCDD activated canonical AhR pathways in infantile and juvenile ovaries.

*

TCDD, but not FICZ, affected estradiol synthesis in juvenile mice only.

Abstract

In female mammals, puberty and fertility are regulated by the synthesis of estradiol (E2) by the ovaries at the infantile stage and at the approach of puberty, a process which may be affected by endocrine disrupting chemicals (EDC)s acting through the Aryl hydrocarbon receptor (AhR). However, there is no information on AhR-mediated regulation of ovarian estrogenic activity during these developmental periods. Here, we assessed in mouse models, the intrinsic and exogenous ligand-induced AhR action on E2 synthesis at the infantile stage (14 days postnatal (dpn)) and at the approach of puberty (28 dpn). Intrinsic AhR pathway became activated in the ovary at the approach of puberty, as suggested by the decreased intra-ovarian expression in prototypical and steroidogenesis-related AhR targets and E2 contents in Ahr knockout ( Ahr ) mice versus Ahr mice exclusively at 28 dpn. Accordingly, AhR nuclear localization in granulosa cells, reflecting its activity in cells responsible for E2 synthesis, was much lower at 14 dpn than at 28 dpn in C57BL/6 mice. However, AhR signaling could be activated by exogenous ligands at both ages, as revealed by FICZ- and TCDD-induced Ahrr and Cyp1a1 expression in C57BL/6 mice. Nevertheless, TCDD impacted ovarian estrogenic activity only at 28 dpn. This age-related AhR action may be ligand-dependent, since FICZ had no effect on E2 synthesis at 28 dpn. In conclusion, AhR would not regulate ovarian estrogenic activity before the approach of puberty. Its activation by EDCs may be more detrimental to reproductive health at this stage than during infancy.

View full text

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Age-dependent vulnerability of the ovary to AhR-mediated TCDD action before puberty: Evidence from mouse models

# data[].attributes.attachment_link.description
Infantile and juvenile mice deleted for Ahr or exposed to FICZ or TCDD were used. * Intrinsic AhR signaling mediated ovarian estradiol synthesis in juvenile mice only. * FICZ and TCDD activated canonical AhR pathways in infantile and juvenile ovaries. * TCDD, but not FICZ, affected estradiol synthesis in juvenile mice only.

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Cha’lie Baker: Early-life exposure to endocrine-disrupting chemicals may be contributing to the increase of females beginning puberty earlier, with no variations in the period of completion of puberty. Exposure to a diverse range of EDCs is common, and alterations in pubertal timing may well be mediated through EDC exposure during crucial developmental windows, according to the study.

# data[].attributes.subject
Cha’lie Baker: Early-life exposure to endocrine-disrupting chemicals may be contributing to the increase of females beginning puberty earlier, with no variations in the period of completion of puberty. Exposure to a diverse range of EDCs is common, and alterations in pubertal timing may well be mediated through EDC exposure during crucial developmental windows, according to the study.

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Abstract

Over the last decades, the onset of puberty in girls has occurred earlier, but the tempo of pubertal progression has been relatively slower, resulting in a younger age at puberty onset without a change in age at menarche. Sufficient energy availability and adiposity contribute to early pubertal development, and environmental factors, such as endocrine-disrupting chemicals (EDCs), may affect not only the control of energy balance, but also puberty and reproduction. EDCs are hormonally active substances that can perturb puberty by acting both peripherally on target organs, such as adipose tissue or adrenal glands, and/or centrally on the hypothalamic-pituitary-gonadal (HPG) axis. Depending on whether the exposure takes place earlier during fetal and neonatal life or later during early childhood, EDCs can lead to different outcomes through different mechanisms. Evidence of associations between exposures to EDCs and altered pubertal timing makes it reasonable to support their relationship. However, human epidemiologic data are limited or inconsistent and cannot provide sufficient evidence for a causal relationship between EDC exposure and changes in pubertal timing. Further investigation is warranted to determine the overall or different effects of EDCs exposure during prenatal or childhood windows on pubertal milestones and to reveal the underlying mechanisms, including epigenetic marks, whereby early-life exposure to EDCs affect the HPG-peripheral tissue axis.

Keywords: Puberty, Endocrine disrupting chemicals, Secular trend

Introduction

Puberty begins with the release of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator from central nervous system inhibition after a quiescent period during childhood [ 1]. The age of menarche has definitely decreased from 16 years in the 1800s to 13 years in the 1960s, after which this downward trend seems to have slowed or even stopped [ 2]. The trend for earlier age at menarche has also been reported in Korea, from 16.6 years for females born between 1925 and 1929 to 12.6 years for females born between 1990 and 1994 [ 3, 4]. While the onset of puberty has been occurring earlier, the overall tempo of pubertal progression is reported to be relatively slower, resulting in skewing towards younger ages for the onset of puberty and towards older ages for the completion of puberty [ 2]. Such increased duration of the pubertal transition may result from gonadotropin-independent estrogenic action at the level of the breast which may originate from peripheral tissues [ 5].

Although genetic factors remain the major determinant of pubertal timing [ 6], the secular trend for earlier onset of puberty has coincided with improved public health and nutrition [ 7- 9]. Sufficient energy availability and early adiposity rebound contribute to early pubertal development. Endocrine-disrupting chemicals (EDCs) may affect not only the control of energy balance and adiposity, but also puberty and reproduction [ 2]. EDCs can act at any level in the 'hypothalamic-pituitary-gonadal (HPG)-peripheral tissues' axis [ 10]. Their effects can manifest right before puberty as well as much earlier, during fetal and neonatal life [ 2]. We reviewed the genetic and environmental factors that have been implicated in pubertal timing. We present the current evidence for the roles of EDCs on pubertal timing in girls based on human epidemiologic data and introduce potential mechanisms depending on the timing of exposure.

Factors implicated in pubertal timing by affecting the hypothalamic-pituitary-gonadal-peripheral tissues axis

Pubertal onset appears to be a highly heritable trait, but earlier sexual maturation in girls may be linked to environmental factors. Some genes implicated in precocious puberty ( DLK1, MKRN3, and KISS1) and in delayed puberty ( TACR3) are associated with variations in pubertal onset. The childhood GnRH pulse generator may be inhibited, in part, by MKRN3. Disinhibition of the HPG axis leads to the progressive amplification of pulsatile GnRH secretion which may be mediated by leptin levels and increased expression of neuropeptides, such as neurokinin B and kisspeptin, and their receptors [ 1]. In addition to central signals, peripheral metabolic and nutritional signals, such as leptin and insulin, play a permissive role in the proper function of the HPG axis [ 11].

Genes involved in pubertal timing or menarche reported by Elks et al. [ 12] and Perry et al. [ 13] include those related to hypothalamic GnRH secretion, pituitary development and function, hormone synthesis and bioactivity, energy homeostasis and growth, and potential peripheral feedback from sex steroids [ 13]. The overlap of genes involved in pubertal onset and obesity from genome wide association studies and human epidemiological data suggests a common pathway connecting early infancy weight gain, earlier pubertal onset, and later obesity in adulthood, although mechanisms whereby early-life changes in adiposity affect growth acceleration and early pubertal onset remain to be determined [ 14, 15].

Obese girls are at risk for early pubertal development. Girls maturing at earlier ages have higher body mass index (BMI) z-scores with advanced Tanner stages [ 16]. Obesity-induced hyperinsulinemia and hyperleptinemia can affect linear growth and advance puberty [ 17]. Since adrenarche starts earlier as childhood BMI and insulin levels gradually increase, the increased adrenal androgen levels in obese children may also be responsible for accelerated growth before puberty [ 17]. In addition, hyperinsulinemia can increase sex steroid bioavailability via stimulation of ovarian growth and steroidogenesis, reduction of sex hormone binding globulin, and increased conversion of androgens to estrogens by stimulating aromatase activity in adipose tissues [ 2]. Breast development can occur from the gonadotropin-independent release of estrogen by peripheral tissues, such as the adrenal glands and adipose tissue. Finally, increased sex steroid levels in obese girls can induce gonadotropin-independent or dependent precocious puberty.

Gene-to-environment interactions during critical periods of development could alter gene expression in the HPG-peripheral tissue axis through epigenetic mechanisms [ 18]. While recent data have started to reveal the epigenetic regulation of KISS1 expression [ 18, 19], epigenetic mechanisms that affect the HPG-peripheral tissue axis remain to be discovered. The intrauterine environment, birth size, nutrition, and (potentially) EDCs can affect adiposity and the timing of puberty. EDCs and adiposity may be the most concerning contributors to changes in puberty and reproduction. While sufficient energy availability provides clues to the mechanism of early pubertal development, changes in the control of both energy balance and reproduction may vary under the influence of common determinants, such as EDCs. These effects can manifest right before puberty as well as much earlier during fetal and neonatal life [ 20, 21]. Although the link between EDCs and pubertal timing does not prove causality, their association is highly suggestive [ 22].

Possible mechanisms for the effect of EDCs on pubertal timing in girls

EDCs are defined as "an exogenous chemical, or mixture of chemicals, that interferes with any aspect of hormone action" by the Endocrine Society's second scientific statement published in 2015 [ 10]. Endocrine disruptors can be naturally occurring or synthetic. This section reviews the effects of synthetic EDCs on pubertal timing in girls.

EDCs can act through several mechanisms at any level in the 'HPG-peripheral tissues' endocrine axis. Directly, EDCs may affect genes or HPG pathways unique to puberty. Indirectly, EDCs can act as obesogens and change metabolic programming during fetal and early childhood development, resulting in alterations in the metabolic and peripheral hormones associated with the onset of puberty [ 15]. A variety of chemical toxins that are ubiquitously present in our environment exert antagonistic and agonistic actions on hormonal axes and pathways at low versus high concentrations, and they demonstrate nonmonotonic dose-response curves [ 10]. Since many EDCs can act as agonists of estrogen receptors (ERs) or antagonists of androgen receptors (ARs), EDCs may mimic naturally occurring estrogens and androgens. In addition, EDCs might bind to a receptor within a cell and block the functions of endogenous hormones, thus acting as antiestrogens and antiandrogens [ 10, 23].

Low-level exposures to a mixture of EDCs may mask the effect of an individual compound. Moreover, interindividual and interspecies variation in susceptibility may be present due to differences in pharmacokinetics and/or genetic polymorphisms in key genes, complicating our ability to translate data from animal studies to human health [ 20]. In humans, the long interval from exposure to endpoints (latent effects) and the setting of mixed exposures over a lifetime make it difficult to analyze the health outcomes of EDCs [ 20]. Furthermore, it is difficult to distinguish the influence of nutrition and adiposity on pubertal outcomes from that of EDCs. Since the prenatal, neonatal, infancy, early childhood and peripubertal periods are critical windows of development, EDCs may have different effects depending on the time of exposure [ 2]. Though the mechanisms whereby fetal or neonatal life changes pubertal timing have not been fully elucidated, EDCs can alter the genes involved in the modification of epigenetic marks [ 2, 15]. Although researching the effect of EDCs on pubertal timing is difficult for the various aforementioned reasons, further investigation is warranted to determine which EDCs can affect pubertal timing, which periods of exposure are critical, and how different mechanisms are involved depending on the timing of exposure in humans [ 24].

Human epidemiologic data on EDCs in regards to pubertal timing in girls

The Endocrine Society's Scientific Statements present a review of the literature on human studies that assessed the associations between EDCs exposure and pubertal timing [ 10]. There have been extensive reviews about the contribution of EDCs on shifts in pubertal timing [ 2, 10, 24, 25] as well as a recent review of publications from 2016 to 2017 that examined EDC associations with pubertal milestones [ 26]. The EDCs most extensively studied in regards to puberty include bisphenol A (BPA), phthalate esters, pesticides (dichlorodiphenyl trichloroethane [DDT], pyrethroids), dioxins and polychlorinated biphenyls (PCBs), and flame retardants (polybrominated biphenyls [PBB], polybrominated diphenyl ethers [PBDEs]) (). This section reviews the human epidemiologic studies regarding the relationship of individual EDCs with pubertal development or menarche in girls.

Table 1.

Phthalates

Phthalates are used as liquid plasticizers in plastics and flooring, personal care products, medical devices and tubing with half-lives of 12 hours. Although the mechanism remains to be further elucidated, phthalates might act as ER agonists or AR antagonists and may also interfere with androgen synthesis [ 53]. Phthalates may be classified into low-molecular-weight phthalates (ester side-chain lengths, 1 to 4 carbons) and high-molecular-weight-phthalates (ester side-chain lengths, 5 or more carbons). Di(2-ethylhexyl) phthalate (DEHP) metabolites (mono(2-ethyl-5-oxohexyl) phthalate and mono(2-ethyl-5-hydroxyhexyl) phthalate), which are classified into high-molecular-weight-metabolites, were the phthalates most commonly studied.

The results from studies regarding the relationship between phthalate and puberty in girls conflict depending on the timing of exposure and/or different phthalate metabolites () [ 31, 33, 34, 36, 42, 45, 47- 49, 54- 65]. Different studies have shown significant associations with premature thelarche [ 54, 56] and precocious or early puberty [ 31, 36, 42, 55, 58, 61, 62, 64, 65], while no relationship with puberty [ 34, 57, 59] or an association with later onset of puberty [ 45, 47- 49, 60] have also been reported. Among several studies on phthalate exposure, nine studies had a longitudinal cohort design [ 33, 45, 47- 49, 59- 61, 63]. Prenatal exposure to high-molecular-weight phthalate metabolites was associated with later onset of puberty [ 48]. Early childhood exposure to phthalate metabolites was not associated to puberty [ 59] or associated with earlier [ 61] or later [ 45, 47, 49, 60] pubertal progression. Meanwhile, differences in pubertal development or age at menarche has been reported depending on the timing of exposure or classes of exposures (high or low-molecular-weight-phthalates) [ 33, 63]. Whereas high-molecular-weight phthalate levels were associated with later pubic hair development, low-molecular-weight phthalate levels were related to advanced breast or pubic hair development [ 33]. While prepubertal exposure to DEHP was associated with later menarche, pubertal exposure to monomethyl phthalate (low-molecular-weight) was related to earlier menarche only among overweight or obese girls and not among those that were of normal weight [ 63].

Revised paradigm of EDCs on pubertal timing and future directions

Parent et al. [ 2] suggested a revised paradigm of EDCs on pubertal timing. Since improvements in public health and nutrition have paralleled a downward trend in pubertal timing and menarche, environmental factors are the predominant determinants in pubertal timing. Classically, environmental factors, including EDCs, might advance pubertal timing via central mechanisms, particularly during the prepubertal period. According to the revised paradigm [ 2], environmental factors can influence puberty and reproduction through not only central but also peripheral mechanisms, depending on whether they take place early during fetal and neonatal life or late during the prepubertal period. Environmental factors can affect energy availability during the fetal and neonatal period or in infancy and early childhood. They can also act as obesogens and promote early adiposity rebound, leading to changes in metabolic or peripheral signals and increases in adrenal androgen levels, with subsequent early pubertal development. Environmental factors, including EDCs, might affect genetic or epigenetic pathways during critical windows of development. Further investigation is warranted in order to reveal the underlying mechanisms whereby early-life exposure to EDCs affects the HPG-peripheral tissue axis.

Conclusion

The onset of puberty in girls is occurring earlier, without changes in the timing of completion of puberty. Physiological variability and multiple other factors affect the initiation of puberty. Exposure to a broad mixture of EDCs is ubiquitous, and the shifts in pubertal timing may be mediated by exposures to EDCs at critical developmental windows. EDCs are hormonally active substances that can act via several mechanisms to perturb puberty either peripherally on target organs (adipose tissue or adrenal glands) or centrally via the HPG axis. EDCs can influence pubertal timing through different mechanisms depending on when the exposure occurs during early-life. Evidence for associations between exposures of EDCs and altered pubertal timing makes it reasonable to support the hypothesis that a relationship between EDCs and pubertal timing exists. However, the current data is insufficient and conflicting to provide sufficient evidence for a causal relationship between EDCs exposure and changes in pubertal timing in humans. Further human epidemiologic studies of prospective and longitudinal design are needed to determine the combined effect of EDC exposure on puberty and reproduction during critical periods. In addition, the underlying mechanisms whereby early-life exposures to EDCs influence puberty, including epigenetic marks, need to be explored in further studies.

Acknowledgments

This study was supported by grants from the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2018R1D1A1B07049806).

Footnotes

Conflict of interest No potential conflict of interest relevant to this article was reported.

References

29. Han EJ, Yim OK, Chung J, Baek SY, Kim Y. The study of relationship between the concentrations of Bisphenol A and DEHP in human plasma and precocious puberty. Anal Sci Technol. 2008;21:375-82. [ Google Scholar]

31. Lee SW, Lee WJ, Chae HW, Lee EB, Kim JH, Kim DH, et al. Determination of serum Di-(2-ethylhexyl) phthalate and bisphenol A level in children with idiopathic central precocious puberty. J Korean Soc Pediatr Endocrinol. 2009;14:154-62. [ Google Scholar]

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Early-life exposure to endocrine-disrupting chemicals and pubertal development in girls

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Over the last decades, the onset of puberty in girls has occurred earlier, but the tempo of pubertal progression has been relatively slower, resulting in a younger age at puberty onset without a change in age at menarche. Sufficient energy availability ...

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Endocrine disruptors in the environment impacting different development stages of children 

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Ximena Herrera: Endocrine disruptor compound in environment: As  a danger for children health 

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Ximena Herrera: Endocrine disruptor compound in environment: As  a danger for children health 

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Endocrine disruptor compounds in environment: As a danger for children health - PubMed (wsu.edu)

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In this article, they talk about the human exposure to environmental EDCs and how recently the awareness has increased because of how it affects human heath. EDC's increase the risks and causes diabetes, obesity, metabolic disorders, thyroid homeostasis and hormone-sensitive cancers. they also talk about the causes of infertility in men that is caused by EDC's and also the roles that EDC has in neurodevelopment disorders. transgenerational effects of EDCs is also included and how EDCs can modify gene expression without mutating DNA.

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National Institute for Research in Environmental Health, Indian Council of Medical Research, Bhopal, India

Department of Stem Cell Research Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India

The exponential growth of pollutant discharges into the environment due to increasing industrial and agricultural activities is a rising threat for human health and a biggest concern for environmental health globally. Several synthetic chemicals, categorized as potential environmental endocrine-disrupting chemicals (EDCs), are evident to affect the health of not only livestock and wildlife but also humankind. In recent years, human exposure to environmental EDCs has received increased awareness due to their association with altered human health as documented by several epidemiological and experimental studies. EDCs are associated with deleterious effects on male and female reproductive health; causes diabetes, obesity, metabolic disorders, thyroid homeostasis and increase the risk of hormone-sensitive cancers. Sewage effluents are a major source of several EDCs, which eventually reach large water bodies and potentially contaminate the drinking water supply. Similarly, water storage material such as different types of plastics also leaches out EDCs in drinking Water. Domestic wastewater containing pharmaceutical ingredients, metals, pesticides and personal care product additives also influences endocrine activity. These EDCs act via various receptors through a variety of known and unknown mechanisms including epigenetic modification. They differ from classic toxins in several ways such as low-dose effect, non-monotonic dose and trans-generational effects. This review aims to highlight the hidden burden of EDCs on human health and discusses the non-classical toxic properties of EDCs in an attempt to understand the magnitude of the exposome on human health. Present data on the environmental EDCs advocate that there may be associations between human exposure to EDCs and several undesirable health outcomes that warrants further human bio-monitoring of EDCs.

Introduction

Effects of environmental pollution on human health are increasingly gaining more attention globally. Environmental pollutants are chemicals that result from human activities, which end up in the environment and subsequently pose risks to human and animal health. Several of these chemicals are collectively known as endocrine-disrupting chemicals (EDCs), which are nowadays gaining more importance in terms of public health due to their widespread effects on human health and potential cause of morbidity.

The Endocrine Society defines EDCs as follows: "an exogenous (non-natural) chemical, or a mixture of chemicals, that interferes with any aspect of hormone action." These chemicals alter the hormonal balance of the body through several different mechanisms; they can mimic hormones, disrupt hormone synthesis or breakdown, alter the development of hormone receptors, act as hormone antagonists or alter hormone binding. Environmental EDCs are mostly released during manufacture and use of human-made materials such as pesticides, plastics/plasticisers, electronic wastes, flame-retardants, metals, food additives, and personal care products. These EDCs can disturb hormonal balance and consequently result in several health disorders, including developmental and reproductive abnormalities, increased prevalence of hormone-sensitive cancers, neurodevelopmental delays and abnormal growth patterns in children, and alterations in immune function.

EDCs are a group of highly heterogeneous synthetic chemicals used in a variety of settings. Some of the common chemicals included in this group are chemicals used in industries and their by-products (polychlorinated biphenyls [PCBs], polybrominated biphenyls [PBBs] and dioxins), plastics (bisphenol A [BPA]), plasticisers (phthalates) and pesticides (methoxychlor [MXC], chlorpyrifos and dichlorodiphenyltrichloroethane [DDT]). Some EDCs were designed to have long half-lives for industrial purposes and are known as "persistent organic pollutants" (POPs) exemplified by PCBs, dichlorodiphenyldichloroethylene (DDE), dioxin, organochlorine pesticides and hexachlorobenzene (HCB) ( Figure 1). The POPs are highly lipophilic and tend to accumulate in the adipose tissue. Due to this property, they also accumulate in the food chain and undergo biomagnification. Many of these substances either do not decay or decay slowly, while others may be metabolized to compounds that are more toxic than the parent chemicals ( 1, 2). Other EDCs such as BPA and phthalates may not be persistent but are used so extensively that environmental exposure is widespread. EDCs are present ubiquitously in the environment and human exposure occurs through intake of water and food, via inhalation, and through the skin.

Figure 1. Sources of environmental EDCs and their effects on metabolism. DEP, Diethyl phthalate; DEHP, Diethylhexyl phthalate; DBP, Dibutyl phthalate; PFOS, Perfluorooctanote; DDT, Dichlorodiphenyltrichloro ethane; PBDE, Polybrominated diphenyl ether; TPTO, bis (triphenyltin) oxide; TBT, Tributyltin.

Drinking water particularly is thought to be a source of significant exposure through leaching, industrial waste discharge and inadequate treatment of water supply for chemicals. EDCs can also be transferred from mother to child through trans-placental route as well as through breast milk. Developing fetuses and children are highly susceptible to environmental exposures, and their effects may not become apparent until a later age. Research shows that such exposures may increase the susceptibility of the child to several non-communicable diseases in their adult life ( Table 1). EDCs can potentially target various hormone systems; however, it has been observed that the effect of these chemicals primarily involves the reproductive system, puberty, embryonic development and sex differentiation in fetal life. Thus, it is likely that the primary mechanism of their action is through interference with sex steroid hormones. Furthermore, there is also a growing apprehension that metabolic disorders of adult hood may be linked with EDCs. Several in vitro, in vivo and epidemiological studies link human-environmental EDC exposures with obesity, metabolic syndrome, type 2 diabetes and possibly with some hormone-responsive cancers ( Figure 2).

Table 1. Studies documenting associations between different EDC exposures and risk to different diseases.

Figure 2. Health Effect of Endocrine disrupting chemicals. Tributyltin (TBT), PerfluorooctaneSulfonate (PFOS), Perfluorinated compounds (PFCs), Perfluorooctanoic acid (PFOA), Bisphenol A (BPA), Diethylstilbestrol (DES), hexachlorobenzene (HCB), dichlorodiphenyltri-chloroethane (DDT), Polybrominated diphenyls ethers (PBDEs), constitutive androstane receptor (CAR), Thyroid Hormone Receptor (ThR), glucocorticoid receptor (GR), Aryl hydrocarbon receptor (Ahr), Androgen receptor (AR), Peroxisome proliferator-activated receptor (PPAR), Estrogen receptor (ER), Liver X Receptor (LXR).

The breadth of this review identifying negative human health impacts of various EDC's strongly suggests that there are significant risks to human health and a precautionary approach and human bio-monitoring of EDC's is warranted.

Effects of EDCs on Glucose Metabolism and Obesity

Over the past several decades, obesity and type two diabetes rates have increased markedly in all age groups worldwide and particularly in developed countries. Obesity is frequently associated with other morbidities such as metabolic disorders (including metabolic syndrome, type 2 diabetes, cardiovascular diseases, pulmonary complications, and liver disease), psychological/social problems, reproductive abnormalities and some forms of cancers. An amalgamation of lifestyle, genetic and environmental factors probably accounts for the quick and considerable increase in obesity rates. An increase in the occurrence of metabolic diseases has been noted in last several decades, which coincide, with the significant changes in the chemical environment around us, which came in practice during the same period. Velmurugan et al. ( 9) has demonstrated that the common risk factors for diabetes may not be the sole contributor, while other factors such as production of EDCs (plastics, plasticizers, pesticides, e-waste, food additives) has been found to be positively associated with the growing trend of the global diabetes incidence in last few decades ( Figures 3, 4).

Figure 3. Total plastic waste generation by country, measured in million tons per year for the year 2010 and disease burden from non-communicable diseases (from 1990 to 2016). Total disease burden from non-communicable diseases (NCDs), measured in DALYs (Disability-Adjusted Life Years) per year. DALYs are used to measure total burden of disease-both from years of life lost and years lived with a disability. One DALY equals 1 lost year of healthy life. Base map courtesy of mapchart.net ( http://www.mapchart.net) [Source: ( 10, 11)].

This change in our chemical environment seems to be a contributing factor in this "obesity pandemic" apart from genetic factors and modern lifestyle that embraces high energy intake, reduced physical activity, sleep deprivation and near stable home temperatures. This has led to the hypothesis that some of these environmental chemicals disrupt the endocrine process and lead to obesity by interfering with various endocrine aspects of metabolism ( 13, 14). This hypothesis garners support from animal as well as epidemiological studies, which have revealed that a diverse array of environmental chemicals can influence adipogenesis and obesity; however, its extent, exact mechanism and epidemiology are yet to be elucidated. The EDCs linked with obesity and adiposity include several POPs ( 15- 18), BPA ( 19, 20), and phthalates ( 21- 23). It has been suggested that in utero or childhood exposure to certain EDCs predisposes some individuals to obesity, highlighting the importance of timing of exposure ( 17, 21, 22, 24, 25). Such EDCs have been referred to as "environmental obesogens" ( 26). The term "metabolic disruptors" has also been suggested as many of these chemicals be associated with other metabolic diseases such as metabolic syndrome, insulin resistance and type 2 diabetes as well ( 17, 27). Several studies have found association of type 2 diabetes and/or its risk factors with PBCs, DDE, organochlorine pesticides, HCB, dioxins, BPA and phthalates ( 28- 37). Some prospective studies have interestingly concluded that no single POP could significantly predict T2D, but some combination of POPs could predict the future risk of T2D ( 28, 31- 33, 38). This also lends support to the concern that exposure to EDCs as mixtures that occur in the environment may have greater significance than that to single chemicals tested in laboratories. In addition, several studies have shown association of gestational diabetes to phthalates, BPA and PCB ( 39- 42). Increased risk of non-alcoholic fatty liver disease has been linked to BPA ( 43) as well as cardiovascular risk factors are found to be linked to both organochlorine pesticides ( 44- 46) and BPA ( 47, 48).

EDCs alter metabolic balance through multiple mechanisms including alterations in peroxisome proliferator-modulated pathways ( 49), adipogenesis ( 50), pancreatic β-cell function ( 51- 53) and hypothalamic neuropeptides ( 54, 55). In addition, decreased physical activity in relation to whole-body energy balance has been reported in mice that were exposed to BPA either during a perinatal period or in adult life ( 56, 57). However, most of the evidence regarding the mechanism of action of EDCs comes from experimental studies. In a recent study, it was shown that prenatal exposure to BPA and certain phthalates might affect the levels of adiponectin and leptin in human fetus ( 58). A novel hypothesis advanced is the role of gut microbiota in EDC-induced type 2 diabetes. It is thought that EDCs not only cause dysbiosis of gut microbiota but also undergo metabolism by gut microbes, and both these processes alter the gut neuroendocrine regulation of metabolism leading to insulin resistance and type 2 diabetes ( 9).

Effect of EDC on Blood Pressure

To elucidate associations of urinary EDC's like phthalate with blood pressure (BP), a cross-sectional analysis of 6-19 years children in the 2003-2008 National Health and Nutrition Examination Survey (NHANES) was carried out which reveals an increase in blood pressure associated with 3-fold increase in urine phthalate (DEHP) ( 59). A study conducted in people living near a dioxin-contaminated area showed that increasing serum dioxin levels are correlated with elevated diastolic blood pressure. In addition, the prevalence of hypertension was also found to be correlated with serum PCDD and PCDF levels in Taiwanese and Florida adults with dioxin exposure ( 60, 61). Similarly, high serum PCB Level was also found to be associated with elevated blood pressure in NHANES 1999-2002 and NHANES 1999-2004 participants ( 62). Studies derive conclusions about the effects of EDCs on hypertension but further studies with larger diverse cohorts are now needed to verify an association between EDC's exposure and risk of hypertension.

EDCs: Female and Male Reproductive Health

Female Reproductive Disorders: Role of Environmental EDCs

Several EDCs are known to exert effects on female reproductive hormones and their receptors through oestrogenic, anti-estrogenic, androgenic and anti-androgenic mechanisms. Some of these EDCs include BPA and phthalate, which are classified as xeno-oestrogens. They act by both receptor-dependent and receptor-independent mechanisms, either directly by binding to estrogen receptors, increasing aromatase activity and increasing estrogen sensitivity, or indirectly by their effect on gonadotropin-releasing hormone, leading to an increase in endogenous estrogen production ( 63- 65). EDCs are thought to affect the menstrual cycle, alter fertility and oogenesis and are implicated in diseases such as polycystic ovary syndrome (PCOS) and endometriosis. Hauser et al. ( 66) documented the first-ever adverse effect of urinary phthalate metabolites by using in vitro fertilization (IVF) where they found that concentrations of urinary metabolites of di(2-ethylhexyl) phthalate (DEHP) and di-isodecyl phthalate (DiDP) was inversely related with oocyte yield and number of metaphase II oocytes at retrieval, whereas only metabolites of di-isononyl phthalate (DiNP) and di-isodecyl phthalate (DiDP) was linked with reduced fertilization rate. However, the exact mechanism of action of these chemicals is not yet clearly understood, but it is likely that multiple pathways are involved including hormone receptors.

Female Puberty

Puberty is a phase of transition between adolescence and adulthood during which the reproductive system reaches full maturity ( 67). The onset of puberty involves a complex neuroendocrine mechanism and influenced by several factors including ethnicity, genetics, nutrition and environmental factors ( 68, 69). Recently early onset of puberty in girls has been noted across several countries and a role of environmental pollutants has been implicated, notably that of BPA, phthalates, and organohalogens ( 70). Puberty is brought about by activation of the hypothalamic-pituitary gonadotropic cells-gonadal axis. The activation of this axis is thought to be regulated by a novel neuropeptide and its receptor, which are in turn modulated through sex steroid receptors in hypothalamic neurons ( 71). It is possible that oestrogenic EDCs or xeno-oestrogens act through the sex steroid receptors to bring about early puberty. During childhood, the levels of sex hormones in blood are very low. However, it has been found that estrogen and androgen receptors are expressed since a very early age ( 72). Therefore, even a small change in these levels through an exogenous source may bring about several pubertal changes ( 73). In several studies, early onset of pubertal changes in girls has been reported to be associated with phthalates ( 74- 77), BPA ( 78, 79), and organohalogens ( 80- 82), whereas in other studies, no such association ( 30, 83, 84) or even inverse association has been reported ( 85, 86). Another study found a non-linear association of BPA and phthalate metabolites with pubertal development ( 87). However, it is possible that these differences result from variation in environmental exposure, timing of exposure and non-linear dose-response patterns of EDC effects. Exposure to EDCs during prenatal and perinatal period has also been thought to be linked with early onset of puberty in girls as well ( 88).

Polycystic Ovary Syndrome (PCOS)

Most of the information on direct effects of EDCs on fertility is available from in vitro and animal studies. A few human studies have been carried out in women attending fertility clinics and have provided variable results. Some of these studies have shown an association of EDCs directly with infertility and its parameters, whereas others have correlated them with PCOS, which is a complex endocrinopathy leading to infertility and insulin resistance. A negative association between urinary concentrations of phthalate metabolites and serum inhibin B levels was found, suggesting a detrimental effect of phthalates on growing antral follicles ( 89). In the Longitudinal Investigation of Fertility and the Environment Study, concentrations of phthalates, but not BPA in women were associated with longer time to pregnancy ( 90). In other studies, BPA was found to be associated with reduced ovarian reserve ( 91), lower antral follicle count ( 92) and PCOS ( 93, 94) in infertile women. Higher quartiles of urinary BPA concentration were also shown to be associated with increased odds of implantation failure ( 95). One of the studies showed that high PCB and pesticide concentrations in the follicular fluid correlated with poor embryological intracytoplasmic sperm injection outcomes ( 96). Another study documented higher serum concentrations of perfluorinated compounds and phthalate metabolites was present in women with PCOS ( 97). DDT was found to be associated with altered hormonal levels in Chinese women with PCOS suggesting a role in its pathogenesis ( 98). In a large American cross-sectional study involving more than 30,000 women, researchers found an association between EDCs and earlier age at menopause, and they also identified 15 EDCs (including 9 PCBs, 3 pesticides, 1 furan and 2 phthalates) that needed further evaluation for potential adverse effects on ovarian function ( 99).

Endometriosis

Endometriosis is the presence of ectopic endometrial tissue, which is a cause of pain and infertility. Relatively few studies have explored the role of EDCs in endometriosis. However, its association has been reported with higher blood or urinary levels of BPA ( 94, 100), various phthalates ( 101- 103) and organochlorine pesticides ( 104, 105). On the other hand, no association or inverse association with phthalates was observed in studies on infertile Japanese women and American women, respectively ( 106, 107). A recent meta-analysis of past epidemiological studies has recorded a significant constant increase in the risk of endometriosis to polychlorinated bisphenyls (PCBs) group (OR = 1.58; 95% CI: 1.18-2.12), organochlorine pesticides (OCPs) group (OR = 1.40; 95% CI: 1.02-1.92) and phthalate esters (PAEs) group (OR = 1.27; 95% CI: 1.00-1.60), while BPA showed no significant association with endometriosis ( 108).

Obstetric Outcomes

In utero exposure to EDCs through trans-placental route has become a concern as it is thought that small endocrine imbalance during fetal development can lead to significant and lasting changes. Several researchers investigated this in the last decade. In utero BPA exposure was found to be associated with decreased birth weight ( 109- 112) and increase in gestational length ( 112, 113). However, Shoaff et al. ( 114), did not find any association between prenatal phthalate exposure and birth size or duration of gestation. In another study, both maternal and paternal BPA and phthalate concentration were found to be associated with smaller birth size and increased gestational age ( 115). Though both maternal and paternal exposure to these chemicals will be similar as they are exposed to similar environment, paternal EDC levels likely reflect maternal exposure and do not directly affect the fetal exposure. Maternal urinary levels of paraben were found to be associated with increased length of male neonates in a Chinese cohort ( 116). An American study on several classes of EDCs showed that gestational exposure to perfluoroalkyl substances and organophosphate pesticides was associated with reduced birth weight while phthalates, BPA, PCBs, PBDEs (Polybrominated diphenyl ethers) or OCPs (organochlorine pesticides) had either small or no association with differences in birth weight ( 117).

Male Reproductive Disorders: Role of Environmental EDCs

EDCs are thought to affect the male reproductive system as well. Human studies indicate that these chemicals affect pubertal growth and semen quality and cause defects such as hypospadias and cryptorchidism. Well over two decades back, Sharpe and Skakkebaek (1993) proposed that several male reproductive disorders such as low sperm counts, cryptorchidism and hypospadias may share a common fetal origin and that environmental exposure to oestrogenic chemicals might play a key role ( 118). Though this estrogen hypothesis was later refuted, except diethylstilbestrol, it propelled scientific research on effects of EDCs on male reproductive system. It is now thought that EDCs act on male reproductive system by disrupting fetal endocrine balance through their action on steroid hormone receptors (SHRs) or interference with the synthesis, kinetics or metabolism natural hormones (World Health Organization [WHO] and United Nations Environment Program 2012). Sperm function affected by reactive oxygen species generated during metabolism of these chemicals is another possible effect of EDCs leading to infertility ( 119).

Male Puberty

Semen Quality

There are numerous causes of infertility in men. Even though sperm count does not associate specifically with fertility, it is documented that men with very low sperm counts frequently have fertility problems. In 1996, in their review article, Toppari et al. expressed concern regarding decreasing semen quality in the last few decades, and they advanced the hypothesis that exposure to environmental chemicals may be responsible for adverse trends in male reproductive health ( 124). Several EDCs have been found to affect semen parameters adversely. These include organophosphate pesticides ( 125- 127), BPA ( 128), perfluorinated compounds ( 129), phthalates ( 130), and organochlorines ( 131). It was observed that adult life exposure to PBC affects mainly sperm motility while perfluorinated compounds alter sperm morphology primarily. However, the overall impact of EDCs on male fertility is not clear so far. Though sperm count and morphology appear to be altered to some extent, the effect on fertility seems to be minor at best.

Cryptorchidism

Cryptorchidism is the condition in which the testis does not descend completely to the bottom of the scrotum. Its incidence has been documented to increase from 1-3% in the late 1950s to 7-9% in the 2000s ( 132). This condition shows familial clustering suggesting a role of genetic and environmental factors. Known risk factors for cryptorchidism include maternal gestational diabetes and maternal use of alcohol, acetaminophen or nicotine-containing substances. In recent research, cryptorchidism has been found to be associated with prenatal or early childhood exposure of several chemicals including PBDE ( 133, 134), BPA ( 135), parabens ( 135), and PBCs ( 134). Other researchers have found no association of cryptorchidism with PBBs ( 136), organochlorine pesticides ( 137), perfluorinated compounds ( 138, 139), or PCBs ( 140). Because several environmental factors are also associated with cryptorchidism, the effects of individual factors are difficult to establish and the possibility of confounders cannot always be eliminated in these studies.

Hypospadias

Hypospadias is a congenital malformation in which the urethral folds do not fuse properly. A meta-analysis suggested an association between pesticide exposure in men and risk of hypospadias in their offspring ( 141). Another study suggests that exposure to HCB and p,p′-DDE during fetal life may be a risk factor for hypospadias ( 142). However, no significant association of exposure to PCBs ( 140, 143), organochlorine pesticides ( 137), PBBs ( 136), PBDE ( 143), or perfluorinated compounds ( 144) with hypospadias was noted in other studies. Hypospadias is a relatively uncommon condition; therefore, most of these studies face the problem of small sample size and are not statistically robust.

Overall, the effect of individual EDCs on reproductive system appears uncertain; however, it is possible that some additive or synergistic effects of mixtures do exist. Conflicting results and differences in study variables make it difficult to establish the role and extent of effects of EDCs either individually or in combinations. Large-scale studies taking into account the exposure of all known EDCs in a uniform manner will be needed to explore such complex association.

Role of EDCs in Neurodevelopmental Disorders

Prenatal exposure to EDCs is thought to affect brain development in the fetus, and some neurodevelopmental abnormalities have been attributed to them. Prenatal BPA exposure has been linked to poor social behavior ( 145, 146), higher anxiety and depressive behaviors ( 147). Some studies have suggested a sex-specific effect of BPA on behavior. A study showed that poor social behavior was found to be more pronounced in girls ( 146), while a study showed that significant positive associations were found between prenatal BPA and emotionally reactive and aggressive behavior in boys ( 148). In another study, prenatal BPA exposure was noted to be associated with increased anxiety and depression in boys but not in girls ( 149). The gender difference is likely due to the close relation of brain development with gonadal hormones as proven by animal studies. The brain, particularly hypothalamus, has been shown to be affected by oestrogenic EDCs in a sex-, time- and exposure-dependent manner ( 150). In utero exposure to phthalates is shown to be associated with adverse cognitive and behavioral outcomes such as lower IQ, attention deficit, hyperactivity and poorer social communication in children ( 151- 153). Other EDCs such as organophosphates ( 152, 154), PBDEs ( 155, 156), perfluorinated compounds ( 157), and PCBs ( 153, 158) have also been linked to adverse neurodevelopmental performance, behavioral problems and lower IQ to varying degrees. There is also some evidence that some PBDEs and PCBs affect neuropsychological status in adults ( 159, 160). Animal studies have provided ample evidence that EDCs act on developing brain through multiple mechanisms: (a) steroid hormones and receptors, (b) neuroendocrine system, and (c) epigenetic changes ( 161). One of the main biological mechanisms proposed for adverse neurodevelopmental effects of prenatal EDC exposure is disturbance of thyroid hormone homeostasis, which is critical for fetal brain development ( 162).

Thyroid Homeostasis and EDCs

Thyroid hormones are controlled through the hypothalamic-pituitary-thyroid axis, and they are vital for the regulation of several biological systems and can be disrupted by EDC exposure. Specifically, the phenols and phthalates are thought to act directly on the thyroid hormone receptor, and studies have shown that these compounds are thyroid receptor antagonists. Epidemiological studies have shown that cognitive functions of the offspring may be adversely affected by even a marginal lowering in thyroxin levels in a pregnant woman ( 163- 165). Thus, exposure to thyroid-disrupting chemicals may have significant consequences for public health even if they bring about a small reduction in thyroid hormone levels. Several studies have evaluated the EDC exposure effect on thyroid hormones in humans. Most of these have explored the effect on pregnant women and new-borns. A study reported that urinary concentrations of phthalate metabolites were seen to be associated with lower T4 and T3 or higher thyroid-stimulating hormone (TSH) levels in adults ( 166). In another study, prenatal exposure to phthalates was found to be inversely associated with both total serum thyroxine in pregnant women and newborns as well as TSH in newborns ( 167). The association with BPA is more variable in several studies. Two studies found no association between thyroid hormone levels and maternal BPA exposure ( 168) and cord blood BPA levels ( 58), respectively. Another study reported no association between maternal urinary BPA concentrations and thyroid hormones measured in cord serum when analyzed for all newborns, but they found that in female newborns, lower cord TSH was associated with the 10-fold increased BPA in maternal urine ( 169). In the analysis of data from the U.S. National Health and Examination Survey, urinary BPA showed a negative relationship with serum TSH levels in adults ( 166), while another European study concluded that a significant positive association was observed between urinary BPA and serum TSH in adult women ( 170). A study found that there was a negative correlation between PCB levels and maternal free T3 but not with cord blood ( 171). However, a systematic analysis did not find any correlation between PCBs and thyroid hormones in either pregnant women or newborns ( 172). In a study including three European cohorts, the researchers concluded that PCB was associated with lower TSH levels in newborns ( 173). In another study, in the case of PBDEs, negative correlation was found with maternal and cord blood thyroid hormones ( 171), while positive correlation was seen with maternal blood thyroid hormone levels in two other studies ( 174, 175). Organochlorine pesticides were noted to be associated with reduction in thyroid hormones ( 176).

Renal Function and EDCs

Chronic kidney disease (CKD) is a worldwide health menace of growing concern, and its prevalence all over world is estimated at 13.4% ( 177). Kidneys serve as the filtration unit and thus are exposed to all compounds in a greater degree compared to other organs. Humans are not exposed to a single compound but to mixture of compounds and they are likely to affect the kidneys as well. Without a doubt, many non-persistent EDCs, e.g., phthalates, bisphenols, benzophenones, and parabens, share common exposure sources such as plasticware and personal care products in various things.

There are growing evidences suggesting that non-persistent endocrine disrupting chemicals (EDCs), are linked with adverse kidney function ( 178). Studies also showed that exposure to di-(2-ethylhexyl) phthalate (DEHP) through the consumption of contaminated milk was associated with micro-albuminuria in children ( 179, 180). The US National Health and Nutrition Examination Survey (NHANES) delineated the association of urinary phthalate metabolites with CKD markers such as albumin-to-creatinine ratio (ACR), estimated glomerular filtration rate (eGFR), and urinary protein-to-creatinine ratio (UPCR) has also been revealed in a population of children ( 181, 182). Urinary BPA levels were also associated with ACR among the US children ( 183), as well as Chinese adults ( 184). Patients on dialysis have more exposure to BPA due to daily use of dialysis tubing. BPA levels are higher in patients undergoing hemodialysis and peritoneal dialysis, than in healthy controls ( 185). NHANES 2003-2006 Estimated Glomerular Filtration Rate (eGFR) among a general population of the US adult females shows positive association with urinary BPA levels but not in the adult males ( 186).

EDCs and Hormone-Dependent Cancers

A large fraction of EDCs acts through oestrogenic and androgenic receptors; therefore, researches have focused on their relation to hormone-dependent cancers. Such neoplasm's include breast, endometrial, ovarian, and cervical cancers in women and prostate cancer in men. The incidence of these cancers has risen at a higher rate over the last few decades ( 187- 189). As other non-communicable disease, this increase is multifactorial, and change in lifestyle, industrialization and EDCs are thought to be responsible at least in part ( 161).

The development and progression of estrogen-dependent malignancies, particularly breast cancer, depend on prolonged or exaggerated estrogen exposure ( 190). Approximately 70% of breast cancers are sensitive to estrogen as they show ER-positivity ( 191). Unopposed estrogen or estrogen-mimicking EDCs can bind to ER, switching on the downstream signaling of estrogen-responsive genes involved in the cell cycle ( 64, 192, 193). Several oestrogenic EDCs, such as BPA and phthalates, have been shown to induce epigenetic modification or genotoxic effects ( 27, 194, 195), which can alter gene products and genetics, eventually predisposing to certain diseases. However, only a few human studies have explored this association so far. Although meta-analysis did not find any association between organochlorine pesticides and breast cancer ( 196), another study linked it to exposure before 14 years of age ( 197). A study in Mexico showed a positive correlation between MEP, a phthalate metabolite, and breast cancer while other phthalates (MBP, MEOHP, and mono [3-carboxypropyl]) had negative correlation with breast cancer risk. The evidence of any relation between BPA exposure and breast cancer is weak, and it is observed only in animal studies. This may be due to its short half-life so that measurement of exposure is difficult ( 161). Agricultural Health Study, USA, has found that several pesticides are linked with increased risk of prostate cancer ( 198, 199). The link between EDCs and other hormone-dependent cancers is tenuous as it is based on a handful of animal and laboratory studies. It has not yet been explored through human studies, and it constitutes a lacuna in our knowledge.

Endocrine Disrupters Accelerate Aging

Growth is the consequence of a multifaceted interaction of genetic, constitutional, nutritional, endocrine and socio-economic factors as well as psychosocial well-being ( 200, 201). The evidence is seen in the fact that over the last century, children have gradually become taller and are reaching puberty earlier. For example, the age of menarche in European girls in the middle of the nineteenth century was 16-17 years, in contrast to the current age of 13 years or less. Rampant use of sex steroids as growth promoters has occurred in animal farming, which continues in several countries of the world.

The timing and evolution of reproductive aging depend mainly on genetic and environmental factors. So far, most research has focused on genetic predispositions ( 202). Nevertheless, the environment probably plays a role, and there is an assumption that EDCs may accelerate reproductive aging, resulting in shortened reproductive lifespan. Recent studies show that exposures to EDC during critical developmental periods, particularly during fetal life and early childhood can cause molecular/cellular changes that modify the function of the affected tissues later in life, a concept referred to as the fetal/developmental basis of adult disease ( 203). These effects are thought to be brought about by epigenetic modifications. Animal studies show that reproductive aging is accelerated by EDC (MXC) ( 204), BPA ( 205), and dioxins ( 206). Recent epidemiological evidence also links EDC exposures during periods of growth to accelerated menopause [diethylstilbestrol ( 207) and perfluorocarbons ( 208)]. BPA has also been shown to promote oxidative stress and inflammation, and it has been suggested that it makes postmenopausal women suffer health effects related to aging ( 209).

Dose-Response Dynamics of EDCs

Several biological effects of EDCs are mediated through SHRs ( 210). Hormone receptors are high-affinity receptors, that is, a small concentration of hormones or hormone mimickers can produce a significant biological effect. In addition, the dose-response curves of these receptors are non-linear; they can be sigmoidal, or more complex, including being non-monotonic. The non-monotonic dose-response (NMDR) curves are often "U-shaped" (with maximal responses observed at low and high doses) or "inverted U-shaped" (with maximal responses observed at intermediate doses) ( 161). EDCs mimic hormones in their actions including low-dose effects and dose-response patterns, which depend on characteristics of their binding receptors and ligands ( 161). Due to this property, environmentally relevant concentrations of these chemicals need to be studied thoroughly for their effects on the general population. Several studies have provided evidence that low-dose environmental exposures to EDCs are associated with disorders in humans as well as wildlife ( 17, 70, 211, 212). Research shows that many SHR-mediated adverse effects of EDCs may be non-linear or even non-monotonic and such effects commonly occur in dose ranges exerting no overt cytotoxicity ( 213- 218). One of the setbacks due to these non-classical responses is in extrapolating the effect from high doses to low doses or vice versa because there is no linear relationship ( 219). Defining safe limits for such chemicals also becomes troublesome.

Most of the evidence for NMDR comes from cell culture and animal studies. Several epidemiological studies have also focused their attention on NMDR in EDCs recently but they differ significantly in their conclusions ( 23, 219). However, these differences in conclusions can arise from several inherent drawbacks in human epidemiological studies, for example, cross-sectional vs. prospective design dichotomy and difference in study analysis methods. In addition, there is a lack of sufficient knowledge about kinetics of these chemicals, their interactions with adiposity and diet, and changes to these factors with age and gender. Furthermore, in epidemiological studies, populations are often exposed to a variable mixture of chemicals at various exposure levels for various durations, and predictably, these interactions are complex. These factors make human studies difficult to interpret.

Epigenetic and Transgenerational Effects of EDCs

More recently, scientists have discovered evidence showing that EDCs can modify gene expression without mutating DNA, which is also known as epigenetic change ( 220). The known possible mechanisms of epigenetic changes include methylation of cytosine residues in DNA, post-translational modification of histones and altered microRNA expression. These changes may lead to transgenerational effects on numerous organs and organ systems and are thought to be tissue-specific as well as dose-dependent ( 221- 223). Epigenetic transgenerational inheritance is the phenomenon where environmental exposures of a woman during gestation lead to germline epimutations, which are then transmitted to subsequent generations with an observable phenotypic expression ( 224, 225). BPA is the most widely studied EDC in terms of epigenetic changes, and several animal studies support this theory ( 226- 228). Other chemicals implicated in epigenetic changes leading to transgenerational effects are dioxins ( 229, 230), DDT ( 231, 232), and phthalates ( 233, 234). In addition, the epigenetic changes are also thought to cause latent effects in case of early life exposure, with manifestation later in life ( 235). However, most of these studies have evaluated only the effects on the reproductive system of experimental animals. The epigenetic changes add another dimension to understand the complexity and mechanism in the studies of environmental exposures and their effects.

Timing of EDC Exposures as a Critical Element

EDCs influence and modify the hormonal homeostasis and, therefore, it is plausible that their effects will be more pronounced in periods during which marked hormonal changes occur, such as perinatal period, puberty and pregnancy. Tissue differentiation and maturation of several organs occur during these "critical windows" which may be influenced by EDCs ( 236). Moreover, the effects of exposure may not be apparent immediately and may take several years to be observable. This is supported by several studies that show the association between several health and behavioral parameters and EDC exposure during these key periods. In utero exposure to several EDCs was found to influence fetal growth and birth weight ( 109- 112, 117) and also predisposed some individuals to obesity and fat gain during later life ( 17, 21, 22). Exposure to EDCs during the prenatal and perinatal period has been thought to influence the timing of puberty as well ( 86, 88). Several studies evaluating the neurological development of children have shown association of prenatal EDC exposure with adverse cognitive and behavioral outcomes ( 145, 146, 148, 151- 153). Prenatal exposure to phthalates was shown to alter thyroid homeostasis in pregnant women as well as the offspring ( 167).

For POPs, latency could be due to long-term continuous exposure. For non-persistent EDCs, exposure could be variable. Apart from the molecular mechanism, epigenetic changes may be important in delayed or latent effects, especially in perinatal exposures.

Risks From Exposure to EDCs Mixture: Cocktail Effect

Gutmicrobiome: A Player in the Toxicity of Environmental Pollutants

Human beings are exposed to different chemicals through air, water and soil, but oral exposure to EDCs, largely via the food chain, is their key pathway into human metabolism. The gut microbiome comprises trillions of bacteria, viruses and fungi, and it is documented as a main participant in the metabolism of nutritional compounds, drugs, antibiotics and environmental toxins ( 259- 264). Bacterial fermentation of undigested carbohydrates and xenobiotic compounds produces short-chain fatty acids, choline, bile metabolites and several volatile compounds including hydrogen sulfide. The gut microbiome also plays a major role in regulation of various hormones, neurotransmitters and ultimately host metabolism. Some metabolites produced during bacterial fermentation in gut behave as hormones that can affect diverse host metabolic functions, and hence the gut microbiome has been anticipated to be a new endocrine organ ( 265- 267). Dysbiosis of gut microbiome is commonly observed in medical conditions. Concerning diabetes, a shift in the Bacteroidetes/ Firmicutes ratio was found to have a key link with plasma glucose concentration ( 268). Microbial metabolism of chemicals, as well as EDCs, by gut microbiome can lead to microbial dysbiosis, stimulation of specific bacterial genes, and altered microbial transformation of molecules ( 260, 268).

Previous researchers had determined how diet and host health could influence the gut microbiome composition ( 269- 273). On the other hand, several acquired factors encountered on an everyday basis can exert extreme effects on the gut microbiome. Environmental chemicals, such as EDCs, heavy metals and air pollutants are increasingly invasive in terrestrial and aquatic environments, and there is a signal that such chemicals will turn out to be even more profuse in near future ( 274- 281). Such chemicals are also found in things that we use daily, such as plastic water bottles, storage containers, and antibacterial supplies. Introduction to such chemicals can result in extensive host effects and concurrently aim commensal microbes harbor within the gut and perhaps other organs. The chemical-induced dysbiosis of the gut microbiome leads to disruptions in various host systems, mainly the central nervous system, via the gut-microbiome-brain axis ( 282- 284). An improved understanding of how environmental chemical-induced gut microbiome dysbiosis changes strengthen host disease is essential. Disorders associated with gut dysbiosis comprise metabolic, neurobehavioral, immunological, cardiovascular, gastrointestinal and several other disease states ( 273, 285- 291). The gut microbiomes have a different class of enzymes such as Phase I and Phase II. They can metabolize environmental xenobiotic chemicals and environmental chemicals may cause dysbiosis of gut microbiome, with potential effects on human health. Based on previous studies we may conclude that gut microbiome plays a major role in the toxicity of environmental pollutants.

Evaluating Health Effects of EDCs in Humans: Is Exposomes Mapping the Key?

Impact of EDCs in the Context of Climate Change

Climate change has been globally acclaimed as a major environmental risk, also when combined with chemical pollution and habitat loss it has the potential to have a severe impact on human health. The impact of escalating climate change has added exigency to the rising problem of EDC's pollutants. A study by Brown et al. ( 297), the first of its kind, showed that climate change could amplify the deleterious effects of pollution from EDCs on aquatic environments. Climate change is influencing the rate at which toxic chemicals are released from plastic materials, stockpiles and polluted sites. Higher temperatures increase the release of persistent organic pollutants (POPs) to air by changing their rate of partitioning between air and soil, and between air, water and sediment. POPs persist in the environment, bioaccumulate through the food chain, and pose a threat of causing detrimental effects to human health ( 298). The fate of environmental POPs in these ecosystems is controlled mainly by temperature and biogeochemical processes. Climate change may increase the re-emissions and redistribution of POPs in different environmental settings via soil-air exchange. Soil-air exchange is a main process of controlling distribution of POPs and terrestrial ecosystem carbon at local and universal scales.

Each POP has a moving potential in air and/or water and can bioaccumulate in biological system such as lipid-rich tissues and biomagnified through terrestrial and aquatic food chains ( 299). Hence, POPs have a potential adverse impact on higher trophic organisms in aquatic as well as environment ( 300- 302) and on human health ( 303). Moreover, intensification of global warming, an increase in POPs levels has been reported in different settings of environment because of the release from reservoirs ( 299, 304). These higher emissions induced by climate change would increase the susceptibility of exposed animals and humans via food chain and lead to greater adverse effects on human health and terrestrial ecosystems ( 305).

While the researcher's grapples with the problem of ever-increasing environmental pollution, at the same time, the causes of climate change and its impacts on the fate and toxicity of chemicals must be addressed.

EDC Regulations in Various Countries

The issue of EDCs is puzzled due to lack of scientific data and many gaps in knowledge, it is still essential to frame regulatory policies to deal with the present scenario. Framing of policies and regulations must go parallel with ongoing research and any measure must be compliant to rapid advances in scientific knowledge. At present countries having regulations addressing the manufacture, use and disposal of various chemicals includes carcinogens, teratogens, mutagens and substances that interrupt reproduction. EDCs represent a relatively new classification, involving diverse chemical classes that are able to mimic endogenous hormones. Although there is no agreement on their regulation, EDCs are addressed in different cases in EU law, such as the Water Framework Directive, Registration, Evaluation and Authorization of Chemicals (REACH), Plant Protection Products Regulation (PPPR) and Cosmetic Regulation ( Table 2). The government and regulatory agencies of developing countries with large population and huge plastic consumers may restrict the use of endocrine disrupting chemicals in the production of cosmetics, food-contact plastics, plastic water tanks and packaging materials and may provide guidelines on the use of EDC's/plastics, awareness on its harmful impacts and the use of risk-free alternatives must be provided, with an intention to ensures the safety of vulnerable group such as infants, children and pregnant women. The challenges that arise from the field of endocrine disruption are the immense diversity of chemicals produced, but not tested, the mixtures and the unknown interactions between them, together with their consequent effects. The lack of an effective and strong legislation and regulation poses a significant threat to humans, animals and plants, and contributes to the exposure to chemicals that may disrupt the endocrine system.

Table 2. Regulations on controlling EDCs in various countries.

Conclusions

Most of the information on the detrimental effect of EDCs comes from animal studies. It is becoming more evident that EDCs may trigger disorders such as metabolic diseases, reproductive abnormalities, endocrine dysfunction and cancers. The sudden escalating rates of these diseases, especially metabolic disorders, correlate with global industrialization and the production and release of EDCs to the environment, drinking water and eventually to food chain. Countries should formulate acts and guidelines including EDCs for the safety of drinking water. To understand the exposure routes of EDCs better, a comprehensive assessment of drinking water supply is required. Exposure to EDC or mixtures, even at low doses, especially during critical window or early development can have an impact on endocrine system by altering transgenerational/epigenetic pathway. Hence, further studies are required to establish the threshold concentrations of EDCs in the environment as well as in human matrixes (bio monitoring) below which its detrimental effects do not occur. Large-scale systematic epidemiology studies need to be carried out taking into consideration the low dose effect and cocktail effect of EDCs.

Author Contributions

MKumar, MKumawat, SS, and DS: manuscript draft. RT and AP: overall review. VV: manuscript drafting and editing. All authors contributed to the article and approved the submitted version.

Funding

This study was supported by Indian Council of Medical Research grant no: NIREH//BPL/PJ-30/2018-19/127. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

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The exponential growth of pollutant discharges into the environment due to increasing industrial and agricultural activities is a rising threat for human health and a biggest concern for environmental health globally. Several synthetic chemicals, categorized as potential environmental endocrine-disrupting chemicals (EDCs), are evident to affect the health of not only livestock and wildlife but also humankind.

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Bhavya Sree Komaravolu: Although it seems as though there is a correlation between EDCs and Constitutional advancements if growth (CAG), a new study shows the pattern is unrelated and CAG is caused by precocious or early puberty. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8226958/ 

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Bhavya Sree Komaravolu: Although it seems as though there is a correlation between EDCs and Constitutional advancements if growth (CAG), a new study shows the pattern is unrelated and CAG is caused by precocious or early puberty. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8226958/ 

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Pierluigi Marzuillo, Academic Editor

Abstract

In recent decades, pubertal onset in girls has been considered to occur at an earlier age than previously. Exposure to endocrine-disrupting chemicals (EDCs) has been associated with alterations in pubertal timing, with several reports suggesting that EDCs may have a role in the secular trend in pubertal maturation, at least in girls. However, relevant studies give inconsistent results. On the other hand, the majority of girls with idiopathic precocious or early puberty present the growth pattern of constitutional advancement of growth (CAG), i.e., growth acceleration soon after birth. Herein, we show that the growth pattern of CAG is unrelated to exposure to endocrine-disrupting chemicals and is the major determinant of precocious or early puberty. Presented data suggest that EDCs, at most, have a minor effect on the timing of pubertal onset in girls.

Keywords: puberty, endocrine-disrupting chemicals, girls, precocious puberty, early puberty, constitutional advancement of growth

1. Introduction

Pubertal timing is multifactorial involving a predominant effect of genetic and epigenetic factors and to a lesser-but still significant-extent, environmental factors. Although genetic factors are considered to explain 50-80% of the onset of puberty [ 1], genes that have been found to play a role in pubertal onset have a minor role in the onset of puberty of the population. Gene mutations, e.g., ESR1, KISS1, KISSR1, MKRN3, are rarely identified as the cause of disordered pubertal timing; thus, these genes do not seem to determine the timing of puberty nor menarche in the female population [ 2]. Recently, it was shown that the onset of puberty is regulated by epigenetic mechanisms, Kiss1 expression is negatively regulated by two polycomb group proteins ( Cbx7 and Eed) [ 3].

Environmental factors are major determinants of the onset of puberty and the age at menarche. Nutrition of the mother or/and of the infant [ 4], chronic diseases and chronic somatic stress, like strenuous exercise or psychological stress, e.g., violence exposure [ 5], or adoption of a girl from an underprivileged environment, exert a major influence on the timing of the pubertal events. Although environmental factors may result in epigenetic modifications in an organism, the epigenetic effects of the environment on the hypothalamic regulation of puberty are still to be discovered.

In girls, when pubertal onset occurs before the age of 8 years, it is considered precocious and when it occurs after 8 years but before 9 years of age, it is considered early. Precocious puberty (PP) when it is gonadotropin dependent is called central or true PP and when it is gonadotropin independent is called peripheral PP. The causes of central precocious puberty may be organic, e.g., due to tumors of the central nervous system (CNS) or most commonly idiopathic, i.e., no etiological factor is identified with appropriate imaging of the CNS. Obviously, it is important to differentiate between organic or idiopathic precocious puberty (IPP) because the former may have dire health consequences. On the other hand, early puberty lies on the extreme of normal variation of timing of pubertal onset [ 6].

In recent decades, there have been reports from several countries detailing that the onset of puberty in girls occurs at a younger age than previously [ 7, 8]. At the same time, endocrine-disrupting chemicals (EDC) have been suggested as affecting the age of pubertal onset, especially in girls. Hence, researchers were led to hypothesize that increasing exposure to EDC had a role in the secular trend for earlier sexual maturation. Moreover, it was suggested that early puberty manifesting in immigrants from developing countries was the result of previous exposure to organochloride pesticides [ 9].

Constitutional advancement of growth (CAG) is the growth pattern of early growth acceleration, which is present in the majority of girls with idiopathic precocious puberty, and in girls with early puberty [ 6, 10]. Herein, we show that CAG is unrelated to EDC exposure and we maintain that EDCs have, at most, a minor role on female pubertal timing.

2. Endocrine-Disrupting Chemicals

Endocrine-disrupting chemicals are compounds that can interfere with the activity of endocrine systems. EDCs action is exerted by imitating or blocking hormone signaling through the relevant hormonal receptor. EDCs may also modulate the synthesis, metabolism, and binding of natural hormones.

EDCs are usually used by industry, as plastics (bisphenol A (BPA)), plasticizers (phthalates), solvents/lubricants (polybrominated biphenyls (PBBs), polychlorinated biphenyls (PCBs), dioxins), pesticides (chlorpyrifos, dichlorodiphenyltrichloroethane (DDT), methoxychlor), fungicides (vinclozolin) and also as flame retardant additives in manufactured materials and pharmaceutical agents, e.g., diethylstilbestrol (DES), a non-steroidal synthetic estrogen [ 11].

EDCs may also be made by nature, e.g., phytoestrogens, which interfere with endogenous endocrine function, are produced by plants and act primarily through estrogen receptors [ 12].

The abundance of EDCs and their ability to interfere with the endocrine system combined with the secular trend for earlier onset of puberty has led many researchers to associate EDCs with early puberty, especially since some EDCs have estrogenic activity.

3. Association between Exposure to Endocrine-Disrupting Chemicals and Timing of Puberty

Commonly used and studied EDCs are phthalates, bisphenol, pesticides and flame retardants.

Phthalates are mainly used as plasticizers, i.e., substances added to plastics to increase their flexibility, transparency, durability, and longevity. They are used primarily to soften polyvinyl chloride (PVC). Human exposure to phthalates [ 13] is extremely prevalent, and it may occur through multiple routes: i.e., oral (via phthalate-contaminated food, water and other liquids and in children through mouthing of toys and teethers), dermal (via cosmetics and other personal care products), inhaled (from breathing phthalate particles in the air) ( https://www.cdc.gov/biomonitoring/Phthalates_FactSheet.html accessed 5 June 2021). Phthalates may have anti-androgenic activity [ 14], and also possess some estrogenic activity [ 15]. In Puerto Rico, high phthalate levels have been linked to premature thelarche [ 16], a normal variant of female premature sexual development. In a study of Danish schoolgirls, high phthalate excretion in urine was associated with delayed pubarche, but not thelarche, which suggests anti-androgenic actions of phthalate [ 17]. Similar results were obtained in a study of US girls [ 18]. Some studies reporting early/precocious puberty associated with phthalate exposure [ 19, 20], whereas in a US study on girls with central precocious puberty, such an association was not found [ 21]. In contrast to the previous reports, in a Chinese study performed in boys, elevated levels of phthalates were associated with constitutional delay of growth and puberty [ 22]. Furthermore, a recent Korean study showed that phthalate metabolites in girls with central precocious puberty were significantly lower than the prepubertal control girls [ 23]. Therefore, more studies are warranted to explore the effect of phthalate exposure on pubertal timing.

BPA (bisphenol A) is found in plastics (e.g., bottles, Tupperware, etc.), and in epoxy resins coating the inside of beverage and food cans, and humans are exposed mainly through food contamination from plastic and can packaging. BPA is the most commonly found estrogen-like endocrine disruptor in the environment. In experimental animals, it has been shown that BPA advances puberty [ 24], but on the other hand, it also has been shown it has no effect on pubertal timing [ 25]. Similar to the experimental animals, results of BPA on human puberty are inconsistent. In a US study of girls, Wolff et al. reported that BPA had no influence on breast development [ 26], however in studies performed in Turkey and in Thailand, idiopathic central precocious puberty was associated with higher levels of BPA than in control girls [ 27, 28]. Watkins et al. studied the in-utero and peripubertal exposure to phthalates and BPA in relation to sexual maturation and did not find any association between BPA and sexual maturation, although in utero phthalate exposure impacted on earlier timing of sexual maturation [ 29].

Pesticides are classified into various classes, e.g., insecticides, herbicides and fungicides. Pesticides enter the human body through water, air, food, and also, they can pass from mother to fetus via the placenta and to the infant through mother's milk. Most of these substances are lipophilic and accumulate in adipose tissue, where they remain for long periods of time. The most widely studied pesticide compounds are dichlorodiphenyltrichloroethane (DDT) and its metabolite dichlorodiethyldichloroethane (DDE). Although the agricultural use of DDT has been banned worldwide, in some developing countries, it is still used against mosquitos. In a study performed in Denmark, mothers who worked in greenhouses in the first trimester of pregnancy were prenatally categorized as exposed or unexposed to pesticides. Female offspring of exposed mothers had decreased age of breast development at 8.9 years, compared with 10.4 years in the unexposed, and 10.0 years in a Danish reference population [ 30]. Other researchers reported decreased menarcheal age to girls exposed in utero to DDE [ 31]. However, the significance of the association disappeared when weight at menarche was controlled for. Adopted or immigrant girls in Belgium, who presented central precocious puberty (CPP), had increased plasma levels of pesticides (DDE), thus CPP could be attributed to pesticide exposure [ 9]. However, other studies did not find an association between DDE levels and the timing of menarche [ 32]. Additionally, in inner-city girls, DDE, Pb, and dietary intakes of phytoestrogens were not significantly associated with breast stage [ 33]. In a study examining prenatal DDT exposure in relation to anthropometric and pubertal measures in adolescent males, no associations between prenatal exposure to any of the DDT compounds and any pubertal measures were noted [ 34].

Flame-retardant chemicals are added to manufactured materials (plastics, textiles, surface finishes and coatings) intended to prevent or slow the further development of ignition with their physical and chemical properties. Among them, organohalogen compounds such as polybrominated diphenyl ethers (PBDEs) are lipophilic persistent endocrine disruptors exhibiting estrogenic as well as androgenic properties. It has been proposed that PBDEs might alter pubertal timing resulting in later menarche in girls [ 35, 36] but earlier pubarche in boys [ 36]. Curiously, girls with idiopathic central precocious puberty, particularly those with higher body mass index (BMI) have been found with higher serum concentrations of PBDEs [ 37].

Thus, the inconsistency of the results of the various studies examining the association of endocrine disruptor chemicals with the onset of puberty [ 38] makes it imperative that more studies on the subject are performed.

4. Constitutional Advancement of Growth

Constitutional advancement of growth (CAG) is a growth pattern characterized by early growth acceleration [ 10]. Children with CAG are born with an average length but present growth acceleration soon after birth, reaching a zenith centile in the first 2 to 4 years of life. Then, the child grows along this centile until the onset of puberty, which is usually early. The growth pattern of CAG is the mirror image of the well-established growth pattern of constitutional delay of growth (CDG), which is characterized by growth deceleration in the first 2 to 3 years of life, although birth length is usually average. As a result of the growth deceleration, the child's height may fall to a nadir centile, which is at or below the third centile, depending on parental height. Then, growth resumes at a normal rate, and the child grows along this centile until the onset of puberty, which is usually delayed.

For a child to be considered as presenting the growth pattern of CAG, his/her Height Standard Deviation Score (SDS) should be ≥1.5 than Target Height (TH) SDS and other conditions that lead to early growth acceleration, such as genetic tall stature, overfeeding, and intrauterine growth restraint have to be excluded. In the case of a typical girl, the growth pattern of CAG is depicted in .

Red bullets depict actual height, orange squares depict bone age. Target Height (TH) derived from the heights of the mother (yellow dot) and father (blue dot) is shown on the right-hand side of the figure and as a blue line within the graph.

5. Constitutional Advancement of Growth and Early Puberty

Based on the observation that girls with precocious puberty (i.e., with breast development before 8 years of age) are tall for age, and also taller for TH, even at the onset of pubertal development, we examined the growth of 47 girls with IPP from birth until diagnosis [ 39]. The vast majority, i.e., 79%, of the girls with IPP manifested the growth pattern of CAG. Thus, we suggested that this pattern may be used as an additional clue in favor of idiopathic precocious puberty in the differential diagnosis of precocious puberty [ 10], a claim that was supported by the fact that all girls with CAG presented no CNS abnormalities in brain magnetic resonance imaging (MRI) [ 39]. We also observed a similar growth pattern in girls with early puberty, i.e., with breast development at an age between 8 and 9 years [ 40]. The greater the difference HSDS-THSDS is above +1.5 SD the greater the possibility for the girl to present precocious than early puberty.

6. Is There an Association between EDC Exposure and CAG?

Since CAG is associated with early growth acceleration, if there was an association between EDCs and CAG it would be related to fetal exposure during pregnancy or early postnatal exposure.

Several studies have examined the association of fetal exposure to EDCs, especially phthalates and BPA, and fetal growth. These compounds, besides hormonal perturbations, may cause oxidative stress and epigenetic modifications that might have a deleterious effect on fetal growth [ 41, 42, 43]. Most relevant studies were cross-sectional, measurements of length and weight were performed at delivery [ 44] and the results were inconsistent [ 45, 46, 47]. In a longitudinal study that examined the relationship between average exposure measures and fetal growth [ 48], researchers observed inverse associations between head and abdominal circumferences, femur length, and estimated fetal weight and Di (2-ethylhexyl) phthalate (DEHP) metabolites. However, no consistent associations were observed for other phthalate metabolites or for BPA. Taken together, these data indicate that it is unlikely EDC exposure in utero has a substantial adverse effect on fetal growth. In our studies on CAG girls, there was no difference in birth weight or length compared to control girls.

What about the effect of postnatal exposure on growth? In a study in which chlordecone (an organochlorine insecticide with estrogenic properties) was measured at cord blood and in breast milk at the age of 3 months, postnatal exposure in girls was associated with lower height at 3, 8 and 18 months [ 49].

Taking into account that girls with CAG present growth acceleration soon after birth, it is unlikely that growth acceleration is induced by estradiol. In neonatal life and early infancy increased estrogen levels are experienced by all normal girls. Estradiol levels peak around the second month after birth and reach levels characteristic of early- to mid-puberty, hence the period of the first 6 months of life is termed as mini-puberty. Despite increased estradiol levels, mini-puberty is not associated with growth acceleration contrary to puberty occurring during childhood.

How could the early growth acceleration characteristic of CAG be explained? Clinical evidence suggests that after the age of 2 to 3 months growth hormone (GH) is necessary for normal growth [ 50]. However, the majority of small for gestational age (SGA) infants present catch-up growth in length since early postnatal life placing them at or above the 3d percentile for length by 6 months of age [ 51]. Catch-up growth in these children is mostly attributed to overnutrition, thereby increasing insulin levels, a hormone with growth-promoting properties. However, from the 3d day of life SGA neonates present functional hypersomatotropism, i.e., increased GH and insulin growth factor 1 (IGF-1) levels relative to appropriate for gestational age neonates, suggesting that the somatotropic axis is fully operational since the first days of life [ 52, 53]. In line with a functional GH/IGF-1 axis from the first days of life is the observation that, in healthy full-term neonates, the postnatal growth velocity is positively related to a spurt in immediate postnatal life IGF-1 levels [ 54]. It is noteworthy that the increased IGF-1 levels may persist in later years [ 55].

SGA children presenting catch-up growth are more prone to insulin resistance and development of metabolic syndrome [ 56]. Recently a study on Sprague-Dawley rats examined whether a post-receptor crosstalk of GH and insulin signaling might affect insulin resistance in catch-up growth SGA animals [ 57]. The authors demonstrated that catch-up growth SGA rats exhibit increased insulin resistance associated with an impaired IRS-1-PI3K-AKT signaling pathway, which resulted from GH signaling-induced upregulation of SOCS3 expression. Thus, these data suggest a link between increased GH levels and insulin resistance in catch-up growth.

Constitutional advancement of growth presents similarities to catch-up growth only that the CAG children are, auxologically, appropriate for gestational age. Accordingly, they are susceptible to developing obesity during childhood, therefore we suggested that the growth pattern of CAG may be a predictor, not only of early puberty, but of childhood obesity as well [ 10]. Moreover, it has been reported that earlier menarche was associated with greater height, adiposity, and significantly increased serum IGF-1 at 8 years of age, even after adjustment for height and BMI [ 58]. Thus, these data allow us to speculate that the growth pattern of CAG is induced by the early activation of the GH/IGF-1 axis.

7. Concluding Remarks

The secular trend of pubertal onset in girls as reflected by the age at menarche has started since the early 20th century with the improvement of the socioeconomic and hygienic conditions. This trend, however, has levelled off in many countries of the developed world [ 59] and it seems that it has reached the age range of menarche occurrence in the paleolithic female [ 60], suggesting that human females evolved to enter puberty at a young age.

There is a growing body of evidence that some EDCs alter the metabolic set-points and increase the risk of obesity [ 61], and that EDC exposure during fetal life is associated with breast cancer [ 62]. It is of interest that the increase in obesity prevalence coincides with increases in endocrine-disrupting chemical production and exposures [ 63]. The rapid increase in the prevalence of obesity in the last three decades suggests that environmental factors are major players in the obesity epidemic. The association between EDCs and early onset of puberty not only is inconsistent but is complicated by the fact that, at least in girls, pubertal events may be advanced by obesity [ 64].

Undoubtedly, there is a great need for the EDC effects on the human body systems to be studied thoroughly. However, from the data presented in this review, it is clear that the major determinant of early puberty, at least in girls, is the presentation of the growth pattern of constitutional advancement of growth, which is unrelated to EDC exposure. Therefore, if there is a role of EDCs on female pubertal timing it seems, at the most, to be a minor one.

Abbreviations

BPA: bisphenol A, CAG: constitutional advancement of growth, CDG: constitutional delay of growth, CNS: central nervous system, CPP: central precocious puberty, DDE: dichlorodiethyldichloroethane, DDT: dichlorodiphenyltrichloroethane, DEHP: di (2-ethylhexyl)phthalate, DES: diethylstilbestrol, EDCs: endocrine-disrupting chemicals, IPP: idiopathic precocious puberty, PBBs: polybrominated biphenyls, PCBs: polychlorinated biphenyls, PP: precocious puberty, SGA: small for gestational age, TH: target height.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

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Endocrine-Disrupting Chemicals and Early Puberty in Girls

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Pierluigi Marzuillo, Academic Editor In recent decades, pubertal onset in girls has been considered to occur at an earlier age than previously. Exposure to endocrine-disrupting chemicals (EDCs) has been associated with alterations in pubertal timing, with several reports suggesting that EDCs may have a role in the secular trend in pubertal maturation, at least in girls.

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Nailah Miller: The Environmental Effects on Puberty.

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Nailah Miller: The Environmental Effects on Puberty.

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The amount of hormones and chemicals in your body determine how early or how late you will start puberty. For children in this new generation children are experiencing puberty a lot earlier than children in the previous generation. Believed to be because the excessive amount of Melatonin in the body.

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Dear EarthTalk: I heard that children are reaching puberty at earlier ages now and that it may have to do with environmental toxins and even their TV viewing habits. Can you enlighten?
-- Mark Abbot, via e-mail

To say that kids are growing up faster than ever these days may be more than just cliché. Recent studies have shown that children are reaching puberty at younger and younger ages, and researchers are starting to see links between this trend and other societal ills such as ubiquitous pollution and sedentary lifestyles.

In a 2007 report for the Breast Cancer Fund entitled "The Falling Age of Puberty in U.S. Girls: What We Know, What We Need to Know," ecologist Sandra Steingraber argues that unfettered access to computers and TVs over the last 30 years has led to an increasingly sedentary lifestyle among kids in the U.S. and beyond. Active kids produce more melatonin, a natural hormone that serves as the body's internal clock and calendar. This could explain why sedentary kids are likely to go through puberty sooner: Their bodies think their decreased melatonin production is a trigger to move into puberty. "[Melatonin is] an inhibitory signal for puberty," says Steingraber. "The more melatonin you have, the later you go into puberty."

Of course, sedentary lifestyles are also linked to childhood obesity, a condition that often continues-along with the many health problems that can accompany it-into adulthood. A recent National Health and Nutrition Examination Survey (NHANES) found that, between 2001 and 2004, 17.5 percent of children ages six to 11 were overweight-an effective doubling of obesity rates three decades ago. A study by the non-profit Obesity Society came up with a slightly higher figure-20 percent-with the percentages higher for Hispanic, African-American and Native American children.

Obesity is certainly one factor in the surge in so-called "precocious" adolescence, but chemicals are also thought to play a role. According to Erin Barnes, writing in E - The Environmental Magazine,a study comparing the body mass index of Danish and American girls found that the former group hit puberty a full year later than the latter even though their weights were in the same range. Another study found that wealthy girls in South Africa reach puberty a full year after their African-American counterparts. "Many researchers," writes Barnes, "are studying the relationship between chemical pollutants like PCBs (polychlorinated bphenyls) and phthalates (commonly used plasticizers) and premature development."

Some researchers believe that the preponderance of synthetic chemicals in more developed societies are interfering with human endocrine development and essentially "tricking" kids' bodies into going through puberty prematurely. Also, precocious puberty in girls has been linked to breast cancer, as well as higher rates of drug abuse, violence, unintended pregnancies, problems in school and mental health issues.

"Shortening childhood means a shortening of the time before the brain's complete re-sculpting occurs," says Steingraber. "Once that happens, the brain doesn't allow for complex learning." She adds that the brain can only build the connections used to learn a language, play a musical instrument or ride a bike before it gets flooded with the sex hormones that come with the onset of puberty.

CONTACTS: Breast Cancer Fund, www.breastcancerfund.org; National Health and Nutrition Examination Survey, www.cdc.gov/nchs/nhanes.htm; Obesity Society, www.obesity.org.

GOT AN ENVIRONMENTAL QUESTION? Send it to: EarthTalk, c/o E/The Environmental Magazine, P.O. Box 5098, Westport, CT 06881; submit it at: www.emagazine.com/earthtalk/thisweek/, or e-mail: earthtalk@emagazine.com. Read past columns at: www.emagazine.com/earthtalk/archives.php.

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Dear EarthTalk: I heard that children are reaching puberty at earlier ages now and that it may have to do with environmental toxins and even their TV viewing habits. Can you enlighten? -- Mark Abbot, via e-mail To say that kids are growing up faster than ever these days may be more than just cliché.

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Alleni Velasco:This article is focused on early exposure of EDC's such as Bisphenol A, Phthalates,Pesticides,Dioxins and PCB's can influence early puberty on certain organs such as adipose tissue or adrenal glands and the HPG axis. 

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Alleni Velasco:This article is focused on early exposure of EDC's such as Bisphenol A, Phthalates,Pesticides,Dioxins and PCB's can influence early puberty on certain organs such as adipose tissue or adrenal glands and the HPG axis. 

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Abstract

Over the last decades, the onset of puberty in girls has occurred earlier, but the tempo of pubertal progression has been relatively slower, resulting in a younger age at puberty onset without a change in age at menarche. Sufficient energy availability and adiposity contribute to early pubertal development, and environmental factors, such as endocrine-disrupting chemicals (EDCs), may affect not only the control of energy balance, but also puberty and reproduction. EDCs are hormonally active substances that can perturb puberty by acting both peripherally on target organs, such as adipose tissue or adrenal glands, and/or centrally on the hypothalamic-pituitary-gonadal (HPG) axis. Depending on whether the exposure takes place earlier during fetal and neonatal life or later during early childhood, EDCs can lead to different outcomes through different mechanisms. Evidence of associations between exposures to EDCs and altered pubertal timing makes it reasonable to support their relationship. However, human epidemiologic data are limited or inconsistent and cannot provide sufficient evidence for a causal relationship between EDC exposure and changes in pubertal timing. Further investigation is warranted to determine the overall or different effects of EDCs exposure during prenatal or childhood windows on pubertal milestones and to reveal the underlying mechanisms, including epigenetic marks, whereby early-life exposure to EDCs affect the HPG-peripheral tissue axis.

Keywords: Puberty, Endocrine disrupting chemicals, Secular trend

Introduction

Puberty begins with the release of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator from central nervous system inhibition after a quiescent period during childhood [ 1]. The age of menarche has definitely decreased from 16 years in the 1800s to 13 years in the 1960s, after which this downward trend seems to have slowed or even stopped [ 2]. The trend for earlier age at menarche has also been reported in Korea, from 16.6 years for females born between 1925 and 1929 to 12.6 years for females born between 1990 and 1994 [ 3, 4]. While the onset of puberty has been occurring earlier, the overall tempo of pubertal progression is reported to be relatively slower, resulting in skewing towards younger ages for the onset of puberty and towards older ages for the completion of puberty [ 2]. Such increased duration of the pubertal transition may result from gonadotropin-independent estrogenic action at the level of the breast which may originate from peripheral tissues [ 5].

Although genetic factors remain the major determinant of pubertal timing [ 6], the secular trend for earlier onset of puberty has coincided with improved public health and nutrition [ 7- 9]. Sufficient energy availability and early adiposity rebound contribute to early pubertal development. Endocrine-disrupting chemicals (EDCs) may affect not only the control of energy balance and adiposity, but also puberty and reproduction [ 2]. EDCs can act at any level in the 'hypothalamic-pituitary-gonadal (HPG)-peripheral tissues' axis [ 10]. Their effects can manifest right before puberty as well as much earlier, during fetal and neonatal life [ 2]. We reviewed the genetic and environmental factors that have been implicated in pubertal timing. We present the current evidence for the roles of EDCs on pubertal timing in girls based on human epidemiologic data and introduce potential mechanisms depending on the timing of exposure.

Factors implicated in pubertal timing by affecting the hypothalamic-pituitary-gonadal-peripheral tissues axis

Pubertal onset appears to be a highly heritable trait, but earlier sexual maturation in girls may be linked to environmental factors. Some genes implicated in precocious puberty ( DLK1, MKRN3, and KISS1) and in delayed puberty ( TACR3) are associated with variations in pubertal onset. The childhood GnRH pulse generator may be inhibited, in part, by MKRN3. Disinhibition of the HPG axis leads to the progressive amplification of pulsatile GnRH secretion which may be mediated by leptin levels and increased expression of neuropeptides, such as neurokinin B and kisspeptin, and their receptors [ 1]. In addition to central signals, peripheral metabolic and nutritional signals, such as leptin and insulin, play a permissive role in the proper function of the HPG axis [ 11].

Genes involved in pubertal timing or menarche reported by Elks et al. [ 12] and Perry et al. [ 13] include those related to hypothalamic GnRH secretion, pituitary development and function, hormone synthesis and bioactivity, energy homeostasis and growth, and potential peripheral feedback from sex steroids [ 13]. The overlap of genes involved in pubertal onset and obesity from genome wide association studies and human epidemiological data suggests a common pathway connecting early infancy weight gain, earlier pubertal onset, and later obesity in adulthood, although mechanisms whereby early-life changes in adiposity affect growth acceleration and early pubertal onset remain to be determined [ 14, 15].

Obese girls are at risk for early pubertal development. Girls maturing at earlier ages have higher body mass index (BMI) z-scores with advanced Tanner stages [ 16]. Obesity-induced hyperinsulinemia and hyperleptinemia can affect linear growth and advance puberty [ 17]. Since adrenarche starts earlier as childhood BMI and insulin levels gradually increase, the increased adrenal androgen levels in obese children may also be responsible for accelerated growth before puberty [ 17]. In addition, hyperinsulinemia can increase sex steroid bioavailability via stimulation of ovarian growth and steroidogenesis, reduction of sex hormone binding globulin, and increased conversion of androgens to estrogens by stimulating aromatase activity in adipose tissues [ 2]. Breast development can occur from the gonadotropin-independent release of estrogen by peripheral tissues, such as the adrenal glands and adipose tissue. Finally, increased sex steroid levels in obese girls can induce gonadotropin-independent or dependent precocious puberty.

Gene-to-environment interactions during critical periods of development could alter gene expression in the HPG-peripheral tissue axis through epigenetic mechanisms [ 18]. While recent data have started to reveal the epigenetic regulation of KISS1 expression [ 18, 19], epigenetic mechanisms that affect the HPG-peripheral tissue axis remain to be discovered. The intrauterine environment, birth size, nutrition, and (potentially) EDCs can affect adiposity and the timing of puberty. EDCs and adiposity may be the most concerning contributors to changes in puberty and reproduction. While sufficient energy availability provides clues to the mechanism of early pubertal development, changes in the control of both energy balance and reproduction may vary under the influence of common determinants, such as EDCs. These effects can manifest right before puberty as well as much earlier during fetal and neonatal life [ 20, 21]. Although the link between EDCs and pubertal timing does not prove causality, their association is highly suggestive [ 22].

Possible mechanisms for the effect of EDCs on pubertal timing in girls

EDCs are defined as "an exogenous chemical, or mixture of chemicals, that interferes with any aspect of hormone action" by the Endocrine Society's second scientific statement published in 2015 [ 10]. Endocrine disruptors can be naturally occurring or synthetic. This section reviews the effects of synthetic EDCs on pubertal timing in girls.

EDCs can act through several mechanisms at any level in the 'HPG-peripheral tissues' endocrine axis. Directly, EDCs may affect genes or HPG pathways unique to puberty. Indirectly, EDCs can act as obesogens and change metabolic programming during fetal and early childhood development, resulting in alterations in the metabolic and peripheral hormones associated with the onset of puberty [ 15]. A variety of chemical toxins that are ubiquitously present in our environment exert antagonistic and agonistic actions on hormonal axes and pathways at low versus high concentrations, and they demonstrate nonmonotonic dose-response curves [ 10]. Since many EDCs can act as agonists of estrogen receptors (ERs) or antagonists of androgen receptors (ARs), EDCs may mimic naturally occurring estrogens and androgens. In addition, EDCs might bind to a receptor within a cell and block the functions of endogenous hormones, thus acting as antiestrogens and antiandrogens [ 10, 23].

Low-level exposures to a mixture of EDCs may mask the effect of an individual compound. Moreover, interindividual and interspecies variation in susceptibility may be present due to differences in pharmacokinetics and/or genetic polymorphisms in key genes, complicating our ability to translate data from animal studies to human health [ 20]. In humans, the long interval from exposure to endpoints (latent effects) and the setting of mixed exposures over a lifetime make it difficult to analyze the health outcomes of EDCs [ 20]. Furthermore, it is difficult to distinguish the influence of nutrition and adiposity on pubertal outcomes from that of EDCs. Since the prenatal, neonatal, infancy, early childhood and peripubertal periods are critical windows of development, EDCs may have different effects depending on the time of exposure [ 2]. Though the mechanisms whereby fetal or neonatal life changes pubertal timing have not been fully elucidated, EDCs can alter the genes involved in the modification of epigenetic marks [ 2, 15]. Although researching the effect of EDCs on pubertal timing is difficult for the various aforementioned reasons, further investigation is warranted to determine which EDCs can affect pubertal timing, which periods of exposure are critical, and how different mechanisms are involved depending on the timing of exposure in humans [ 24].

Human epidemiologic data on EDCs in regards to pubertal timing in girls

The Endocrine Society's Scientific Statements present a review of the literature on human studies that assessed the associations between EDCs exposure and pubertal timing [ 10]. There have been extensive reviews about the contribution of EDCs on shifts in pubertal timing [ 2, 10, 24, 25] as well as a recent review of publications from 2016 to 2017 that examined EDC associations with pubertal milestones [ 26]. The EDCs most extensively studied in regards to puberty include bisphenol A (BPA), phthalate esters, pesticides (dichlorodiphenyl trichloroethane [DDT], pyrethroids), dioxins and polychlorinated biphenyls (PCBs), and flame retardants (polybrominated biphenyls [PBB], polybrominated diphenyl ethers [PBDEs]) (). This section reviews the human epidemiologic studies regarding the relationship of individual EDCs with pubertal development or menarche in girls.

Table 1.

Phthalates

Phthalates are used as liquid plasticizers in plastics and flooring, personal care products, medical devices and tubing with half-lives of 12 hours. Although the mechanism remains to be further elucidated, phthalates might act as ER agonists or AR antagonists and may also interfere with androgen synthesis [ 53]. Phthalates may be classified into low-molecular-weight phthalates (ester side-chain lengths, 1 to 4 carbons) and high-molecular-weight-phthalates (ester side-chain lengths, 5 or more carbons). Di(2-ethylhexyl) phthalate (DEHP) metabolites (mono(2-ethyl-5-oxohexyl) phthalate and mono(2-ethyl-5-hydroxyhexyl) phthalate), which are classified into high-molecular-weight-metabolites, were the phthalates most commonly studied.

The results from studies regarding the relationship between phthalate and puberty in girls conflict depending on the timing of exposure and/or different phthalate metabolites () [ 31, 33, 34, 36, 42, 45, 47- 49, 54- 65]. Different studies have shown significant associations with premature thelarche [ 54, 56] and precocious or early puberty [ 31, 36, 42, 55, 58, 61, 62, 64, 65], while no relationship with puberty [ 34, 57, 59] or an association with later onset of puberty [ 45, 47- 49, 60] have also been reported. Among several studies on phthalate exposure, nine studies had a longitudinal cohort design [ 33, 45, 47- 49, 59- 61, 63]. Prenatal exposure to high-molecular-weight phthalate metabolites was associated with later onset of puberty [ 48]. Early childhood exposure to phthalate metabolites was not associated to puberty [ 59] or associated with earlier [ 61] or later [ 45, 47, 49, 60] pubertal progression. Meanwhile, differences in pubertal development or age at menarche has been reported depending on the timing of exposure or classes of exposures (high or low-molecular-weight-phthalates) [ 33, 63]. Whereas high-molecular-weight phthalate levels were associated with later pubic hair development, low-molecular-weight phthalate levels were related to advanced breast or pubic hair development [ 33]. While prepubertal exposure to DEHP was associated with later menarche, pubertal exposure to monomethyl phthalate (low-molecular-weight) was related to earlier menarche only among overweight or obese girls and not among those that were of normal weight [ 63].

Revised paradigm of EDCs on pubertal timing and future directions

Parent et al. [ 2] suggested a revised paradigm of EDCs on pubertal timing. Since improvements in public health and nutrition have paralleled a downward trend in pubertal timing and menarche, environmental factors are the predominant determinants in pubertal timing. Classically, environmental factors, including EDCs, might advance pubertal timing via central mechanisms, particularly during the prepubertal period. According to the revised paradigm [ 2], environmental factors can influence puberty and reproduction through not only central but also peripheral mechanisms, depending on whether they take place early during fetal and neonatal life or late during the prepubertal period. Environmental factors can affect energy availability during the fetal and neonatal period or in infancy and early childhood. They can also act as obesogens and promote early adiposity rebound, leading to changes in metabolic or peripheral signals and increases in adrenal androgen levels, with subsequent early pubertal development. Environmental factors, including EDCs, might affect genetic or epigenetic pathways during critical windows of development. Further investigation is warranted in order to reveal the underlying mechanisms whereby early-life exposure to EDCs affects the HPG-peripheral tissue axis.

Conclusion

The onset of puberty in girls is occurring earlier, without changes in the timing of completion of puberty. Physiological variability and multiple other factors affect the initiation of puberty. Exposure to a broad mixture of EDCs is ubiquitous, and the shifts in pubertal timing may be mediated by exposures to EDCs at critical developmental windows. EDCs are hormonally active substances that can act via several mechanisms to perturb puberty either peripherally on target organs (adipose tissue or adrenal glands) or centrally via the HPG axis. EDCs can influence pubertal timing through different mechanisms depending on when the exposure occurs during early-life. Evidence for associations between exposures of EDCs and altered pubertal timing makes it reasonable to support the hypothesis that a relationship between EDCs and pubertal timing exists. However, the current data is insufficient and conflicting to provide sufficient evidence for a causal relationship between EDCs exposure and changes in pubertal timing in humans. Further human epidemiologic studies of prospective and longitudinal design are needed to determine the combined effect of EDC exposure on puberty and reproduction during critical periods. In addition, the underlying mechanisms whereby early-life exposures to EDCs influence puberty, including epigenetic marks, need to be explored in further studies.

Acknowledgments

This study was supported by grants from the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2018R1D1A1B07049806).

Footnotes

Conflict of interest No potential conflict of interest relevant to this article was reported.

References

29. Han EJ, Yim OK, Chung J, Baek SY, Kim Y. The study of relationship between the concentrations of Bisphenol A and DEHP in human plasma and precocious puberty. Anal Sci Technol. 2008;21:375-82. [ Google Scholar]

31. Lee SW, Lee WJ, Chae HW, Lee EB, Kim JH, Kim DH, et al. Determination of serum Di-(2-ethylhexyl) phthalate and bisphenol A level in children with idiopathic central precocious puberty. J Korean Soc Pediatr Endocrinol. 2009;14:154-62. [ Google Scholar]

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Early-life exposure to endocrine-disrupting chemicals and pubertal development in girls

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Over the last decades, the onset of puberty in girls has occurred earlier, but the tempo of pubertal progression has been relatively slower, resulting in a younger age at puberty onset without a change in age at menarche. Sufficient energy availability ...

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Kirsten Secena: Exposure to EDCs like BPAs,  phthalates and pesticides have caused puberty to prematurely start in girls.

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Kirsten Secena: Exposure to EDCs like BPAs,  phthalates and pesticides have caused puberty to prematurely start in girls.

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https://www.mdpi.com/2227-9067/8/6/492/htm

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by 1,* and 2,3

Pediatric Endocrinology Unit, Third Department of Pediatrics, National and Kapodistrian University of Athens, "Attikon" University Hospital, Haidari, 12462 Athens, Greece

Pediatric-Adolescent Endocrinology and Diabetes, Athens Medical Center, 15125 Marousi, Greece

Endocrine Unit, Aretaeion University Hospital, 11528 Athens, Greece

Author to whom correspondence should be addressed.

Academic Editor: Pierluigi Marzuillo

Received: 28 May 2021 / Revised: 7 June 2021 / Accepted: 8 June 2021 / Published: 10 June 2021

In recent decades, pubertal onset in girls has been considered to occur at an earlier age than previously. Exposure to endocrine-disrupting chemicals (EDCs) has been associated with alterations in pubertal timing, with several reports suggesting that EDCs may have a role in the secular trend in pubertal maturation, at least in girls. However, relevant studies give inconsistent results. On the other hand, the majority of girls with idiopathic precocious or early puberty present the growth pattern of constitutional advancement of growth (CAG), i.e., growth acceleration soon after birth. Herein, we show that the growth pattern of CAG is unrelated to exposure to endocrine-disrupting chemicals and is the major determinant of precocious or early puberty. Presented data suggest that EDCs, at most, have a minor effect on the timing of pubertal onset in girls.

1. Introduction

Pubertal timing is multifactorial involving a predominant effect of genetic and epigenetic factors and to a lesser-but still significant-extent, environmental factors. Although genetic factors are considered to explain 50-80% of the onset of puberty [ 1], genes that have been found to play a role in pubertal onset have a minor role in the onset of puberty of the population. Gene mutations, e.g., ESR1, KISS1, KISSR1, MKRN3, are rarely identified as the cause of disordered pubertal timing; thus, these genes do not seem to determine the timing of puberty nor menarche in the female population [ 2]. Recently, it was shown that the onset of puberty is regulated by epigenetic mechanisms, Kiss1 expression is negatively regulated by two polycomb group proteins (Cbx7 and Eed) [ 3].

Environmental factors are major determinants of the onset of puberty and the age at menarche. Nutrition of the mother or/and of the infant [ 4], chronic diseases and chronic somatic stress, like strenuous exercise or psychological stress, e.g., violence exposure [ 5], or adoption of a girl from an underprivileged environment, exert a major influence on the timing of the pubertal events. Although environmental factors may result in epigenetic modifications in an organism, the epigenetic effects of the environment on the hypothalamic regulation of puberty are still to be discovered.

In girls, when pubertal onset occurs before the age of 8 years, it is considered precocious and when it occurs after 8 years but before 9 years of age, it is considered early. Precocious puberty (PP) when it is gonadotropin dependent is called central or true PP and when it is gonadotropin independent is called peripheral PP. The causes of central precocious puberty may be organic, e.g., due to tumors of the central nervous system (CNS) or most commonly idiopathic, i.e., no etiological factor is identified with appropriate imaging of the CNS. Obviously, it is important to differentiate between organic or idiopathic precocious puberty (IPP) because the former may have dire health consequences. On the other hand, early puberty lies on the extreme of normal variation of timing of pubertal onset [ 6].

In recent decades, there have been reports from several countries detailing that the onset of puberty in girls occurs at a younger age than previously [ 7, 8]. At the same time, endocrine-disrupting chemicals (EDC) have been suggested as affecting the age of pubertal onset, especially in girls. Hence, researchers were led to hypothesize that increasing exposure to EDC had a role in the secular trend for earlier sexual maturation. Moreover, it was suggested that early puberty manifesting in immigrants from developing countries was the result of previous exposure to organochloride pesticides [ 9].

Constitutional advancement of growth (CAG) is the growth pattern of early growth acceleration, which is present in the majority of girls with idiopathic precocious puberty, and in girls with early puberty [ 6, 10]. Herein, we show that CAG is unrelated to EDC exposure and we maintain that EDCs have, at most, a minor role on female pubertal timing.

2. Endocrine-Disrupting Chemicals

Endocrine-disrupting chemicals are compounds that can interfere with the activity of endocrine systems. EDCs action is exerted by imitating or blocking hormone signaling through the relevant hormonal receptor. EDCs may also modulate the synthesis, metabolism, and binding of natural hormones.

EDCs are usually used by industry, as plastics (bisphenol A (BPA)), plasticizers (phthalates), solvents/lubricants (polybrominated biphenyls (PBBs), polychlorinated biphenyls (PCBs), dioxins), pesticides (chlorpyrifos, dichlorodiphenyltrichloroethane (DDT), methoxychlor), fungicides (vinclozolin) and also as flame retardant additives in manufactured materials and pharmaceutical agents, e.g., diethylstilbestrol (DES), a non-steroidal synthetic estrogen [ 11].

EDCs may also be made by nature, e.g., phytoestrogens, which interfere with endogenous endocrine function, are produced by plants and act primarily through estrogen receptors [ 12].

The abundance of EDCs and their ability to interfere with the endocrine system combined with the secular trend for earlier onset of puberty has led many researchers to associate EDCs with early puberty, especially since some EDCs have estrogenic activity.

3. Association between Exposure to Endocrine-Disrupting Chemicals and Timing of Puberty

Commonly used and studied EDCs are phthalates, bisphenol, pesticides and flame retardants.

Phthalates are mainly used as plasticizers, i.e., substances added to plastics to increase their flexibility, transparency, durability, and longevity. They are used primarily to soften polyvinyl chloride (PVC). Human exposure to phthalates [ 13] is extremely prevalent, and it may occur through multiple routes: i.e., oral (via phthalate-contaminated food, water and other liquids and in children through mouthing of toys and teethers), dermal (via cosmetics and other personal care products), inhaled (from breathing phthalate particles in the air) ( https://www.cdc.gov/biomonitoring/Phthalates_FactSheet.html accessed 5 June 2021). Phthalates may have anti-androgenic activity [ 14], and also possess some estrogenic activity [ 15]. In Puerto Rico, high phthalate levels have been linked to premature thelarche [ 16], a normal variant of female premature sexual development. In a study of Danish schoolgirls, high phthalate excretion in urine was associated with delayed pubarche, but not thelarche, which suggests anti-androgenic actions of phthalate [ 17]. Similar results were obtained in a study of US girls [ 18]. Some studies reporting early/precocious puberty associated with phthalate exposure [ 19, 20], whereas in a US study on girls with central precocious puberty, such an association was not found [ 21]. In contrast to the previous reports, in a Chinese study performed in boys, elevated levels of phthalates were associated with constitutional delay of growth and puberty [ 22]. Furthermore, a recent Korean study showed that phthalate metabolites in girls with central precocious puberty were significantly lower than the prepubertal control girls [ 23]. Therefore, more studies are warranted to explore the effect of phthalate exposure on pubertal timing.

BPA (bisphenol A) is found in plastics (e.g., bottles, Tupperware, etc.), and in epoxy resins coating the inside of beverage and food cans, and humans are exposed mainly through food contamination from plastic and can packaging. BPA is the most commonly found estrogen-like endocrine disruptor in the environment. In experimental animals, it has been shown that BPA advances puberty [ 24], but on the other hand, it also has been shown it has no effect on pubertal timing [ 25]. Similar to the experimental animals, results of BPA on human puberty are inconsistent. In a US study of girls, Wolff et al. reported that BPA had no influence on breast development [ 26], however in studies performed in Turkey and in Thailand, idiopathic central precocious puberty was associated with higher levels of BPA than in control girls [ 27, 28]. Watkins et al. studied the in-utero and peripubertal exposure to phthalates and BPA in relation to sexual maturation and did not find any association between BPA and sexual maturation, although in utero phthalate exposure impacted on earlier timing of sexual maturation [ 29].

Pesticides are classified into various classes, e.g., insecticides, herbicides and fungicides. Pesticides enter the human body through water, air, food, and also, they can pass from mother to fetus via the placenta and to the infant through mother's milk. Most of these substances are lipophilic and accumulate in adipose tissue, where they remain for long periods of time. The most widely studied pesticide compounds are dichlorodiphenyltrichloroethane (DDT) and its metabolite dichlorodiethyldichloroethane (DDE). Although the agricultural use of DDT has been banned worldwide, in some developing countries, it is still used against mosquitos. In a study performed in Denmark, mothers who worked in greenhouses in the first trimester of pregnancy were prenatally categorized as exposed or unexposed to pesticides. Female offspring of exposed mothers had decreased age of breast development at 8.9 years, compared with 10.4 years in the unexposed, and 10.0 years in a Danish reference population [ 30]. Other researchers reported decreased menarcheal age to girls exposed in utero to DDE [ 31]. However, the significance of the association disappeared when weight at menarche was controlled for. Adopted or immigrant girls in Belgium, who presented central precocious puberty (CPP), had increased plasma levels of pesticides (DDE), thus CPP could be attributed to pesticide exposure [ 9]. However, other studies did not find an association between DDE levels and the timing of menarche [ 32]. Additionally, in inner-city girls, DDE, Pb, and dietary intakes of phytoestrogens were not significantly associated with breast stage [ 33]. In a study examining prenatal DDT exposure in relation to anthropometric and pubertal measures in adolescent males, no associations between prenatal exposure to any of the DDT compounds and any pubertal measures were noted [ 34].

Flame-retardant chemicals are added to manufactured materials (plastics, textiles, surface finishes and coatings) intended to prevent or slow the further development of ignition with their physical and chemical properties. Among them, organohalogen compounds such as polybrominated diphenyl ethers (PBDEs) are lipophilic persistent endocrine disruptors exhibiting estrogenic as well as androgenic properties. It has been proposed that PBDEs might alter pubertal timing resulting in later menarche in girls [ 35, 36] but earlier pubarche in boys [ 36]. Curiously, girls with idiopathic central precocious puberty, particularly those with higher body mass index (BMI) have been found with higher serum concentrations of PBDEs [ 37].

Thus, the inconsistency of the results of the various studies examining the association of endocrine disruptor chemicals with the onset of puberty [ 38] makes it imperative that more studies on the subject are performed.

4. Constitutional Advancement of Growth

Constitutional advancement of growth (CAG) is a growth pattern characterized by early growth acceleration [ 10]. Children with CAG are born with an average length but present growth acceleration soon after birth, reaching a zenith centile in the first 2 to 4 years of life. Then, the child grows along this centile until the onset of puberty, which is usually early. The growth pattern of CAG is the mirror image of the well-established growth pattern of constitutional delay of growth (CDG), which is characterized by growth deceleration in the first 2 to 3 years of life, although birth length is usually average. As a result of the growth deceleration, the child's height may fall to a nadir centile, which is at or below the third centile, depending on parental height. Then, growth resumes at a normal rate, and the child grows along this centile until the onset of puberty, which is usually delayed.

For a child to be considered as presenting the growth pattern of CAG, his/her Height Standard Deviation Score (SDS) should be ≥1.5 than Target Height (TH) SDS and other conditions that lead to early growth acceleration, such as genetic tall stature, overfeeding, and intrauterine growth restraint have to be excluded. In the case of a typical girl, the growth pattern of CAG is depicted in Figure 1.

Red bullets depict actual height, orange squares depict bone age. Target Height (TH) derived from the heights of the mother (yellow dot) and father (blue dot) is shown on the right-hand side of the figure and as a blue line within the graph.

5. Constitutional Advancement of Growth and Early Puberty

Based on the observation that girls with precocious puberty (i.e., with breast development before 8 years of age) are tall for age, and also taller for TH, even at the onset of pubertal development, we examined the growth of 47 girls with IPP from birth until diagnosis [ 39]. The vast majority, i.e., 79%, of the girls with IPP manifested the growth pattern of CAG. Thus, we suggested that this pattern may be used as an additional clue in favor of idiopathic precocious puberty in the differential diagnosis of precocious puberty [ 10], a claim that was supported by the fact that all girls with CAG presented no CNS abnormalities in brain magnetic resonance imaging (MRI) [ 39]. We also observed a similar growth pattern in girls with early puberty, i.e., with breast development at an age between 8 and 9 years [ 40]. The greater the difference HSDS-THSDS is above +1.5 SD the greater the possibility for the girl to present precocious than early puberty.

6. Is There an Association between EDC Exposure and CAG?

Since CAG is associated with early growth acceleration, if there was an association between EDCs and CAG it would be related to fetal exposure during pregnancy or early postnatal exposure.

Several studies have examined the association of fetal exposure to EDCs, especially phthalates and BPA, and fetal growth. These compounds, besides hormonal perturbations, may cause oxidative stress and epigenetic modifications that might have a deleterious effect on fetal growth [ 41, 42, 43]. Most relevant studies were cross-sectional, measurements of length and weight were performed at delivery [ 44] and the results were inconsistent [ 45, 46, 47]. In a longitudinal study that examined the relationship between average exposure measures and fetal growth [ 48], researchers observed inverse associations between head and abdominal circumferences, femur length, and estimated fetal weight and Di (2-ethylhexyl) phthalate (DEHP) metabolites. However, no consistent associations were observed for other phthalate metabolites or for BPA. Taken together, these data indicate that it is unlikely EDC exposure in utero has a substantial adverse effect on fetal growth. In our studies on CAG girls, there was no difference in birth weight or length compared to control girls.

What about the effect of postnatal exposure on growth? In a study in which chlordecone (an organochlorine insecticide with estrogenic properties) was measured at cord blood and in breast milk at the age of 3 months, postnatal exposure in girls was associated with lower height at 3, 8 and 18 months [ 49].

Taking into account that girls with CAG present growth acceleration soon after birth, it is unlikely that growth acceleration is induced by estradiol. In neonatal life and early infancy increased estrogen levels are experienced by all normal girls. Estradiol levels peak around the second month after birth and reach levels characteristic of early- to mid-puberty, hence the period of the first 6 months of life is termed as mini-puberty. Despite increased estradiol levels, mini-puberty is not associated with growth acceleration contrary to puberty occurring during childhood.

How could the early growth acceleration characteristic of CAG be explained? Clinical evidence suggests that after the age of 2 to 3 months growth hormone (GH) is necessary for normal growth [ 50]. However, the majority of small for gestational age (SGA) infants present catch-up growth in length since early postnatal life placing them at or above the 3d percentile for length by 6 months of age [ 51]. Catch-up growth in these children is mostly attributed to overnutrition, thereby increasing insulin levels, a hormone with growth-promoting properties. However, from the 3d day of life SGA neonates present functional hypersomatotropism, i.e., increased GH and insulin growth factor 1 (IGF-1) levels relative to appropriate for gestational age neonates, suggesting that the somatotropic axis is fully operational since the first days of life [ 52, 53]. In line with a functional GH/IGF-1 axis from the first days of life is the observation that, in healthy full-term neonates, the postnatal growth velocity is positively related to a spurt in immediate postnatal life IGF-1 levels [ 54]. It is noteworthy that the increased IGF-1 levels may persist in later years [ 55].

SGA children presenting catch-up growth are more prone to insulin resistance and development of metabolic syndrome [ 56]. Recently a study on Sprague-Dawley rats examined whether a post-receptor crosstalk of GH and insulin signaling might affect insulin resistance in catch-up growth SGA animals [ 57]. The authors demonstrated that catch-up growth SGA rats exhibit increased insulin resistance associated with an impaired IRS-1-PI3K-AKT signaling pathway, which resulted from GH signaling-induced upregulation of SOCS3 expression. Thus, these data suggest a link between increased GH levels and insulin resistance in catch-up growth.

Constitutional advancement of growth presents similarities to catch-up growth only that the CAG children are, auxologically, appropriate for gestational age. Accordingly, they are susceptible to developing obesity during childhood, therefore we suggested that the growth pattern of CAG may be a predictor, not only of early puberty, but of childhood obesity as well [ 10]. Moreover, it has been reported that earlier menarche was associated with greater height, adiposity, and significantly increased serum IGF-1 at 8 years of age, even after adjustment for height and BMI [ 58]. Thus, these data allow us to speculate that the growth pattern of CAG is induced by the early activation of the GH/IGF-1 axis.

7. Concluding Remarks

The secular trend of pubertal onset in girls as reflected by the age at menarche has started since the early 20th century with the improvement of the socioeconomic and hygienic conditions. This trend, however, has levelled off in many countries of the developed world [ 59] and it seems that it has reached the age range of menarche occurrence in the paleolithic female [ 60], suggesting that human females evolved to enter puberty at a young age.

There is a growing body of evidence that some EDCs alter the metabolic set-points and increase the risk of obesity [ 61], and that EDC exposure during fetal life is associated with breast cancer [ 62]. It is of interest that the increase in obesity prevalence coincides with increases in endocrine-disrupting chemical production and exposures [ 63]. The rapid increase in the prevalence of obesity in the last three decades suggests that environmental factors are major players in the obesity epidemic. The association between EDCs and early onset of puberty not only is inconsistent but is complicated by the fact that, at least in girls, pubertal events may be advanced by obesity [ 64].

Undoubtedly, there is a great need for the EDC effects on the human body systems to be studied thoroughly. However, from the data presented in this review, it is clear that the major determinant of early puberty, at least in girls, is the presentation of the growth pattern of constitutional advancement of growth, which is unrelated to EDC exposure. Therefore, if there is a role of EDCs on female pubertal timing it seems, at the most, to be a minor one.

Funding

This research received no external funding.

Institutional Review Board Statement

Informed Consent Statement

Data Availability Statement

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

BPA: bisphenol A, CAG: constitutional advancement of growth, CDG: constitutional delay of growth, CNS: central nervous system, CPP: central precocious puberty, DDE: dichlorodiethyldichloroethane, DDT: dichlorodiphenyltrichloroethane, DEHP: di (2-ethylhexyl)phthalate, DES: diethylstilbestrol, EDCs: endocrine-disrupting chemicals, IPP: idiopathic precocious puberty, PBBs: polybrominated biphenyls, PCBs: polychlorinated biphenyls, PP: precocious puberty, SGA: small for gestational age, TH: target height.

References

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Figure 1. This girl presented at the clinic at the age of 8.1 years with a Tanner stage 2 breast and pubic hair development. Breast developed at the age of 7.4 years and pubic hair at the age of 7.8 years. Before the appearance of any signs of puberty, her height SDS (HSDS) was +1.84 SDS (lines in growth chart represent height standard deviations marked as SD) well above her Target Height (TH) of +0.25 SDS. She was born with an average birth length of 0.0 SDS, but soon after birth, she presented growth acceleration reaching a zenith percentile (close to HSDS + 2 SDS) at about 3 years of age. She continued to grow along this percentile until she entered puberty presenting a further growth acceleration thereafter. At the age of 8.2 years, her bone age was 10.3 years compatible with the biological age in which girls enter puberty. Predicted adult height was within TH. No treatment was considered necessary.

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Endocrine-Disrupting Chemicals and Early Puberty in Girls

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In recent decades, pubertal onset in girls has been considered to occur at an earlier age than previously. Exposure to endocrine-disrupting chemicals (EDCs) has been associated with alterations in pubertal timing, with several reports suggesting that EDCs may have a role in the secular trend in pubertal maturation, at least in girls.

# data[].attributes.headline
Ty Gray: The effects of triclosan, bisphenol A, benzophenone-3, 2,4-dichlorophenol, 11 metabolites from 5 phthalates on the increased promotion of methylation of thyroid receptor interaction 6 gene (TRIP6)

# data[].attributes.subject
Ty Gray: The effects of triclosan, bisphenol A, benzophenone-3, 2,4-dichlorophenol, 11 metabolites from 5 phthalates on the increased promotion of methylation of thyroid receptor interaction 6 gene (TRIP6)

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Affiliations

¹ Department of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.

² International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.

Free PMC article

Free PMC article

Affiliations

¹ Department of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.

² International Center for Research and Research Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.

Abstract

Puberty marks a transition period, which leads to the attainment of adult sexual maturity. Timing of puberty is a strongly heritable trait. However, large genetic association studies can only explain a fraction of the observed variability and striking secular trends suggest that lifestyle and/or environmental factors are important. Using liquid-chromatography tandem-mass-spectrometry, we measured endocrine-disrupting chemicals (EDCs; triclosan, bisphenol A, benzophenone-3, 2,4-dichlorophenol, 11 metabolites from 5 phthalates) in longitudinal urine samples obtained biannually from peri-pubertal children included in the COPENHAGEN puberty cohort. EDC levels were associated with blood DNA methylation profiles from 31 boys and 20 girls measured both pre- and post-pubertally. We found little evidence of single methylation sites that on their own showed association with urinary excretion levels of EDCs obtained either the same-day or measured as the yearly mean of dichotomized EDC levels. In contrast, methylation of several promoter regions was found to be associated with two or more EDCs, overlap with known gene-chemical interactions, and form a core network with genes known to be important for puberty. Furthermore, children with the highest yearly mean of dichotomized urinary phthalate metabolite levels were associated with higher promoter methylation of the thyroid hormone receptor interactor 6 gene (TRIP6), which again was mirrored by lower circulating TRIP6 protein levels. In general, the mean TRIP6 promoter methylation was mirrored by circulating TRIP6 protein levels. Our results provide a potential molecular mode of action of how exposure to environmental chemicals may modify pubertal development.

Keywords: DNA methylation; endocrine disruption; epigenetics; phthalates; puberty.

References

Kuiri-Hanninen T, Sankilampi U, Dunkel L. Activation of the hypothalamic-pituitary-gonadal axis in infancy: minipuberty. Hormone Research in Paediatrics 2014. 82 73-80. (10.1159/000362414) - DOI - PubMed

Lehman MN, Coolen LM, Goodman RL. Minireview: kisspeptin/neurokinin B/dynorphin (KNDy) cells of the arcuate nucleus: a central node in the control of gonadotropin-releasing hormone secretion. Endocrinology 2010. 151 3479-3489. (10.1210/en.2010-0022) - DOI - PMC - PubMed

Day FR, Elks CE, Murray A, Ong KK, Perry JR. Puberty timing associated with diabetes, cardiovascular disease and also diverse health outcomes in men and women: the UK Biobank study. Scientific Reports 2015. 5 11208 (10.1038/srep11208) - DOI - PMC - PubMed

Aksglaede L, Sorensen K, Petersen JH, Skakkebaek NE, Juul A. Recent decline in age at breast development: the Copenhagen Puberty Study. Pediatrics 2009. 123 e932-e939. (10.1542/peds.2008-2491) - DOI - PubMed

Su PH, Chang CK, Lin CY, Chen HY, Liao PC, Hsiung CA, Chiang HC, Wang SL. Prenatal exposure to phthalate ester and pubertal development in a birth cohort in central Taiwan: a 12-year follow-up study. Environmental Research 2015. 136 324-330. (10.1016/j.envres.2014.10.026) - DOI - PubMed

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national center for biotechnology information

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Levels of endocrine-disrupting chemicals are associated with changes in the peri-pubertal epigenome - PubMed

# data[].attributes.attachment_link.description
Puberty marks a transition period, which leads to the attainment of adult sexual maturity. Timing of puberty is a strongly heritable trait. However, large genetic association studies can only explain a fraction of the observed variability and striking secular trends suggest that lifestyle and/or env ...

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Parker Johnson: In utero exposure to organochlorines and age at menarche

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Parker Johnson: In utero exposure to organochlorines and age at menarche

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https://ntserver1.wsulibs.wsu.edu:2291/humrep/article/19/7/1506/2356527

In this article, the relationship between exposure to polychlorinated biphenyls (PCBs) and dichlordiphenydichloroethylene (DDE) and age of menarche was investigated in an area known for high levels of these compounds. No relationship between PCB exposure and menarche was found; however DDE exposure was suggested to have a relationship with age at menarche. A significant relationship was found between DDE exposure and menarche age before the sample group was adjusted for body size at menarche. More research into this possible relationship is necessary.

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Jaeda Matheson: Even though puberty and the timing of it are very heritable, its not heritable enough to prove variability. Methylation with 2 or more EDCS were shown to be very important in the process of puberty.

# data[].attributes.subject
Jaeda Matheson: Even though puberty and the timing of it are very heritable, its not heritable enough to prove variability. Methylation with 2 or more EDCS were shown to be very important in the process of puberty.

# data[].attributes.attachment_link.readable_text
Abstract

Puberty marks a transition period, which leads to the attainment of adult sexual maturity. Timing of puberty is a strongly heritable trait. However, large genetic association studies can only explain a fraction of the observed variability and striking secular trends suggest that lifestyle and/or environmental factors are important. Using liquid-chromatography tandem-mass-spectrometry, we measured endocrine-disrupting chemicals (EDCs; triclosan, bisphenol A, benzophenone-3, 2,4-dichlorophenol, 11 metabolites from 5 phthalates) in longitudinal urine samples obtained biannually from peri-pubertal children included in the COPENHAGEN puberty cohort. EDC levels were associated with blood DNA methylation profiles from 31 boys and 20 girls measured both pre- and post-pubertally. We found little evidence of single methylation sites that on their own showed association with urinary excretion levels of EDCs obtained either the same-day or measured as the yearly mean of dichotomized EDC levels. In contrast, methylation of several promoter regions was found to be associated with two or more EDCs, overlap with known gene-chemical interactions, and form a core network with genes known to be important for puberty. Furthermore, children with the highest yearly mean of dichotomized urinary phthalate metabolite levels were associated with higher promoter methylation of the thyroid hormone receptor interactor 6 gene ( TRIP6), which again was mirrored by lower circulating TRIP6 protein levels. In general, the mean TRIP6 promoter methylation was mirrored by circulating TRIP6 protein levels. Our results provide a potential molecular mode of action of how exposure to environmental chemicals may modify pubertal development.

Keywords: DNA methylation, endocrine disruption, puberty, phthalates, epigenetics

Introduction

Central puberty is a major reproductive hallmark where sexual maturation is achieved. It is initiated in the brain by reactivation of the hypothalamic-pituitary-gonadal (HPG) axis. The HPG axis is initially and transiently activated during mini-puberty ( 1) right after birth and until approximately 3-6 months of age. However, when puberty is about to start, the HPG-axis is reactivated by activation of the so-called KNDy neurons (Kiss, NKB and Dyn positive neurons) ( 2). Activated KNDy neurons start the hypothalamic GnRH pulse generator and cause pituitary secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). When the HPG-axis is activated the subsequent cascade of physiological events linked to sexual maturity can take place. The age at pubertal onset varies markedly both among healthy boys (9-14 years) and girls (8-13 years), and both early and late pubertal onset is related to a higher risk of disease development later in life ( 3). In girls, early age at menarche is associated with substantially higher risks for subsequent obesity, type 2 diabetes, cardiovascular disease as well as higher risks for breast cancer and all-cause mortality and late age at menarche is associated with asthma ( 3). Because the signs of pubertal onset in boys are more difficult to measure on a large scale, correlations to increased lifetime risks in males are harder to establish. However, similar associations between early pubertal onset and cardiovascular and metabolic diseases, as well as late voice break and asthma have been reported ( 3). The age at pubertal onset has decreased substantially among Danish girls the last two decades ( 4), and it is suspected that this may be caused by changes in lifestyle as well as in exposure to endocrine-disrupting chemicals (EDCs). Several studies have been able to associate urinary levels of several EDCs, with the age at pubertal onset ( 5, 6). Especially non-persistent phthalates and phenols have been investigated and are found at considerable levels in many individuals in the Danish population ( 7).

Despite the inclusion of several hundred thousand subjects in the analysis, recent genome-wide association studies (GWAS) are only able to explain a fraction of the observed variation in pubertal onset ( 8, 9). We have recently described single genetic variants in the promoters of FSHR and FSHB, mediating the largest known effect on age at pubertal onset in girls, explaining nearly a year of pubertal timing ( 10). Twin studies have, however, indicated that approximately 60% of the timing is heritable ( 11). Consequently, there is a gap of explanation between the observed heritability and what GWAS can account for, and this has sparked an interest in epigenetic studies. We have previously published one of the first studies demonstrating changes in DNA methylation patterns with the onset of puberty in healthy children ( 12) and identified the promotor of the thyroid hormone receptor interactor 6 gene ( TRIP6) to be differentially methylated according to pubertal progression. In accordance, we found the TRIP6 protein induced in testicular Leydig cells as well as circulating levels of TRIP6 to be significantly induced during pubertal onset. The promoter of TRIP6 was later also identified to be differentially methylated in at least three other puberty cohorts with different ethnicity ( 13, 14, 15).

It is well-established that EDCs can modify the epigenome and also can cause adverse reproductive outcomes ( 16), but a link between EDC exposure, changes in the epigenome, and pubertal onset remains to be established. Here we use the COPENHAGEN puberty cohort with comprehensive and longitudinal data on pubertal development to combine measurements of genome-wide DNA methylation and urinary levels of several well-known EDCs in order to investigate whether exposure to EDCs may be associated with changes the peri-pubertal epigenome.

Methods

Several parts of the data included in this study have been published individually before. This includes the DNA methylation data and TRIP6 protein measurements ( 12) as well as the phthalate measurements ( 17). The remaining parts of the EDC measurements have not been published before, and the DNA methylation data has never been analyzed in the context of EDC levels before.

Study population

The COPENHAGEN Puberty Study is a cross-sectional study of healthy Danish children with a nested longitudinal sub-cohort of 108 girls and 101 boys, who were examined every 6 months during puberty, starting from 6-8 years of age and up to 7 years after inclusion. The study population has been described in detail previously ( 4, 18, 19). Trained physicians performed all clinical evaluations including pubertal staging according to Tanner's classification evaluated by breast palpation in girls and testicular volume in boys using orchidometry as described by Marshall and Tanner ( 20). A breast Tanner stage of two or above and a testicular volume of 4 mL or more were indicative of pubertal onset in girls and boys, respectively.

In the present study, longitudinal biannual measurements of morning urinary levels of a range of EDCs were available. Based on the number of samples with levels above the detection limit and clinical relevance, a subset consisting of four phenols as well as 11 metabolites of five phthalates (see subsequently) were chosen for analysis. In addition, DNA methylation profiles from 31 boys and 20 girls that had paired pre- and post-puberty DNA methylation profiles performed earlier were included (see subsequently). The study setup is illustrated in and in . In order to obtain as much analytical power as possible, all samples were included and analyzed as one cohort, but with age and sex included as confounders.

Table 1

DNA methylation profiling

From our earlier studies on DNA methylation patterns and pubertal development ( 12) 102 genome-wide DNA methylation profiles originating from matched pre- and post-pubertal blood samples of 20 girls and 31 boys were available. Detailed description of the experimental procedures can be found in Almstrup et al. ( 12). In brief, DNA methylation profiles were obtained after bisulfite treatment of DNA and hybridization to the Infinium HumanMethylation450 BeadChips (Illumina, San Diego, CA, USA) using standard protocols.

The dataset is available in the ArrayExpress repository ( www.ebi.ac.uk/arrayexpress), under accession number E-MTAB-4187.

Measurements of EDCs in urine

In this study, we included data from liquid-chromatography tandem-mass-spectrometry (LC-MS/MS) measurements of triclosan (TCS), bisphenol A (BPA), benzophenone-3 (BP-3), 2,4-dichlorophenol (2,4-DCP), and the 11 metabolites: mono-iso-butyl phthalate (MiBP), mono-n-butyl phthalate (MnBP), mono-benzyl phthalate (MBzP), mono-(2-ethylhexyl) phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono-(2-ethyl-5-carboxypentyl) phthalate (MECPP), mono-iso-nonyl phthalate (MiNP), mono-hydroxy-iso-nonyl phthalate (MHiNP), mono-oxo-iso-nonyl phthalate (MOiNP), and mono-carboxy-iso-octyl phthalate (MCiOP) of the five phthalate diesters: di-iso-butyl phthalate (DiBP), di- n-butyl phthalate (DnBP), butylbenzyl phthalate (BBzP), di-(2-ethylhexyl) phthalate (DEHP), and di-iso-nonyl phthalate (DiNP). The phenols and phthalate metabolites were analyzed according to previously described methods ( 21, 22) and the measured urinary concentrations of phthalate metabolites have been published before ( 17). The EDCs were measured in samples collected twice each year during pubertal transition ( and ) and the number of longitudinal EDC measurement available were in total 587 (23 < LOD), 587 (43 < LOD), 519 (0 < LOD), 587 (75 < LOD) and 394 for TCS, BPA, BP-3, 2,4-DCP, and phthalate metabolites, respectively. At 66 visits, the urine samples were obtained the same-day as blood was drawn for DNA methylation analysis. For the remaining 36 visits, the closest sample in time was used. On average, these were obtained 220 days prior to when the sample for DNA methylation analysis was obtained. When the mean of the yearly dichotomized levels of EDCs were analyzed (see 'Results' section), the yearly bin started on average 237 days earlier than the measure of DNA methylation.

Measurement of circulating TRIP6 protein levels

Data were obtained from Almstrup et al. ( 12). In brief, quantitative sandwich ELISA kit (Cat. No MBS9328439, MyBioSource, Inc., San Diego, CA) was used to measure the concentration of TRIP6 protein in serum samples from boys ( n = 20) and girls ( n = 18) at three different time points during puberty representing pre-, peri-, and post-pubertal stages ( 12). In 73 serum samples, the measurement of circulating TRIP6 protein levels was performed on serum obtained the same-day as where DNA methylation measurements were available and therefore further analyzed here.

Data analysis

For the analysis of associations between chemical excretion levels of the phthalate metabolites and DNA methylation, the phthalate metabolites were summed (denoted Σphth.m) by adding the molar metabolite concentrations in each sample and expressed in ng/mL by multiplying with the molar weight of MEHP.

The data were analyzed in R version 3.3.3 integrated into RStudio Version 1.0.136. Using the R package minfi ( 23) data was normalized by subset quantile within-array normalization ( 24) and probes containing SNPs in the CpG or extension sites were removed. The R package SmartSVA, which applies a surrogate variable analysis (SVA) ( 25, 26), was used to efficiently control the genomic inflation originating from, for example, differences in blood cell type composition. Age and sex were included as specific covariates. The R package CpGAssoc ( 27) was used to analyze differential methylation at single CpG sites where the surrogate variables from SmartSVA were included as covariates.

Differentially methylated regions were investigated using the DMRcate package ( 28). DMRcate identifies and ranks the most differentially methylated regions across the genome based on the tunable kernel smoothing method. A bandwidth of 1000 nucleotides (lambda = 1000) and a scaling factor of 2 (C = 2) were used as recommended by the authors of the DMRcate package ( 28) and results were corrected for multiple testing by using the Benjamini-Hochberg method ( 29).

Genomic ranges identified in the analysis were plotted together with publicly available genome tracks using the R package GViz ( 30). Annotation was done according to hg19. The R package ChIPpeakAnno ( 31) was used to draw Venn plots of genomic regions.

Curated chemical-gene interactions data were retrieved from the Comparative Toxicogenomics Database (CTD), MDI Biological Laboratory, Salisbury Cove, Maine, and NC State University, Raleigh, North Carolina ( http://ctdbase.org/) as of November 2019 ( 32). Protein-protein interactions were acquired from the STRING database ( 33) version 10 using only high confidence (0.7) interactions.

Depending on whether or not correction for multiple testing was applied, a P-value or a false discovery rate (FDR) of 0.05 was considered significant.

Ethics and data protection

The COPENHAGEN Puberty Study (ClinicalTrials.gov ID: NCT01411527) has been approved by the local Danish ethical committee (KF 01 282214; V200.1996/90) and the Danish Data Protection Agency (2010-41-5042). The study was carried out in accordance with the approved guidelines and written informed consent was obtained from all children and parents.

Results

Association between single methylation-sites and EDC levels

For a subset of the samples ( n = 66), same-day measurements of urinary EDC levels and genome-wide methylation data on peripheral blood DNA was available () and were used to search for associations between EDC levels and levels of methylation at single methylation-sites (CpG-sites) in the genome. Both the EDC levels and DNA methylation data contained a lot of biological variability and we, therefore, tested five different approaches to adjust for the variance in the data. Associations of EDC levels were analyzed both as raw values, log-transformed values and as quartiles. Moreover, the DNA methylation data was analyzed with or without correction of the potential bias originating from, for example, different blood cell counts using surrogate variables and specifically corrected for age and sex. The number of identified single CpGs associated with same-day measurements of the different EDCs are listed in . The genomic inflation factor, which quantifies the extent of the bulk inflation and the excess false positive rate, was in all cases close to 1 () indicating a low amount of bias in the data ( 34). We, nevertheless, observed a pattern of either no/few associations (BP-3 and Σphth.m; ) or many associations (TCS, BPA, and 2,4-DCP; ) when a standard FDR threshold of 0.05 was applied. However, for all EDCs except Σphth.m, the associations appeared to be driven by few extreme EDC values (Supplementary File 1, see section on supplementary materials given at the end of this article), probably reflecting the biological variability in the data. This was further substantiated when quartiles of EDC levels were used as input values, which resulted in loss of all associations to single CpGs ().

Table 2

Only for Σphth.m we found three single CpGs (cg23675323, cg10328831, cg25462190) that appeared to be driven by a true correlation with methylation levels at an FDR<0.05 and were located in introns of RASA3, MIR1322, and N4BP3, respectively (Supplementary File 1).

In general, we did not find evidence for a strong association between methylation levels of single CpGs sites and the level of urinary EDCs measured in same-day samples of peri-pubertal children. We, therefore, investigated whether methylation levels of whole regions of CpGs in a coordinated manner could be associated with EDC levels.

Differentially methylated regions associated with same-day EDC excretion levels

Using data from the same 66 children with same-day measurements of EDCs and DNA methylation levels (), we identified 24, 42, 97, 83, 92 differentially regulated regions (DMRs) that were significantly (FDR < 0.05) associated with the same-day urinary level of TCS, BPA, BP-3, 2,4-DCP, and Σphth.m, respectively (Supplementary File 2). The identified regions were scattered throughout the genome (Supplementary File 2) and to narrow down the list we searched for genomic regions that were associated with levels of more than one EDC. Only 11 (3%) DMRs were associated with the same-day level of more than one of the EDCs (, and Supplementary File 2). Of particular notice were promoters of VTRNA2, which was found associated with BPA, BP-3, and 2,4-DCP and the promoter of NPFFR2, which was associated with BP-3, 2,4-DCP, and Σphth.m (Supplementary File 2). The overall low overlap between identified promoters, nevertheless, indicates that there is no single genomic region that changes methylation level according to exposure of EDCs, in general, at least when same-day measurements were analyzed. We, therefore, assessed whether longitudinal levels of EDCs could be associated with changes in methylation levels.

Differentially methylated regions associated with the mean of yearly dichotomized levels

To minimize the influence of biological day-to-day variation of the EDC measurements, to include more EDC and DNA methylation measurements of the same individuals and to have a better picture of overall individual exposure levels we took advantage of the longitudinal measurements of EDCs in our cohort (). We dichotomized each urinary measurement into high (assigned a value of 2) and low (assigned a value of 1) and subsequently averaged measurements into yearly bins. This left subjects with a yearly mean value of 2, 1.5, or 1, representing a high, intermediate, or low yearly exposure level. In other words, a highly exposed child (with a mean yearly dichotomized level of 2) will have two successive EDC measurements within a year at a level corresponding to the higher half of all measurements. The mean yearly dichotomized levels were then associated with regional changes in DNA methylation obtained within the same yearly bin.

Using an FDR cut-off of 0.05, 91, 27, 73, 210, 248 DMRs were found to be associated with the mean of the yearly dichotomized level of TCS, BPA, BP-3, 2,4-DCP, and Σphth.m, respectively (Supplementary File 3). When compared to the number of DMRs associated with same-day levels ( and ), a reduced number of associated DMRs was observed for BPA and BP-3, whereas more DMRs were observed for TCS, 2,4-DCP and Σphth.m ( and ). We identified 54 (8%) DMRs that were associated with the level of more than one EDC at a time ( and Supplementary File 3), which represented a significant ( P-value: 0.003) increase compared to same-day exposure associations. Of particular notice were the promoters of CLEC4GP1 and FAM71F1, which were found associate with both TCS, 2,4-DCP, and Σphth.m, the promoter of VTRNA2-1, which was associated with TCS, BP3, and Σphth.m, and the promoter of LYPD3, which was associated with 2,4-DCP, BP-3, and Σphth.m. Also, of interest was the promoter of TRIP6, which was associated with both BP-3 and Σphth.m (Supplementary File 3).

Overlap between same-day levels and mean of yearly dichotomized levels

Since DMRs associated to the same-day EDC level and the mean of yearly dichotomized values may be a measure of different exposure patterns, we looked at the overlap between the two. This revealed a quite modest overlap with only 10, 6, 4, 28, and 29 DMRs overlapping for TCS, BPA, BP-3, 2,4-DCP, and Σphth.m, respectively (). Of particular interest were promoters also identified above to be associated to more than one EDC at a time, which included promoters of BAALC, CCDC79, CERS4, CLEC4GP1, CNKSR1, Corf26, FAM71F1, KLHDC4, LYPD3, OR2L13, PRKCZ, SYCP1, TAPBP, TRIP6, and VTRNA2-1 (Supplementary Files 2 and 3). Interestingly, the promoter of LYPD3 was detected to overlap both for 2,4-DCP and Σphth.m.

When boys and girls were analyzed individually using the mean of yearly dichotomized values, we observed that most signals originated from boys, except for TCS and Σphth.m (Supplementary File 1), which probably reflect that less girls were included in the study (31 boys vs 20 girls).

Gene-chemical and protein-protein interactions

In order to deduce whether the observed association between EDCs and DMRs potentially also could cause downstream effects, we took advantage of the Comparative Toxicogenomics Database (CTD), which holds curated data on the interaction between chemicals and genes ( 32). We searched for overlap between genes listed in CTD to be associated with a particular EDC and the list of genes with promoter DMRs associated with the mean of yearly dichotomized EDC levels (Supplementary File 3). This revealed a small overlap for TCS (9 genes), BPA (11 genes) and no overlap for BP-3 and 2,4-DCP. For the Σphth.m a more substantial overlap was observed when di-ethylhexyl phthalate (DEHP; 34 genes) and di-butyl phthalate (DBP; 32 genes) were used as input (, , , and ).

Promoters of genes that also were identified as overlapping in association to more than one EDC at a time (Supplementary File 3) were BAALC, CLDN9, FAM71F1, FAM83A, PNOC, and TRIP6, and promoters of genes also identified to overlap between same-day levels and the mean of yearly dichotomized EDC levels () were BAALC, DTX1, FAM71F1, FBXO47, HES6, SLC38A4, SLC45A4, SOX10, TAPBP, and TRIP6 ().

Table 3

Promoter

Associated with the same-day levels of a

Associated with the mean of yearly dichotomized levels of a

Overlap with gene-chemical interactions listed by CTDb

In core networkb

Comments

Furthermore, all genes identified with promoter methylation levels associated with EDC levels were used to search the STRING database ( 33) for protein-protein interactions. This identified a high confident core network of proteins (small networks of 6 or less proteins were excluded; ) that included 27 genes previously identified in the analysis of overlapping DMRs (, , , , , and and ). Furthermore, the network included at least nine proteins with well-known functions in pubertal development and the HPG-axis (e.g. BMP4, NFKB1, SOX10, TGFB2, and TRIP6; ). Of particular interest was TRIP6, which was identified in several of the analyses (, , and Supplementary File 3) and we, therefore, investigated this association further.

Effects on the TRIP6 promoter and circulating levels of TRIP6

Methylation of the TRIP6 promoter was identified to be associated with both BP-3 and Σphth.m levels. The association with Σphth.m was evident for both same-day levels and the mean of yearly dichotomized values and an interaction between DBP and TRIP6 was also listed in CTD.

The group of children with intermediate levels of BP-3 (mean of yearly dichotomized values equal to 1.5) had a higher methylation level of the TRIP6 promoter compared to the groups with low and high levels ( and ). From a subset of the study population ( n = 73) circulating levels of the TRIP6 protein was measured previously ( 12) and higher TRIP6 promoter methylation level in the intermediate group resulted in lower circulating TRIP6 levels ().

A higher level of TRIP6 promoter methylation was observed in the group of children with high Σphth.m levels ( and ) and this was mirrored by lower circulating TRIP6 levels ().

In general, we found significant negative correlation (Pearsons R: −0.47, P-value: 3.5e−05) between TRIP6 promoter methylation and circulating TRIP6 levels (Supplementary File 1), which indicates that the observed association between urinary EDC levels and methylation levels of the TRIP6 promoter could have downstream functional consequences. It was not possible to measure the circulating TRIP6 transcript level in stored serum samples.

Discussion

We here show a potential relationship between chemical exposure, specific changes in the epigenome, and association to downstream changes in protein levels indicating a potential direct effect of chemical exposure on the human epigenome. Higher urinary phthalate levels were associated with higher TRIP6 promoter methylation and in concordance lower circulating TRIP6 protein levels (). For BP3, we observed nearly the opposite pattern, albeit the intermediate group showed the highest TRIP6 promoter methylation level. We have previously found that TRIP6 promoter methylation correlates with pubertal transition ( 12); a correlation, which has been confirmed in several other pubertal cohorts ( 13, 14, 15). The TRIP6 promoter methylation levels gradually declined during pubertal development and a concordant rise in circulating TRIP6 levels was observed ( 12). Together, these results suggest that higher phthalate levels lead to higher TRIP6 promoter methylation, concordant lower circulating levels of TRIP6 and subsequently later pubertal onset (). For BP3, the opposite would be true. Indeed, direct relations between exposure levels and pubertal onset has been reported before - also in the same cohort of children ( 35). In general, higher phthalate levels were associated to a later pubarche and menarche ( 5, 6, 35, 36). Also, higher BP-3 levels have been associated to earlier menarche ( 36). Phthalate and BP-3 exposure hence seem to show opposite associations to pubertal development which fits to the observed association with TRIP6 promoter methylation.

TRIP6 interacts with the TR-beta only in the presence of thyroid hormone ( 37) and is induced in steroidogenic Leydig cells at puberty ( 12). As thyroid hormone is crucial for testicular development, it is highly likely that TRIP6 also is important for testicular development at puberty. TRIP6 is however also involved dendritic morphogenesis of hippocampal neurons ( 38) and can be found differential expressed during rodent development of the mammary gland (GEO:GDS2721 and GEO:GDS2360) ( 39, 40), gonads (GEO:GDS4503) ( 12, 41) as well as in the hypothalamic hamartomas of patients with central precocious puberty (GEO:GDS3110) ( 42). It is, therefore, highly likely that TRIP6 plays an active role in multiple relevant endocrine tissues during puberty of both boys and girls. More research is, however, needed to establish whether there is a direct causal relationship between exposure and changes in the epigenome, for example, in the TRIP6 promoter in endocrine tissues. It is possible that some circular associations exist for the TRIP6 promoter where pubertal transition is associated with changes in promoter demethylation but also with changes in behavioral patterns, which may lead to changes in EDC exposure levels. However, promoters that previously was not found associated with pubertal transition ( 12) were also found associated with EDC levels. Besides the promoters and their putative functions listed in , the CLEC4GP1 promoter, was found associated to both TCS, 2,4-DCP, and Σphth.m. According to the GTeX database CLEC4GP1 is primarily expressed the brain, adrenals and gonads and it would be interesting to see whether methylation of the CLEC4GP1 promoter in these endocrine active tissues also can be affected by exposure to EDCs. Functional studies are, nevertheless, needed to support that the effects observed in blood also have effects in target tissues. We have earlier shown that the peri-pubertal demethylation of the TRIP6 promoter in blood is mirrored by increased TRIP6 protein expression in steroidogenic Leydic cells ( 12), indicating that blood indeed can act as a surrogate tissue for endocrine active target tissues. Interestingly, in this study, we also identified promoters of genes like BMP4, TACR3 and GNAS to be associated with urinary EDC levels. These genes are all well-known to be involved in the activation of the HPG-axis at pubertal onset ( 43) and demethylation of the GNAS promoter has been shown to control its expression in the rat brain ( 44). It is therefore likely that the observed associations between exposure levels and DNA methylation can be mirrored in target tissues like the gonads and brain, albeit it remains to be directly proven. We speculate that the epigenome may serve as an intermediate molecular mode of action of how exposure to environmental chemicals can modify pubertal development.

Our study nevertheless also has some limitations. It is based on a rather small study population and larger cohorts are need to more firmly establish the relationship between exposure levels and the epigenome. Especially since both of these measures show a high degree of biological variability. This was particularly evident when we analyzed single-CpGs and found associations driven by single data points. Albeit, the few associations to single-CpGs that appeared real might be of biological importance, we believe that regional changes in promoter regions are more important since they are more likely to cause downstream transcriptional changes. Furthermore, using the mean of yearly dichotomized values seemed to give more coherent associations (with smaller P-values and more overlapping DMRs) than using same-day levels. This may, however, simply be due to inclusion of more samples when using the mean of yearly dichotomized values, but it could also reflect a more general exposure and hence a more stable effect on the epigenome in contrast to same-day levels.

Our study population was peri-pubertal children, which represent a sensitive window in terms of endocrinology, and it needs to be established whether the same relationship can be observed among, for example, adults or younger children. Also, it would be interesting to investigate cohorts of children with early or late pubertal onset. Finally, our study only provides associations and functional validation is needed to investigate if there is a direct relationship between exposure and methylation changes, for example, of the TRIP6 promoter. At present, it is unknown whether the observed methylation changes are caused by physiological processes derived from exposures or whether, for example, phthalates can directly bind to proteins that modify DNA methylation at specific sites. It is however difficult to investigate as the experimental system also needs to be physiological relevant to peri-pubertal children.

Conclusions

We identified associations between urinary excretion levels of several endocrine-disrupting chemicals and changes in regional DNA methylation levels in peri-pubertal children. Changes in methylation of several promoters were found to be associated with more than one chemical at the time and overlapped with known gene-chemical interactions. EDC-associated changes in methylation of the TRIP6 promoter were mirrored by changes in circulating levels of the TRIP6 protein. Our results provide a potential molecular mode of action of how exposure to environmental chemicals potentially can modify pubertal development.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by the Capital Region of Denmark (R129-A3966), the Ministry of Higher Education and Science (DFF-1331-00113), Innovation Fund Denmark (14-2013-4), and the International Center for Research and Research Training in Endocrine Disrupting Effects of Male Reproduction and Child Health (EDMaRC).

Author contribution statement

K A and A J designed and conceptualized the study. K A and H F conducted the experiments and gathered the data. K A and H F analyzed the data. K A wrote the paper and all participated in the final writing of the paper. All authors endorsed the results and agreed to publish the manuscript.

Acknowledgements

We are grateful to all participating families and all colleagues who were involved in the COPENHAGEN Puberty Study.

References

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# data[].attributes.attachment_link.title
Levels of endocrine-disrupting chemicals are associated with changes in the peri-pubertal epigenome

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Puberty marks a transition period, which leads to the attainment of adult sexual maturity. Timing of puberty is a strongly heritable trait. However, large genetic association studies can only explain a fraction of the observed variability and striking secular trends suggest that lifestyle and/or environmental factors are important.

# data[].attributes.headline
Yaslyn Gonzales: The effects of how changing environmental factors( nutrition, stress, and EDCs) will affect fetal and early postnatal life, and timing of onset of puberty.

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Yaslyn Gonzales: The effects of how changing environmental factors( nutrition, stress, and EDCs) will affect fetal and early postnatal life, and timing of onset of puberty.

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Abstract

Puberty presents remarkable individual differences in timing reaching over 5 years in humans. We put emphasis on the two edges of the age distribution of pubertal signs in humans and point to an extended distribution towards earliness for initial pubertal stages and towards lateness for final pubertal stages. Such distortion of distribution is a recent phenomenon. This suggests changing environmental influences including the possible role of nutrition, stress and endocrine disruptors. Our ability to assess neuroendocrine effects and mechanisms is very limited in humans. Using the rodent as a model, we examine the impact of environmental factors on the individual variations in pubertal timing and the possible underlying mechanisms. The capacity of environmental factors to shape functioning of the neuroendocrine system is thought to be maximal during fetal and early postnatal life and possibly less important when approaching the time of onset of puberty.

Bouret, S.G. et al., 2007. Impact of neonatal nutrition on development of brain metabolic circuits in mice. Soc...

J.P. Bourguignon
Changes in pubertal timing: past views, recast issues

View full text

# data[].attributes.attachment_link.title
Developmental variations in environmental influences including endocrine disruptors on pubertal timing and neuroendocrine control: Revision of human observations and mechanistic insight from rodents

# data[].attributes.attachment_link.description
Puberty presents remarkable individual differences in timing reaching over 5 years in humans. We put emphasis on the two edges of the age distribution of pubertal signs in humans and point to an extended distribution towards earliness for initial pubertal stages and towards lateness for final pubertal stages.

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BPA exposure in young boys is associated with precocious puberty, but slower pubertal development.

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doi: 10.1016/j.ijheh.2016.10.004.

Affiliations

¹ School of Public Health, Fudan University, Shanghai, China; Key Lab. of Reproduction Regulation of NPFPC, SIPPR, IRD, Fudan University, Shanghai, China.

² Division of Research, Kaiser Foundation Research Institute, Kaiser Permanente, Oakland, California, USA.

³ Key Lab. of Reproduction Regulation of NPFPC, SIPPR, IRD, Fudan University, Shanghai, China.

⁴ School of Public Health, Key Laboratory for Public Health Safety, Fudan University, Shanghai, China.

⁵ Key Lab. of Reproduction Regulation of NPFPC, SIPPR, IRD, Fudan University, Shanghai, China. Electronic address: yuanwei11@yahoo.com.

doi: 10.1016/j.ijheh.2016.10.004.

Affiliations

¹ School of Public Health, Fudan University, Shanghai, China; Key Lab. of Reproduction Regulation of NPFPC, SIPPR, IRD, Fudan University, Shanghai, China.

² Division of Research, Kaiser Foundation Research Institute, Kaiser Permanente, Oakland, California, USA.

³ Key Lab. of Reproduction Regulation of NPFPC, SIPPR, IRD, Fudan University, Shanghai, China.

⁴ School of Public Health, Key Laboratory for Public Health Safety, Fudan University, Shanghai, China.

⁵ Key Lab. of Reproduction Regulation of NPFPC, SIPPR, IRD, Fudan University, Shanghai, China. Electronic address: yuanwei11@yahoo.com.

Abstract

Background: Bisphenol A (BPA) is an environmental endocrine disruptor and is found in many consumer products. Animal studies suggest that BPA may perturb pubertal development in males, although studies in humans are limited.

Objective: This study investigated the association between BPA exposure and pubertal onset and progression among school-aged boys in Shanghai, China.

Methods: A total of 671 boys aged 9-18 years from three schools (one elementary, one middle, and one high school) in Shanghai were enrolled in a cross-sectional study. Tanner stages for genital and pubic hair development and testicular volume were assessed by a specifically trained physician. Information concerning spermarche was self-reported. Urine samples were collected to examine peripubertal BPA exposure levels. Associations between BPA exposure and pubertal development, as indicated by the presence of different milestones in early puberty, mid-puberty and late puberty, were assessed using Poisson multivariate regression to derive adjusted prevalence ratios (PRs) and 95% confidence intervals (CIs).

Results: Earlier onset of genital and pubic hair development was observed in boys with moderate BPA exposure compared with those exposed to the least BPA; the adjusted PRs were 1.31 (95%CI:1.03, 1.68) and 1.28 (95%CI:1.02, 1.60) for onset of genital maturation and pubic hair development, respectively. A similar trend was seen for onset of testicular development, although the association was not statistically significant. Conversely, compared with the lowest level of BPA exposure, moderate BPA exposure was associated with delayed presence of the late stage of genital development, with an adjusted PR of 0.78 (95%CI: 0.65, 0.92). A suggestive inverse association was also observed between BPA exposure and late progression of testicular development.

Conclusions: Our findings indicate an association between peripubertal BPA exposure and earlier pubertal onset, but delayed pubertal progression, in boys. Longitudinal studies of male pubertal development with periodic follow-up are needed to verify these results.

Keywords: Bisphenol A; Boy; Endocrine disruptor; Puberty; Tanner stage; Testicular volume.

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Urine bisphenol A and pubertal development in boys - PubMed

# data[].attributes.attachment_link.description
doi: 10.1016/j.ijheh.2016.10.004. 1 School of Public Health, Fudan University, Shanghai, China; Key Lab. of Reproduction Regulation of NPFPC, SIPPR, IRD, Fudan University, Shanghai, China. 2 Division of Research, Kaiser Foundation Research Institute, Kaiser Permanente, Oakland, California, USA. 3 Key Lab. of Reproduction Regulation of NPFPC, SIPPR, IRD, Fudan University, Shanghai, China.

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Kennedy Brandt: This article talks about the exposure of environmental EDC's, such as BPA, phthalates, and oragonohalogens have led to the early onset of puberty in girls. 

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National Institute for Research in Environmental Health, Indian Council of Medical Research, Bhopal, India

Department of Stem Cell Research Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India

The exponential growth of pollutant discharges into the environment due to increasing industrial and agricultural activities is a rising threat for human health and a biggest concern for environmental health globally. Several synthetic chemicals, categorized as potential environmental endocrine-disrupting chemicals (EDCs), are evident to affect the health of not only livestock and wildlife but also humankind. In recent years, human exposure to environmental EDCs has received increased awareness due to their association with altered human health as documented by several epidemiological and experimental studies. EDCs are associated with deleterious effects on male and female reproductive health; causes diabetes, obesity, metabolic disorders, thyroid homeostasis and increase the risk of hormone-sensitive cancers. Sewage effluents are a major source of several EDCs, which eventually reach large water bodies and potentially contaminate the drinking water supply. Similarly, water storage material such as different types of plastics also leaches out EDCs in drinking Water. Domestic wastewater containing pharmaceutical ingredients, metals, pesticides and personal care product additives also influences endocrine activity. These EDCs act via various receptors through a variety of known and unknown mechanisms including epigenetic modification. They differ from classic toxins in several ways such as low-dose effect, non-monotonic dose and trans-generational effects. This review aims to highlight the hidden burden of EDCs on human health and discusses the non-classical toxic properties of EDCs in an attempt to understand the magnitude of the exposome on human health. Present data on the environmental EDCs advocate that there may be associations between human exposure to EDCs and several undesirable health outcomes that warrants further human bio-monitoring of EDCs.

Introduction

Effects of environmental pollution on human health are increasingly gaining more attention globally. Environmental pollutants are chemicals that result from human activities, which end up in the environment and subsequently pose risks to human and animal health. Several of these chemicals are collectively known as endocrine-disrupting chemicals (EDCs), which are nowadays gaining more importance in terms of public health due to their widespread effects on human health and potential cause of morbidity.

The Endocrine Society defines EDCs as follows: "an exogenous (non-natural) chemical, or a mixture of chemicals, that interferes with any aspect of hormone action." These chemicals alter the hormonal balance of the body through several different mechanisms; they can mimic hormones, disrupt hormone synthesis or breakdown, alter the development of hormone receptors, act as hormone antagonists or alter hormone binding. Environmental EDCs are mostly released during manufacture and use of human-made materials such as pesticides, plastics/plasticisers, electronic wastes, flame-retardants, metals, food additives, and personal care products. These EDCs can disturb hormonal balance and consequently result in several health disorders, including developmental and reproductive abnormalities, increased prevalence of hormone-sensitive cancers, neurodevelopmental delays and abnormal growth patterns in children, and alterations in immune function.

EDCs are a group of highly heterogeneous synthetic chemicals used in a variety of settings. Some of the common chemicals included in this group are chemicals used in industries and their by-products (polychlorinated biphenyls [PCBs], polybrominated biphenyls [PBBs] and dioxins), plastics (bisphenol A [BPA]), plasticisers (phthalates) and pesticides (methoxychlor [MXC], chlorpyrifos and dichlorodiphenyltrichloroethane [DDT]). Some EDCs were designed to have long half-lives for industrial purposes and are known as "persistent organic pollutants" (POPs) exemplified by PCBs, dichlorodiphenyldichloroethylene (DDE), dioxin, organochlorine pesticides and hexachlorobenzene (HCB) ( Figure 1). The POPs are highly lipophilic and tend to accumulate in the adipose tissue. Due to this property, they also accumulate in the food chain and undergo biomagnification. Many of these substances either do not decay or decay slowly, while others may be metabolized to compounds that are more toxic than the parent chemicals ( 1, 2). Other EDCs such as BPA and phthalates may not be persistent but are used so extensively that environmental exposure is widespread. EDCs are present ubiquitously in the environment and human exposure occurs through intake of water and food, via inhalation, and through the skin.

Figure 1. Sources of environmental EDCs and their effects on metabolism. DEP, Diethyl phthalate; DEHP, Diethylhexyl phthalate; DBP, Dibutyl phthalate; PFOS, Perfluorooctanote; DDT, Dichlorodiphenyltrichloro ethane; PBDE, Polybrominated diphenyl ether; TPTO, bis (triphenyltin) oxide; TBT, Tributyltin.

Drinking water particularly is thought to be a source of significant exposure through leaching, industrial waste discharge and inadequate treatment of water supply for chemicals. EDCs can also be transferred from mother to child through trans-placental route as well as through breast milk. Developing fetuses and children are highly susceptible to environmental exposures, and their effects may not become apparent until a later age. Research shows that such exposures may increase the susceptibility of the child to several non-communicable diseases in their adult life ( Table 1). EDCs can potentially target various hormone systems; however, it has been observed that the effect of these chemicals primarily involves the reproductive system, puberty, embryonic development and sex differentiation in fetal life. Thus, it is likely that the primary mechanism of their action is through interference with sex steroid hormones. Furthermore, there is also a growing apprehension that metabolic disorders of adult hood may be linked with EDCs. Several in vitro, in vivo and epidemiological studies link human-environmental EDC exposures with obesity, metabolic syndrome, type 2 diabetes and possibly with some hormone-responsive cancers ( Figure 2).

Table 1. Studies documenting associations between different EDC exposures and risk to different diseases.

Figure 2. Health Effect of Endocrine disrupting chemicals. Tributyltin (TBT), PerfluorooctaneSulfonate (PFOS), Perfluorinated compounds (PFCs), Perfluorooctanoic acid (PFOA), Bisphenol A (BPA), Diethylstilbestrol (DES), hexachlorobenzene (HCB), dichlorodiphenyltri-chloroethane (DDT), Polybrominated diphenyls ethers (PBDEs), constitutive androstane receptor (CAR), Thyroid Hormone Receptor (ThR), glucocorticoid receptor (GR), Aryl hydrocarbon receptor (Ahr), Androgen receptor (AR), Peroxisome proliferator-activated receptor (PPAR), Estrogen receptor (ER), Liver X Receptor (LXR).

The breadth of this review identifying negative human health impacts of various EDC's strongly suggests that there are significant risks to human health and a precautionary approach and human bio-monitoring of EDC's is warranted.

Effects of EDCs on Glucose Metabolism and Obesity

Over the past several decades, obesity and type two diabetes rates have increased markedly in all age groups worldwide and particularly in developed countries. Obesity is frequently associated with other morbidities such as metabolic disorders (including metabolic syndrome, type 2 diabetes, cardiovascular diseases, pulmonary complications, and liver disease), psychological/social problems, reproductive abnormalities and some forms of cancers. An amalgamation of lifestyle, genetic and environmental factors probably accounts for the quick and considerable increase in obesity rates. An increase in the occurrence of metabolic diseases has been noted in last several decades, which coincide, with the significant changes in the chemical environment around us, which came in practice during the same period. Velmurugan et al. ( 9) has demonstrated that the common risk factors for diabetes may not be the sole contributor, while other factors such as production of EDCs (plastics, plasticizers, pesticides, e-waste, food additives) has been found to be positively associated with the growing trend of the global diabetes incidence in last few decades ( Figures 3, 4).

Figure 3. Total plastic waste generation by country, measured in million tons per year for the year 2010 and disease burden from non-communicable diseases (from 1990 to 2016). Total disease burden from non-communicable diseases (NCDs), measured in DALYs (Disability-Adjusted Life Years) per year. DALYs are used to measure total burden of disease-both from years of life lost and years lived with a disability. One DALY equals 1 lost year of healthy life. Base map courtesy of mapchart.net ( http://www.mapchart.net) [Source: ( 10, 11)].

This change in our chemical environment seems to be a contributing factor in this "obesity pandemic" apart from genetic factors and modern lifestyle that embraces high energy intake, reduced physical activity, sleep deprivation and near stable home temperatures. This has led to the hypothesis that some of these environmental chemicals disrupt the endocrine process and lead to obesity by interfering with various endocrine aspects of metabolism ( 13, 14). This hypothesis garners support from animal as well as epidemiological studies, which have revealed that a diverse array of environmental chemicals can influence adipogenesis and obesity; however, its extent, exact mechanism and epidemiology are yet to be elucidated. The EDCs linked with obesity and adiposity include several POPs ( 15- 18), BPA ( 19, 20), and phthalates ( 21- 23). It has been suggested that in utero or childhood exposure to certain EDCs predisposes some individuals to obesity, highlighting the importance of timing of exposure ( 17, 21, 22, 24, 25). Such EDCs have been referred to as "environmental obesogens" ( 26). The term "metabolic disruptors" has also been suggested as many of these chemicals be associated with other metabolic diseases such as metabolic syndrome, insulin resistance and type 2 diabetes as well ( 17, 27). Several studies have found association of type 2 diabetes and/or its risk factors with PBCs, DDE, organochlorine pesticides, HCB, dioxins, BPA and phthalates ( 28- 37). Some prospective studies have interestingly concluded that no single POP could significantly predict T2D, but some combination of POPs could predict the future risk of T2D ( 28, 31- 33, 38). This also lends support to the concern that exposure to EDCs as mixtures that occur in the environment may have greater significance than that to single chemicals tested in laboratories. In addition, several studies have shown association of gestational diabetes to phthalates, BPA and PCB ( 39- 42). Increased risk of non-alcoholic fatty liver disease has been linked to BPA ( 43) as well as cardiovascular risk factors are found to be linked to both organochlorine pesticides ( 44- 46) and BPA ( 47, 48).

EDCs alter metabolic balance through multiple mechanisms including alterations in peroxisome proliferator-modulated pathways ( 49), adipogenesis ( 50), pancreatic β-cell function ( 51- 53) and hypothalamic neuropeptides ( 54, 55). In addition, decreased physical activity in relation to whole-body energy balance has been reported in mice that were exposed to BPA either during a perinatal period or in adult life ( 56, 57). However, most of the evidence regarding the mechanism of action of EDCs comes from experimental studies. In a recent study, it was shown that prenatal exposure to BPA and certain phthalates might affect the levels of adiponectin and leptin in human fetus ( 58). A novel hypothesis advanced is the role of gut microbiota in EDC-induced type 2 diabetes. It is thought that EDCs not only cause dysbiosis of gut microbiota but also undergo metabolism by gut microbes, and both these processes alter the gut neuroendocrine regulation of metabolism leading to insulin resistance and type 2 diabetes ( 9).

Effect of EDC on Blood Pressure

To elucidate associations of urinary EDC's like phthalate with blood pressure (BP), a cross-sectional analysis of 6-19 years children in the 2003-2008 National Health and Nutrition Examination Survey (NHANES) was carried out which reveals an increase in blood pressure associated with 3-fold increase in urine phthalate (DEHP) ( 59). A study conducted in people living near a dioxin-contaminated area showed that increasing serum dioxin levels are correlated with elevated diastolic blood pressure. In addition, the prevalence of hypertension was also found to be correlated with serum PCDD and PCDF levels in Taiwanese and Florida adults with dioxin exposure ( 60, 61). Similarly, high serum PCB Level was also found to be associated with elevated blood pressure in NHANES 1999-2002 and NHANES 1999-2004 participants ( 62). Studies derive conclusions about the effects of EDCs on hypertension but further studies with larger diverse cohorts are now needed to verify an association between EDC's exposure and risk of hypertension.

EDCs: Female and Male Reproductive Health

Female Reproductive Disorders: Role of Environmental EDCs

Several EDCs are known to exert effects on female reproductive hormones and their receptors through oestrogenic, anti-estrogenic, androgenic and anti-androgenic mechanisms. Some of these EDCs include BPA and phthalate, which are classified as xeno-oestrogens. They act by both receptor-dependent and receptor-independent mechanisms, either directly by binding to estrogen receptors, increasing aromatase activity and increasing estrogen sensitivity, or indirectly by their effect on gonadotropin-releasing hormone, leading to an increase in endogenous estrogen production ( 63- 65). EDCs are thought to affect the menstrual cycle, alter fertility and oogenesis and are implicated in diseases such as polycystic ovary syndrome (PCOS) and endometriosis. Hauser et al. ( 66) documented the first-ever adverse effect of urinary phthalate metabolites by using in vitro fertilization (IVF) where they found that concentrations of urinary metabolites of di(2-ethylhexyl) phthalate (DEHP) and di-isodecyl phthalate (DiDP) was inversely related with oocyte yield and number of metaphase II oocytes at retrieval, whereas only metabolites of di-isononyl phthalate (DiNP) and di-isodecyl phthalate (DiDP) was linked with reduced fertilization rate. However, the exact mechanism of action of these chemicals is not yet clearly understood, but it is likely that multiple pathways are involved including hormone receptors.

Female Puberty

Puberty is a phase of transition between adolescence and adulthood during which the reproductive system reaches full maturity ( 67). The onset of puberty involves a complex neuroendocrine mechanism and influenced by several factors including ethnicity, genetics, nutrition and environmental factors ( 68, 69). Recently early onset of puberty in girls has been noted across several countries and a role of environmental pollutants has been implicated, notably that of BPA, phthalates, and organohalogens ( 70). Puberty is brought about by activation of the hypothalamic-pituitary gonadotropic cells-gonadal axis. The activation of this axis is thought to be regulated by a novel neuropeptide and its receptor, which are in turn modulated through sex steroid receptors in hypothalamic neurons ( 71). It is possible that oestrogenic EDCs or xeno-oestrogens act through the sex steroid receptors to bring about early puberty. During childhood, the levels of sex hormones in blood are very low. However, it has been found that estrogen and androgen receptors are expressed since a very early age ( 72). Therefore, even a small change in these levels through an exogenous source may bring about several pubertal changes ( 73). In several studies, early onset of pubertal changes in girls has been reported to be associated with phthalates ( 74- 77), BPA ( 78, 79), and organohalogens ( 80- 82), whereas in other studies, no such association ( 30, 83, 84) or even inverse association has been reported ( 85, 86). Another study found a non-linear association of BPA and phthalate metabolites with pubertal development ( 87). However, it is possible that these differences result from variation in environmental exposure, timing of exposure and non-linear dose-response patterns of EDC effects. Exposure to EDCs during prenatal and perinatal period has also been thought to be linked with early onset of puberty in girls as well ( 88).

Polycystic Ovary Syndrome (PCOS)

Most of the information on direct effects of EDCs on fertility is available from in vitro and animal studies. A few human studies have been carried out in women attending fertility clinics and have provided variable results. Some of these studies have shown an association of EDCs directly with infertility and its parameters, whereas others have correlated them with PCOS, which is a complex endocrinopathy leading to infertility and insulin resistance. A negative association between urinary concentrations of phthalate metabolites and serum inhibin B levels was found, suggesting a detrimental effect of phthalates on growing antral follicles ( 89). In the Longitudinal Investigation of Fertility and the Environment Study, concentrations of phthalates, but not BPA in women were associated with longer time to pregnancy ( 90). In other studies, BPA was found to be associated with reduced ovarian reserve ( 91), lower antral follicle count ( 92) and PCOS ( 93, 94) in infertile women. Higher quartiles of urinary BPA concentration were also shown to be associated with increased odds of implantation failure ( 95). One of the studies showed that high PCB and pesticide concentrations in the follicular fluid correlated with poor embryological intracytoplasmic sperm injection outcomes ( 96). Another study documented higher serum concentrations of perfluorinated compounds and phthalate metabolites was present in women with PCOS ( 97). DDT was found to be associated with altered hormonal levels in Chinese women with PCOS suggesting a role in its pathogenesis ( 98). In a large American cross-sectional study involving more than 30,000 women, researchers found an association between EDCs and earlier age at menopause, and they also identified 15 EDCs (including 9 PCBs, 3 pesticides, 1 furan and 2 phthalates) that needed further evaluation for potential adverse effects on ovarian function ( 99).

Endometriosis

Endometriosis is the presence of ectopic endometrial tissue, which is a cause of pain and infertility. Relatively few studies have explored the role of EDCs in endometriosis. However, its association has been reported with higher blood or urinary levels of BPA ( 94, 100), various phthalates ( 101- 103) and organochlorine pesticides ( 104, 105). On the other hand, no association or inverse association with phthalates was observed in studies on infertile Japanese women and American women, respectively ( 106, 107). A recent meta-analysis of past epidemiological studies has recorded a significant constant increase in the risk of endometriosis to polychlorinated bisphenyls (PCBs) group (OR = 1.58; 95% CI: 1.18-2.12), organochlorine pesticides (OCPs) group (OR = 1.40; 95% CI: 1.02-1.92) and phthalate esters (PAEs) group (OR = 1.27; 95% CI: 1.00-1.60), while BPA showed no significant association with endometriosis ( 108).

Obstetric Outcomes

In utero exposure to EDCs through trans-placental route has become a concern as it is thought that small endocrine imbalance during fetal development can lead to significant and lasting changes. Several researchers investigated this in the last decade. In utero BPA exposure was found to be associated with decreased birth weight ( 109- 112) and increase in gestational length ( 112, 113). However, Shoaff et al. ( 114), did not find any association between prenatal phthalate exposure and birth size or duration of gestation. In another study, both maternal and paternal BPA and phthalate concentration were found to be associated with smaller birth size and increased gestational age ( 115). Though both maternal and paternal exposure to these chemicals will be similar as they are exposed to similar environment, paternal EDC levels likely reflect maternal exposure and do not directly affect the fetal exposure. Maternal urinary levels of paraben were found to be associated with increased length of male neonates in a Chinese cohort ( 116). An American study on several classes of EDCs showed that gestational exposure to perfluoroalkyl substances and organophosphate pesticides was associated with reduced birth weight while phthalates, BPA, PCBs, PBDEs (Polybrominated diphenyl ethers) or OCPs (organochlorine pesticides) had either small or no association with differences in birth weight ( 117).

Male Reproductive Disorders: Role of Environmental EDCs

EDCs are thought to affect the male reproductive system as well. Human studies indicate that these chemicals affect pubertal growth and semen quality and cause defects such as hypospadias and cryptorchidism. Well over two decades back, Sharpe and Skakkebaek (1993) proposed that several male reproductive disorders such as low sperm counts, cryptorchidism and hypospadias may share a common fetal origin and that environmental exposure to oestrogenic chemicals might play a key role ( 118). Though this estrogen hypothesis was later refuted, except diethylstilbestrol, it propelled scientific research on effects of EDCs on male reproductive system. It is now thought that EDCs act on male reproductive system by disrupting fetal endocrine balance through their action on steroid hormone receptors (SHRs) or interference with the synthesis, kinetics or metabolism natural hormones (World Health Organization [WHO] and United Nations Environment Program 2012). Sperm function affected by reactive oxygen species generated during metabolism of these chemicals is another possible effect of EDCs leading to infertility ( 119).

Male Puberty

Semen Quality

There are numerous causes of infertility in men. Even though sperm count does not associate specifically with fertility, it is documented that men with very low sperm counts frequently have fertility problems. In 1996, in their review article, Toppari et al. expressed concern regarding decreasing semen quality in the last few decades, and they advanced the hypothesis that exposure to environmental chemicals may be responsible for adverse trends in male reproductive health ( 124). Several EDCs have been found to affect semen parameters adversely. These include organophosphate pesticides ( 125- 127), BPA ( 128), perfluorinated compounds ( 129), phthalates ( 130), and organochlorines ( 131). It was observed that adult life exposure to PBC affects mainly sperm motility while perfluorinated compounds alter sperm morphology primarily. However, the overall impact of EDCs on male fertility is not clear so far. Though sperm count and morphology appear to be altered to some extent, the effect on fertility seems to be minor at best.

Cryptorchidism

Cryptorchidism is the condition in which the testis does not descend completely to the bottom of the scrotum. Its incidence has been documented to increase from 1-3% in the late 1950s to 7-9% in the 2000s ( 132). This condition shows familial clustering suggesting a role of genetic and environmental factors. Known risk factors for cryptorchidism include maternal gestational diabetes and maternal use of alcohol, acetaminophen or nicotine-containing substances. In recent research, cryptorchidism has been found to be associated with prenatal or early childhood exposure of several chemicals including PBDE ( 133, 134), BPA ( 135), parabens ( 135), and PBCs ( 134). Other researchers have found no association of cryptorchidism with PBBs ( 136), organochlorine pesticides ( 137), perfluorinated compounds ( 138, 139), or PCBs ( 140). Because several environmental factors are also associated with cryptorchidism, the effects of individual factors are difficult to establish and the possibility of confounders cannot always be eliminated in these studies.

Hypospadias

Hypospadias is a congenital malformation in which the urethral folds do not fuse properly. A meta-analysis suggested an association between pesticide exposure in men and risk of hypospadias in their offspring ( 141). Another study suggests that exposure to HCB and p,p′-DDE during fetal life may be a risk factor for hypospadias ( 142). However, no significant association of exposure to PCBs ( 140, 143), organochlorine pesticides ( 137), PBBs ( 136), PBDE ( 143), or perfluorinated compounds ( 144) with hypospadias was noted in other studies. Hypospadias is a relatively uncommon condition; therefore, most of these studies face the problem of small sample size and are not statistically robust.

Overall, the effect of individual EDCs on reproductive system appears uncertain; however, it is possible that some additive or synergistic effects of mixtures do exist. Conflicting results and differences in study variables make it difficult to establish the role and extent of effects of EDCs either individually or in combinations. Large-scale studies taking into account the exposure of all known EDCs in a uniform manner will be needed to explore such complex association.

Role of EDCs in Neurodevelopmental Disorders

Prenatal exposure to EDCs is thought to affect brain development in the fetus, and some neurodevelopmental abnormalities have been attributed to them. Prenatal BPA exposure has been linked to poor social behavior ( 145, 146), higher anxiety and depressive behaviors ( 147). Some studies have suggested a sex-specific effect of BPA on behavior. A study showed that poor social behavior was found to be more pronounced in girls ( 146), while a study showed that significant positive associations were found between prenatal BPA and emotionally reactive and aggressive behavior in boys ( 148). In another study, prenatal BPA exposure was noted to be associated with increased anxiety and depression in boys but not in girls ( 149). The gender difference is likely due to the close relation of brain development with gonadal hormones as proven by animal studies. The brain, particularly hypothalamus, has been shown to be affected by oestrogenic EDCs in a sex-, time- and exposure-dependent manner ( 150). In utero exposure to phthalates is shown to be associated with adverse cognitive and behavioral outcomes such as lower IQ, attention deficit, hyperactivity and poorer social communication in children ( 151- 153). Other EDCs such as organophosphates ( 152, 154), PBDEs ( 155, 156), perfluorinated compounds ( 157), and PCBs ( 153, 158) have also been linked to adverse neurodevelopmental performance, behavioral problems and lower IQ to varying degrees. There is also some evidence that some PBDEs and PCBs affect neuropsychological status in adults ( 159, 160). Animal studies have provided ample evidence that EDCs act on developing brain through multiple mechanisms: (a) steroid hormones and receptors, (b) neuroendocrine system, and (c) epigenetic changes ( 161). One of the main biological mechanisms proposed for adverse neurodevelopmental effects of prenatal EDC exposure is disturbance of thyroid hormone homeostasis, which is critical for fetal brain development ( 162).

Thyroid Homeostasis and EDCs

Thyroid hormones are controlled through the hypothalamic-pituitary-thyroid axis, and they are vital for the regulation of several biological systems and can be disrupted by EDC exposure. Specifically, the phenols and phthalates are thought to act directly on the thyroid hormone receptor, and studies have shown that these compounds are thyroid receptor antagonists. Epidemiological studies have shown that cognitive functions of the offspring may be adversely affected by even a marginal lowering in thyroxin levels in a pregnant woman ( 163- 165). Thus, exposure to thyroid-disrupting chemicals may have significant consequences for public health even if they bring about a small reduction in thyroid hormone levels. Several studies have evaluated the EDC exposure effect on thyroid hormones in humans. Most of these have explored the effect on pregnant women and new-borns. A study reported that urinary concentrations of phthalate metabolites were seen to be associated with lower T4 and T3 or higher thyroid-stimulating hormone (TSH) levels in adults ( 166). In another study, prenatal exposure to phthalates was found to be inversely associated with both total serum thyroxine in pregnant women and newborns as well as TSH in newborns ( 167). The association with BPA is more variable in several studies. Two studies found no association between thyroid hormone levels and maternal BPA exposure ( 168) and cord blood BPA levels ( 58), respectively. Another study reported no association between maternal urinary BPA concentrations and thyroid hormones measured in cord serum when analyzed for all newborns, but they found that in female newborns, lower cord TSH was associated with the 10-fold increased BPA in maternal urine ( 169). In the analysis of data from the U.S. National Health and Examination Survey, urinary BPA showed a negative relationship with serum TSH levels in adults ( 166), while another European study concluded that a significant positive association was observed between urinary BPA and serum TSH in adult women ( 170). A study found that there was a negative correlation between PCB levels and maternal free T3 but not with cord blood ( 171). However, a systematic analysis did not find any correlation between PCBs and thyroid hormones in either pregnant women or newborns ( 172). In a study including three European cohorts, the researchers concluded that PCB was associated with lower TSH levels in newborns ( 173). In another study, in the case of PBDEs, negative correlation was found with maternal and cord blood thyroid hormones ( 171), while positive correlation was seen with maternal blood thyroid hormone levels in two other studies ( 174, 175). Organochlorine pesticides were noted to be associated with reduction in thyroid hormones ( 176).

Renal Function and EDCs

Chronic kidney disease (CKD) is a worldwide health menace of growing concern, and its prevalence all over world is estimated at 13.4% ( 177). Kidneys serve as the filtration unit and thus are exposed to all compounds in a greater degree compared to other organs. Humans are not exposed to a single compound but to mixture of compounds and they are likely to affect the kidneys as well. Without a doubt, many non-persistent EDCs, e.g., phthalates, bisphenols, benzophenones, and parabens, share common exposure sources such as plasticware and personal care products in various things.

There are growing evidences suggesting that non-persistent endocrine disrupting chemicals (EDCs), are linked with adverse kidney function ( 178). Studies also showed that exposure to di-(2-ethylhexyl) phthalate (DEHP) through the consumption of contaminated milk was associated with micro-albuminuria in children ( 179, 180). The US National Health and Nutrition Examination Survey (NHANES) delineated the association of urinary phthalate metabolites with CKD markers such as albumin-to-creatinine ratio (ACR), estimated glomerular filtration rate (eGFR), and urinary protein-to-creatinine ratio (UPCR) has also been revealed in a population of children ( 181, 182). Urinary BPA levels were also associated with ACR among the US children ( 183), as well as Chinese adults ( 184). Patients on dialysis have more exposure to BPA due to daily use of dialysis tubing. BPA levels are higher in patients undergoing hemodialysis and peritoneal dialysis, than in healthy controls ( 185). NHANES 2003-2006 Estimated Glomerular Filtration Rate (eGFR) among a general population of the US adult females shows positive association with urinary BPA levels but not in the adult males ( 186).

EDCs and Hormone-Dependent Cancers

A large fraction of EDCs acts through oestrogenic and androgenic receptors; therefore, researches have focused on their relation to hormone-dependent cancers. Such neoplasm's include breast, endometrial, ovarian, and cervical cancers in women and prostate cancer in men. The incidence of these cancers has risen at a higher rate over the last few decades ( 187- 189). As other non-communicable disease, this increase is multifactorial, and change in lifestyle, industrialization and EDCs are thought to be responsible at least in part ( 161).

The development and progression of estrogen-dependent malignancies, particularly breast cancer, depend on prolonged or exaggerated estrogen exposure ( 190). Approximately 70% of breast cancers are sensitive to estrogen as they show ER-positivity ( 191). Unopposed estrogen or estrogen-mimicking EDCs can bind to ER, switching on the downstream signaling of estrogen-responsive genes involved in the cell cycle ( 64, 192, 193). Several oestrogenic EDCs, such as BPA and phthalates, have been shown to induce epigenetic modification or genotoxic effects ( 27, 194, 195), which can alter gene products and genetics, eventually predisposing to certain diseases. However, only a few human studies have explored this association so far. Although meta-analysis did not find any association between organochlorine pesticides and breast cancer ( 196), another study linked it to exposure before 14 years of age ( 197). A study in Mexico showed a positive correlation between MEP, a phthalate metabolite, and breast cancer while other phthalates (MBP, MEOHP, and mono [3-carboxypropyl]) had negative correlation with breast cancer risk. The evidence of any relation between BPA exposure and breast cancer is weak, and it is observed only in animal studies. This may be due to its short half-life so that measurement of exposure is difficult ( 161). Agricultural Health Study, USA, has found that several pesticides are linked with increased risk of prostate cancer ( 198, 199). The link between EDCs and other hormone-dependent cancers is tenuous as it is based on a handful of animal and laboratory studies. It has not yet been explored through human studies, and it constitutes a lacuna in our knowledge.

Endocrine Disrupters Accelerate Aging

Growth is the consequence of a multifaceted interaction of genetic, constitutional, nutritional, endocrine and socio-economic factors as well as psychosocial well-being ( 200, 201). The evidence is seen in the fact that over the last century, children have gradually become taller and are reaching puberty earlier. For example, the age of menarche in European girls in the middle of the nineteenth century was 16-17 years, in contrast to the current age of 13 years or less. Rampant use of sex steroids as growth promoters has occurred in animal farming, which continues in several countries of the world.

The timing and evolution of reproductive aging depend mainly on genetic and environmental factors. So far, most research has focused on genetic predispositions ( 202). Nevertheless, the environment probably plays a role, and there is an assumption that EDCs may accelerate reproductive aging, resulting in shortened reproductive lifespan. Recent studies show that exposures to EDC during critical developmental periods, particularly during fetal life and early childhood can cause molecular/cellular changes that modify the function of the affected tissues later in life, a concept referred to as the fetal/developmental basis of adult disease ( 203). These effects are thought to be brought about by epigenetic modifications. Animal studies show that reproductive aging is accelerated by EDC (MXC) ( 204), BPA ( 205), and dioxins ( 206). Recent epidemiological evidence also links EDC exposures during periods of growth to accelerated menopause [diethylstilbestrol ( 207) and perfluorocarbons ( 208)]. BPA has also been shown to promote oxidative stress and inflammation, and it has been suggested that it makes postmenopausal women suffer health effects related to aging ( 209).

Dose-Response Dynamics of EDCs

Several biological effects of EDCs are mediated through SHRs ( 210). Hormone receptors are high-affinity receptors, that is, a small concentration of hormones or hormone mimickers can produce a significant biological effect. In addition, the dose-response curves of these receptors are non-linear; they can be sigmoidal, or more complex, including being non-monotonic. The non-monotonic dose-response (NMDR) curves are often "U-shaped" (with maximal responses observed at low and high doses) or "inverted U-shaped" (with maximal responses observed at intermediate doses) ( 161). EDCs mimic hormones in their actions including low-dose effects and dose-response patterns, which depend on characteristics of their binding receptors and ligands ( 161). Due to this property, environmentally relevant concentrations of these chemicals need to be studied thoroughly for their effects on the general population. Several studies have provided evidence that low-dose environmental exposures to EDCs are associated with disorders in humans as well as wildlife ( 17, 70, 211, 212). Research shows that many SHR-mediated adverse effects of EDCs may be non-linear or even non-monotonic and such effects commonly occur in dose ranges exerting no overt cytotoxicity ( 213- 218). One of the setbacks due to these non-classical responses is in extrapolating the effect from high doses to low doses or vice versa because there is no linear relationship ( 219). Defining safe limits for such chemicals also becomes troublesome.

Most of the evidence for NMDR comes from cell culture and animal studies. Several epidemiological studies have also focused their attention on NMDR in EDCs recently but they differ significantly in their conclusions ( 23, 219). However, these differences in conclusions can arise from several inherent drawbacks in human epidemiological studies, for example, cross-sectional vs. prospective design dichotomy and difference in study analysis methods. In addition, there is a lack of sufficient knowledge about kinetics of these chemicals, their interactions with adiposity and diet, and changes to these factors with age and gender. Furthermore, in epidemiological studies, populations are often exposed to a variable mixture of chemicals at various exposure levels for various durations, and predictably, these interactions are complex. These factors make human studies difficult to interpret.

Epigenetic and Transgenerational Effects of EDCs

More recently, scientists have discovered evidence showing that EDCs can modify gene expression without mutating DNA, which is also known as epigenetic change ( 220). The known possible mechanisms of epigenetic changes include methylation of cytosine residues in DNA, post-translational modification of histones and altered microRNA expression. These changes may lead to transgenerational effects on numerous organs and organ systems and are thought to be tissue-specific as well as dose-dependent ( 221- 223). Epigenetic transgenerational inheritance is the phenomenon where environmental exposures of a woman during gestation lead to germline epimutations, which are then transmitted to subsequent generations with an observable phenotypic expression ( 224, 225). BPA is the most widely studied EDC in terms of epigenetic changes, and several animal studies support this theory ( 226- 228). Other chemicals implicated in epigenetic changes leading to transgenerational effects are dioxins ( 229, 230), DDT ( 231, 232), and phthalates ( 233, 234). In addition, the epigenetic changes are also thought to cause latent effects in case of early life exposure, with manifestation later in life ( 235). However, most of these studies have evaluated only the effects on the reproductive system of experimental animals. The epigenetic changes add another dimension to understand the complexity and mechanism in the studies of environmental exposures and their effects.

Timing of EDC Exposures as a Critical Element

EDCs influence and modify the hormonal homeostasis and, therefore, it is plausible that their effects will be more pronounced in periods during which marked hormonal changes occur, such as perinatal period, puberty and pregnancy. Tissue differentiation and maturation of several organs occur during these "critical windows" which may be influenced by EDCs ( 236). Moreover, the effects of exposure may not be apparent immediately and may take several years to be observable. This is supported by several studies that show the association between several health and behavioral parameters and EDC exposure during these key periods. In utero exposure to several EDCs was found to influence fetal growth and birth weight ( 109- 112, 117) and also predisposed some individuals to obesity and fat gain during later life ( 17, 21, 22). Exposure to EDCs during the prenatal and perinatal period has been thought to influence the timing of puberty as well ( 86, 88). Several studies evaluating the neurological development of children have shown association of prenatal EDC exposure with adverse cognitive and behavioral outcomes ( 145, 146, 148, 151- 153). Prenatal exposure to phthalates was shown to alter thyroid homeostasis in pregnant women as well as the offspring ( 167).

For POPs, latency could be due to long-term continuous exposure. For non-persistent EDCs, exposure could be variable. Apart from the molecular mechanism, epigenetic changes may be important in delayed or latent effects, especially in perinatal exposures.

Risks From Exposure to EDCs Mixture: Cocktail Effect

Gutmicrobiome: A Player in the Toxicity of Environmental Pollutants

Human beings are exposed to different chemicals through air, water and soil, but oral exposure to EDCs, largely via the food chain, is their key pathway into human metabolism. The gut microbiome comprises trillions of bacteria, viruses and fungi, and it is documented as a main participant in the metabolism of nutritional compounds, drugs, antibiotics and environmental toxins ( 259- 264). Bacterial fermentation of undigested carbohydrates and xenobiotic compounds produces short-chain fatty acids, choline, bile metabolites and several volatile compounds including hydrogen sulfide. The gut microbiome also plays a major role in regulation of various hormones, neurotransmitters and ultimately host metabolism. Some metabolites produced during bacterial fermentation in gut behave as hormones that can affect diverse host metabolic functions, and hence the gut microbiome has been anticipated to be a new endocrine organ ( 265- 267). Dysbiosis of gut microbiome is commonly observed in medical conditions. Concerning diabetes, a shift in the Bacteroidetes/ Firmicutes ratio was found to have a key link with plasma glucose concentration ( 268). Microbial metabolism of chemicals, as well as EDCs, by gut microbiome can lead to microbial dysbiosis, stimulation of specific bacterial genes, and altered microbial transformation of molecules ( 260, 268).

Previous researchers had determined how diet and host health could influence the gut microbiome composition ( 269- 273). On the other hand, several acquired factors encountered on an everyday basis can exert extreme effects on the gut microbiome. Environmental chemicals, such as EDCs, heavy metals and air pollutants are increasingly invasive in terrestrial and aquatic environments, and there is a signal that such chemicals will turn out to be even more profuse in near future ( 274- 281). Such chemicals are also found in things that we use daily, such as plastic water bottles, storage containers, and antibacterial supplies. Introduction to such chemicals can result in extensive host effects and concurrently aim commensal microbes harbor within the gut and perhaps other organs. The chemical-induced dysbiosis of the gut microbiome leads to disruptions in various host systems, mainly the central nervous system, via the gut-microbiome-brain axis ( 282- 284). An improved understanding of how environmental chemical-induced gut microbiome dysbiosis changes strengthen host disease is essential. Disorders associated with gut dysbiosis comprise metabolic, neurobehavioral, immunological, cardiovascular, gastrointestinal and several other disease states ( 273, 285- 291). The gut microbiomes have a different class of enzymes such as Phase I and Phase II. They can metabolize environmental xenobiotic chemicals and environmental chemicals may cause dysbiosis of gut microbiome, with potential effects on human health. Based on previous studies we may conclude that gut microbiome plays a major role in the toxicity of environmental pollutants.

Evaluating Health Effects of EDCs in Humans: Is Exposomes Mapping the Key?

Impact of EDCs in the Context of Climate Change

Climate change has been globally acclaimed as a major environmental risk, also when combined with chemical pollution and habitat loss it has the potential to have a severe impact on human health. The impact of escalating climate change has added exigency to the rising problem of EDC's pollutants. A study by Brown et al. ( 297), the first of its kind, showed that climate change could amplify the deleterious effects of pollution from EDCs on aquatic environments. Climate change is influencing the rate at which toxic chemicals are released from plastic materials, stockpiles and polluted sites. Higher temperatures increase the release of persistent organic pollutants (POPs) to air by changing their rate of partitioning between air and soil, and between air, water and sediment. POPs persist in the environment, bioaccumulate through the food chain, and pose a threat of causing detrimental effects to human health ( 298). The fate of environmental POPs in these ecosystems is controlled mainly by temperature and biogeochemical processes. Climate change may increase the re-emissions and redistribution of POPs in different environmental settings via soil-air exchange. Soil-air exchange is a main process of controlling distribution of POPs and terrestrial ecosystem carbon at local and universal scales.

Each POP has a moving potential in air and/or water and can bioaccumulate in biological system such as lipid-rich tissues and biomagnified through terrestrial and aquatic food chains ( 299). Hence, POPs have a potential adverse impact on higher trophic organisms in aquatic as well as environment ( 300- 302) and on human health ( 303). Moreover, intensification of global warming, an increase in POPs levels has been reported in different settings of environment because of the release from reservoirs ( 299, 304). These higher emissions induced by climate change would increase the susceptibility of exposed animals and humans via food chain and lead to greater adverse effects on human health and terrestrial ecosystems ( 305).

While the researcher's grapples with the problem of ever-increasing environmental pollution, at the same time, the causes of climate change and its impacts on the fate and toxicity of chemicals must be addressed.

EDC Regulations in Various Countries

The issue of EDCs is puzzled due to lack of scientific data and many gaps in knowledge, it is still essential to frame regulatory policies to deal with the present scenario. Framing of policies and regulations must go parallel with ongoing research and any measure must be compliant to rapid advances in scientific knowledge. At present countries having regulations addressing the manufacture, use and disposal of various chemicals includes carcinogens, teratogens, mutagens and substances that interrupt reproduction. EDCs represent a relatively new classification, involving diverse chemical classes that are able to mimic endogenous hormones. Although there is no agreement on their regulation, EDCs are addressed in different cases in EU law, such as the Water Framework Directive, Registration, Evaluation and Authorization of Chemicals (REACH), Plant Protection Products Regulation (PPPR) and Cosmetic Regulation ( Table 2). The government and regulatory agencies of developing countries with large population and huge plastic consumers may restrict the use of endocrine disrupting chemicals in the production of cosmetics, food-contact plastics, plastic water tanks and packaging materials and may provide guidelines on the use of EDC's/plastics, awareness on its harmful impacts and the use of risk-free alternatives must be provided, with an intention to ensures the safety of vulnerable group such as infants, children and pregnant women. The challenges that arise from the field of endocrine disruption are the immense diversity of chemicals produced, but not tested, the mixtures and the unknown interactions between them, together with their consequent effects. The lack of an effective and strong legislation and regulation poses a significant threat to humans, animals and plants, and contributes to the exposure to chemicals that may disrupt the endocrine system.

Table 2. Regulations on controlling EDCs in various countries.

Conclusions

Most of the information on the detrimental effect of EDCs comes from animal studies. It is becoming more evident that EDCs may trigger disorders such as metabolic diseases, reproductive abnormalities, endocrine dysfunction and cancers. The sudden escalating rates of these diseases, especially metabolic disorders, correlate with global industrialization and the production and release of EDCs to the environment, drinking water and eventually to food chain. Countries should formulate acts and guidelines including EDCs for the safety of drinking water. To understand the exposure routes of EDCs better, a comprehensive assessment of drinking water supply is required. Exposure to EDC or mixtures, even at low doses, especially during critical window or early development can have an impact on endocrine system by altering transgenerational/epigenetic pathway. Hence, further studies are required to establish the threshold concentrations of EDCs in the environment as well as in human matrixes (bio monitoring) below which its detrimental effects do not occur. Large-scale systematic epidemiology studies need to be carried out taking into consideration the low dose effect and cocktail effect of EDCs.

Author Contributions

MKumar, MKumawat, SS, and DS: manuscript draft. RT and AP: overall review. VV: manuscript drafting and editing. All authors contributed to the article and approved the submitted version.

Funding

This study was supported by Indian Council of Medical Research grant no: NIREH//BPL/PJ-30/2018-19/127. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

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The exponential growth of pollutant discharges into the environment due to increasing industrial and agricultural activities is a rising threat for human health and a biggest concern for environmental health globally. Several synthetic chemicals, categorized as potential environmental endocrine-disrupting chemicals (EDCs), are evident to affect the health of not only livestock and wildlife but also humankind.

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Lauryn Cameron:  exposure to  Endocrine disrupting chemicals (EDCs) during critical periods of development can result in delayed/advanced puberty and long-term reproductive consequences

# data[].attributes.subject
Lauryn Cameron:  exposure to  Endocrine disrupting chemicals (EDCs) during critical periods of development can result in delayed/advanced puberty and long-term reproductive consequences

# data[].attributes.attachment_link.readable_text
The onset of puberty strongly depends on organizational processes taking place during the fetal and early postnatal life. Therefore, exposure to environmental pollutants such as Endocrine disrupting chemicals (EDCs) during critical periods of development can result in delayed/advanced puberty and long-term reproductive consequences. Human evidence of altered pubertal timing after exposure to endocrine disrupting chemicals is equivocal. However, the age distribution of pubertal signs points to a skewed distribution towards earliness for initial pubertal stages and towards lateness for final pubertal stages. Such distortion of distribution is a recent phenomenon and suggests environmental influences including the possible role of nutrition, stress and endocrine disruptors. Rodent and ovine studies indicate a role of fetal and neonatal exposure to EDCs, along the concept of early origin of health and disease. Such effects involve neuroendocrine mechanisms at the level of the hypothalamus where homeostasis of reproduction is programmed and regulated but also peripheral effects at the level of the gonads or the mammary gland.

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Endocrine disrupters and possible contribution to pubertal changes

# data[].attributes.attachment_link.description
The onset of puberty strongly depends on organizational processes taking place during the fetal and early postnatal life. Therefore, exposure to environmental pollutants such as Endocrine disrupting chemicals (EDCs) during critical periods of development can result in delayed/advanced puberty and long-term reproductive consequences. Human evidence of altered pubertal timing after exposure to endocrine disrupting chemicals is equivocal.

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Dinorha Vidrio: Exposure to EDC can lead to early onset of puberty. Estrogen-mimicking disruptors promote breast tissue growth that later leads to breast cancer in many women. 

# data[].attributes.subject
Dinorha Vidrio: Exposure to EDC can lead to early onset of puberty. Estrogen-mimicking disruptors promote breast tissue growth that later leads to breast cancer in many women. 

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Abstract

Puberty is the process of physical changes between childhood and adulthood during which adolescents reach sexual maturity and become capable of reproduction. It is considered one of the main temporal windows of susceptibility for the influence of the endocrine-disrupting chemicals (EDCs). EDCs may act as single chemical agents or as chemical mixtures; they can be pubertal influencers, accelerating and anticipating the processing of maturation of secondary sexual characteristics. Moreover, recent studies have started to point out how exposure to EDCs during puberty may predispose to breast cancer later in life. In fact, the estrogen-mimicking endocrine disruptors (EEDs) may influence breast tissue development during puberty in two main ways: the first is the action on the proliferation of the breast stromal cells, the second concerns epigenetic mechanisms. The aim of this mini-review was to better highlight what is new and what is not completely known regarding the role of EDCs during puberty.

Keywords: puberty, Endocrine disrupting chemicals (EDCs), Window of susceptibility, Early Puberty, Estrogen-mimicking Endocrine Disruptors (EEDs), breast development, breast cancer

1. Background

Endocrine-disrupting chemicals (EDCs) influence one or more functions of the endocrine system and cause several adverse health effects in human beings. They are ubiquitous in nature, being found in industrial chemicals, drugs, plastics, and other materials common in everyday life [ 1].

EDCs may represent a risk for our health, working through many different mechanisms of action. In fact, they may take several routes of absorption (oral, dermal, etc.), affect different temporal windows (prenatal and postnatal life, puberty), and be made up of many combinations of chemicals (mixtures), leading to extremely difficulty in interpreting their influence on human wellbeing.

Moreover, their effect seems to be transgenerational, occurring over at least two or three generations. As the window of susceptibility, puberty is considered one of the hot spots in the lifetime when EDCs may exert their effects [ 2].

The main aims of this mini-review were (1) to provide an update of what we know about the relationships between EDCs and pubertal trends, and (2) to highlight the new role of EDCs as influencers of breast cancer predisposition later in life.

2. Puberty and Pubertal Onset

Puberty is the process of physical change that occurs between childhood and adulthood, during which adolescents reach sexual maturity and become capable of reproduction.

The main clinical manifestations of puberty are the growth spurt, progression of secondary sexual characteristics, development of gonads, and changes in body composition. These features lead to an increase in strength and endurance [ 3].

During pubertal development, changes in the brain activity are also detectable: the frontal lobes increase their volume and activity, leading to more complex psychological understanding [ 4]. The whole process involves years of development on both a mind and a body level.

In the last two centuries, pubertal timing has changed dramatically. Data show that the age of the menarche is experiencing a declining trend, from 17 years old at the beginning of nineteenth century to 13 years old by the 50s [ 5]. In the United States, the onset of puberty in girls is occurring earlier than previous studies have documented, with breast and pubic hair development appearing on average 1 year earlier in white girls and 2 years earlier in African-American girls. Beyond these data, it is also underlined how this early onset of puberty seems not to be correlated with a parallel anticipation of the menarche age. According to the most recent studies, the age of menarche has decreased by only few months [ 6].

As far as timing is concerned, in the 60s, Tanner et al. identified the normal range of pubertal onset as between 8 and 13 years in girls, and between 9 years and 6 months and 13 years and 8 months in boys [ 3, 4]. Therefore, the development of secondary sexual characteristics is considered precocious when it happens in girls under the age of 8 years old and in boys younger than 9 years old.

The timing of pubertal onset depends on genetic and environmental factors. The genes involved in this process are the ones that control GnRH hypothalamic secretion, pituitary development and its functioning, hormone synthesis and bioactivity, energy homeostasis and growth, and potential peripheral feedback from sex steroids [ 7].

Studies show that some populations are much more likely to develop precocious puberty: girls; African-Americans; individuals presenting other medical conditions (McCune-Albright syndrome, congenital adrenal hyperplasia) or experiencing or having experienced radiation therapy of the central nervous system; those exposed to sex hormones; children that may have been in contact with estrogen or testosterone cream or ointment, or with other substances such as adults' medications, dietary supplements, or food containing phytoestrogens.

The nutritional status of the pediatric population influences the age of pubertal onset. Adolescents with anorexia nervosa usually reach menarche later [ 8], highlighting a correlation between nutritional state and development. Girls who are overweight or obese have a higher probability of being diagnosed with central precocious puberty or early puberty compared to the general population [ 9]. Genomic studies and human epidemiological data suggest a correlation between early infancy weight gain, early pubertal onset, and later obesity in adulthood, which can be explained by the overlapping of genes involved in pubertal onset and obesity [ 10]. Moreover, obesity induces hyperinsulinemia and hyperleptinemia, which can affect linear growth and advanced puberty [ 11].

Both events, pubertal timing and obesity, are influenced by the environment, and there is a strong interaction between genes and environments during critical periods of development such as puberty. This interaction is due to substances, EDCs, which derail the normal hormonal process.

3. EDCs and Puberty

In 2015, the Endocrine Society defined an EDC as "an exogenous chemical or mixture of chemicals, which interferes with any aspect of hormone action". The EDCs affect the control of energy balance, reproduction, hormone-sensitive cancer, thyroid, neurodevelopment, and the neuroendocrine system. They can act at any level in the hypothalamic-pituitary-gonadal system or the peripheral tissue. During a lifespan, there are some critical points (windows of development) that are important because during them, cells are rapidly proliferating and epigenetic changes are more likely to occur. These periods are fetal life, neonatal life, and puberty [ 12].

Moreover, EDCs act at any level in the hypothalamic-pituitary-gonadal-peripheral tissue-endocrine axis. Indeed, they can act not only as agonists or antagonists of estrogen receptors, but can also target and act against androgen and progesterone receptors. They may also mimic the natural occurrence of estrogens and androgens. Furthermore, EDCs bind to cell receptors and block the functions of endogenous hormones, thus acting as anti-estrogens and anti-androgens [ 13].

The most important effect of EDCs is considered to be the anticipation of or, more generally, impact on the timing of female puberty [ 14]. Nevertheless, this effect is difficult to demonstrate since low-level exposures to a mixture of EDCs may mask the effects of some of them. Moreover, inter-individual variation in susceptibility might interfere with an objective analysis. Finally, the effects on humans to EDCs exposure may show up with a considerable delay.

We analyzed the most important and well-known EDCs that might interfere with natural pubertal timing ().

Table 1

Atrazine

Herbicide

<1 day

Dermal absorption, inhalation

Prenatal life

ER agonist

Early puberty

↑incidence of breast cancer

BPA

Polycarbonate plastics, epoxy resins, plastic toys and bottles, lining of food cans

4-5 h

Ingestion, dermal absorption, inhalation

Prenatal life
Puberty

ER agonist
Antiandrogen

Premature thelarche

Induce neoplastic transformation

DES

Nonsteroidal estrogen medication used in the past

3-6 h

Ingestion

Prenatal life
Puberty

ER agonist
Epigenetic silencing of mRNA

Not known

↑breast cancer

DDT/DDE

Contaminated water, soil crops, fish

6-10 years

Ingestion, dermal absorption, inhalation

Prenatal life and lactation period

ER agonist
Antiandrogen
Antiprogestin
Induction of aromatase

Precocious puberty
Anticipated menarche
Later onset of puberty

↑cells proliferation index and mammary tumor cells proliferation

Dioxin

By-product of chlorinated herbicide production, smelting, chlorine bleaching of paper

7-11 years

Ingestion, inhalation

Prenatal and lactation
Puberty

ER agonist
Antiandrogen
Interfere with sex-steroid synthesis

Delayed breast development

Induce epigenetic silencing of key tumor suppressor (TCDD)

Parabens

Cosmetics and pharmaceutical products, toothpaste and food preservatives.

<24 h

Dermal absorption, ingestion

Pre- post-natal life and puberty

ER agonist

Not known

↑incidence of breast cancer

PBDEs/PBB

Furniture, mattresses, carpet pads, automobile seats, flame-retardant textiles

2 days-3 months

Ingestion, dermal abruption, inhalation

Prenatal life, perinatal period, peripuberty

ER agonist/antagonist
Antiandrogen

Early pubic hair in boys
Premature thelarche
Early/late menarche
Early puberty

Not known

PCBs

Contaminated air and food, skin contact with old electrical equipment

12 days-16 years

Ingestion, dermal absorption, inhalation

Prenatal life

ER agonist/antagonist
Antiandrogen

Early onset of menarche
Delayed pubertal development

Accumulates in breast adipose tissue

PFOA

Carpeting, upholstery, floor wax, firefighting foam, sealants

3.8 years

Ingestion, inhalation

Prenatal life
Puberty

Induced estrogen

Delayed pubertal development

↑susceptibility to carcinogenesis in future generations

Phthalates

Contaminated food, PVC plastics and flooring, personal care, medical devices and tubing

12 h

Ingestion, dermal absorption, inhalation

Prenatal life

ER agonist/antagonist
Antiandrogen
Interfere with androgen synthesis

Early puberty
Premature thelarche
Delayed pubic hair development

↑cells'proliferation in the breast

Pyrethroids

Contaminated water, soil, food

10 h

Ingestion, dermal abruption, inhalation

Androgen agonist

Delayed puberty

Not known

Vinclozolin

Fungicide

1-3 months

Ingestion, dermal absorption, inhalation

Prenatal life
Puberty

ER agonist
Antiandrogen
Epigenetic alteration

Not known

Dysregulates the gland development
↑formation of mammary tumor

3.1. BPA

Bisphenol A (BPA) is a precursor of plastics, polycarbonates, and epoxy resins. BPA-based plastic is clear and tough, and it is used in a lot of common objects such as plastic bottles, food storage containers, baby bottles, and CDs. Epoxy resins are utilized to line water pipes, as coating on the insides of many food and beverage containers, and in making thermal paper. This chemical is almost ubiquitous, which means that people are at great risk for exposure and, even if the estrogen receptor agonist activity is weak, its potential should not be underestimated. BPA also acts as an anti-androgen.

Studies demonstrate how this EDC is associated with premature thelarche, precocious puberty and pubertal development [ 15, 16, 17].

3.2. DDT/DDE

Dichlorodiphenyltrichloroethane, commonly known as DDT, is an organochlorine. It was originally developed as an insecticide for use in agriculture and it is colorless, tasteless, and odorless. Because of these features, exposure to DDT is imperceptible, but being exposed during fetal life and lactation can affect sexual development. Even if DDT is banned from the market, it will remain present in the environment as a persistent organic pollutant (POP). Moreover, in some low-income countries, it is still widely used.

DDT and its metabolite dichlorodiphenyldichloroethane (DDE) are the most studied of the pesticides that have been identified as EDCs. Both have estrogenic, anti-androgenic, and antiprogestin effect and induce aromatase. According to Vasiliu et al. (2004), there is an association between exposure to these chemicals and precocious puberty, delayed puberty, and earlier age of menarche [ 18, 19].

3.3. Dioxins

Dioxins are byproducts of burning or various industrial processes such as chlorinated herbicide production, smelting, or chlorine bleaching of paper. They have an estrogenic and anti-androgenic effect, and they interfere with sex steroid synthesis. Exposure to dioxins during the prenatal and lactation period is associated with delayed breast development. On the other hand, exposure to these chemicals has not been correlated with age of menarche or pubic hair development [ 20].

3.4. PBDEs and PBB

Flame retardants are a group of chemicals added to manufactured materials (plastics, textiles, surface finishes, and coatings). They are intended to prevent or slow down the further development of ignition. Some examples of such chemicals are polybrominated diphenyl ethers (PBDEs) and polybrominated biphenyl (PBB). They act as estrogens and anti-androgens. Data show an association between PBDEs and premature thelarche, earlier menarche, and earlier pubertal development [ 21, 22]. Furthermore, prenatal exposure to PBDEs is associated with later menarche in girls and earlier development of pubic hair in boys [ 23].

Moreover, exposure during the peri-pubertal period seems to interfere with reproductive development [ 24].

3.5. PCBs

Polychlorinated biphenyl (PCBs) is a dioxin-like compound derived from biphenyl. This chemical is widely used as a dielectric and coolant fluid in electrical apparatuses, in carbonless copy papers, and in heat transfer fluids. Its mechanism of action is rather similar to that of dioxins, but compared to dioxins, there is evidence that exposure during the prenatal period leads to early onset of menarche and to delayed pubertal development [ 25].

3.6. Phthalates

Phthalates are esters of phthalic anhydride. They are used as liquid plasticizers in plastic, flooring, personal care products, medical devices, and tubing. They increase the flexibility, transparency, durability, and longevity of materials. Their most common use is to soften polyvinyl chloride (PVC).

Their endocrine-disrupting mechanism is not fully clarified, but they either act as estrogen receptor agonists and antagonists, or androgen receptor antagonists. Moreover, they can also disrupt androgen synthesis.

Different studies have demonstrated a significant association with premature thelarche and precocious or early puberty [ 26, 27].

Phthalates can be classified as low- or high-molecular-weight phthalates. Depending on the class and the timing of exposure, different outcome have been observed. High-molecular-weight phthalate levels several years before puberty are associated with later pubic hair development and younger age of menarche; in contrast, low-molecular-weight phthalate levels are related to advanced breast or pubic hair development [ 28, 29].

3.7. Pyrethroids

A pyrethroid is an organic compound similar to a natural pyrethrin, which comes from the pyrethrum flower. This chemical is found in most common commercial household insecticides. It has been reported both as a weak androgen and for its association with late onset of puberty in girls [ 30, 31].

4. Puberty and EDCs: A Temporal Window for Breast Cancer

In girls, the first physical sign of puberty is thelarche, with physical breast changes in both the stroma and the epithelium. The amount of fibrous and fatty tissue in the stroma increases (the adult non-lactating breast is more than 80% composed of stroma) [ 32]. The ducts first elongate, proliferate, and form lobular structures composed of the growth ductal tree; with the achievement of sexual maturity, they decrease in size, the proliferative phase reducing [ 33]. Thus, puberty is the time in which the breast tissue undergoes rapid changes regulated by small fluctuations of endogenous hormones [ 34]. These rapid structural and cellular modifications are thought to create a "window of susceptibility" [ 35].

Breast tissue during this period of morphogenesis and re-modeling is particularly sensitive to carcinogenic effects; studies have demonstrated that exposure to radiation, DDT, medications, and disease before and during puberty increases breast cancer risk in adulthood [ 36, 37, 38, 39, 40].

The role of estrogen in breast development and tumorigenesis is confirmed. Prolonged and uninterrupted exposure to endogenous estrogen and administration of exogenous estrogens to postmenopausal women increases the risk of cancer. In contrast, gonadectomy in premenopausal women reduces the risk of breast cancer, while pharmacological agents serve to treat the same condition, blocking estrogen biosynthesis or estrogen receptors.

It is not entirely clear how human exposure to specific EDCs can be linked to breast cancer. The EDCs of interest in this mechanism are those with estrogenic activity: they are called estrogen-mimicking endocrine disruptors (EEDs).

The main effects of EEDs are two-fold: the first is their action on the proliferation of the stromal cells, the second concerns epigenetic mechanisms. EEDs affect stromal cells, interfering with the estrogen signal pathway [ 40, 41].

Moreover, EEDs may modify the breast matrix composition. All these changes macroscopically create a different breast density, which is one of the strongest predictors of premenopausal breast cancer risk [ 42].

Breast cancer is also associated with epigenetic changes such as DNA methylation, histone modifications, and non-coding RNAs (ncRNAs). These mechanisms work together to determine whether the epigenome may expresses itself and how, facilitating gene expression control, X chromosome inactivation, and/or genomic imprinting [ 43].

The most important chemical EEDs and their activities to promote or facilitate breast cancer are analyzed below ().

4.1. Atrazine

Atrazine is an herbicide used to prevent pre- and post-emergence broadleaf weeds in crops (corn and sugarcane) and turf. It can induce aromatase activity, increasing the level of estrogen in the body. Atrazine is not classified as a carcinogen or mutagen because its link with breast cancer is not totally clear. On this issue, studies are contradictory. Some studies on human reaction to atrazine exposure have seemed to detect a weak association between atrazine and breast cancer [ 44].

4.2. BPA

BPA increases the methylation levels of key genes associated with tumor development (e.g., BRCA1, CDKN2A, etc.), altering the epigenome in order to promote proliferation, senescence, and tumor development [ 45].

3D cultures of breast cells have shown that BPA reduces the number of tubules, increases spherical masses, and causes more deformed acini, indicating an ability to induce neoplastic transformation [ 46]. This because BPA exposure increases HOXB9 expression in breast cells, both in vitro and in vivo, through a mechanism that involves increased recruitment of transcription and chromatin modification factors. HOXB9 is a homeobox-containing gene that plays a key role in mammary gland development, and is associated with breast cancer. BPA acts as a competitor for estrogen receptors (ERs), which leads to the activation of this gene through chromatin modifications (histone methylation and acetylation) [ 47].

4.3. DDT/DDE

The estrogenic property of these compounds and their prominent role in mRNA regulation increase epithelial cells' proliferation index and mammary tumor cell proliferation [ 48]. Moreover, a prospective study showed that girls exposed to high levels of DDT and DDE were more likely to develop breast cancer than those who were exposed to a lower dose. On top of that, the younger the age at which a person was exposed to these compounds, the higher the risk of developing breast cancer [ 38]. These data support the hypothesis that the age of exposure must be considered for risk assessment.

4.4. DES

Diethylstilbestrol (DES) is a nonsteroidal estrogen medication that was used in the past to support during pregnancy women with a history of recurrent miscarriage, as hormone therapy for menopausal symptoms and estrogen deficiency, and as a treatment for breast cancer.

The development of breast cancer in women exposed to DES is mediated by the proliferative effects of estrogens, and by a differential expression of ncRNAs. In particular, epigenetically silenced mRNA causes disordered regulation of the p53-mediated apoptotic pathway [ 49].

4.5. Parabens

Parabens are a widely used class of preservatives in cosmetic and pharmaceutical products. They are used for their bactericidal and fungicidal properties. They are commonly found in shampoos, commercial moisturizers, topical/parenteral pharmaceuticals, suntan products, make-up, and toothpaste, and are also used as food preservatives.

They have estrogen-like effects and they may be associated with breast cancer etiology. This is demonstrated by the association between the utilization of paraben-containing body care products in the Western world and the increased incidence of breast cancer [ 50].

4.6. PCBs

Studies have demonstrated that polychlorinated biphenyls accumulate in breast adipose tissue, but so far, an association has not been proven between PCB exposure and breast cancer [ 51]. This is probably because PCBs are mixtures of congeners with varied properties, and because breast cancers of different subtypes are etiologically distinct diseases. The absence of stratified subgroup analysis on individual PCB exposure and patients with specific biological subtypes of breast cancer may have resulted in an underestimation of the correlations between PCBs and breast cancer [ 52]. Moreover, PCBs' mechanisms of action are different. In fact, the natures of the biochemical and toxic effects of PCB congeners are largely determined by their structure, as well as different PCB congeners sometimes exerting conflicting actions. Some PCBs act as estrogen agonists both in vitro and in experimental animal systems; some others have been shown to be anti-estrogenic, while others are enzyme inducers of phenobarbital (PB)-type cytochrome P450 [ 53]. The main mechanism involved in breast cancer seems to be the estrogen agonist mechanism.

4.7. PFOA

Perfluorooctatonic acid (PFOA) serves as a surfactant in the emulsion polymerization of fluoropolymers and as a building block for polymeric materials. It is used for various industrial applications (carpeting, upholstery, floor wax, firefighting foam, sealants etc.). Data show how in CD-1 and C57B1/6 mice, early PFOA exposure alters the mammary gland without changing other pubertal endpoints [ 54]. This development may increase susceptibility to carcinogenesis in future generations.

4.8. Phthalates

One characteristic of phthalates is their lipophilicity, so they accumulate in breast tissue where they cause modification of the mammary gland [ 55]. Epigenetic changes due to exposure to phthalates increase the proliferation of cells in the breast [ 56].

4.9. TCDD

2,3,7,8-Tetracloridibenzo-p-dioxin, or TCDD, is a famous organochlorine mainly known as a contaminant in Agent Orange, an herbicide used in the Vietnam War. The scientific community is aware of its catastrophic effects, including birth defects, pregnancy loss, and increased rates of cancer. In 2012, a retrospective study showed a significantly higher prevalence of breast cancers in women working in a chemical pesticide establishment due to their exposure to high levels of TCDD [ 57].

TCDD has a significant effect on the epigenome in breast and other tissues. It is interesting to note that TCDD induces an epigenetic silencing of key tumor suppressors, which may have a role in breast cancer development [ 58].

4.10. Vinclozolin

Vincozolin is a common dicarboximide fungicide used to control diseases in vineyards and on fruits and vegetables. Exposure to this pesticide in female rats causes the formation of mammary tumors and dysregulates gland development [ 59]. It promotes an epigenetic alteration in the germ line that creates a transgenerational disease state [ 60].

5. Conclusions and Perspectives

Most of these EDCs are present in everyday objects and in various common solutions; it is almost impossible not to be exposed to these chemicals, even if laws were to ban the use of most of them and restrict the others. It is known that environmental factors, including EDCs, are the most important determinant in explaining the puberty secular trend and the timing of menarche. The anticipation of these two endpoints of development can be explained by the action of environment during the pubertal period, and how it may advance and activate the central axis.

According to Parent et al., this paradigm of EDCs could be revised; in fact, environmental factors can affect puberty and reproduction through central and peripheral mechanisms, taking advantage of the critical windows of development [ 61]. During these periods, EDCs can modify genetic or epigenetic pathways.

The epigenetic pathways are interesting because of their long-term action. Studies on animals have demonstrated the effects of a mixture of EDCs on female sexual development over three generations, and that the effects are even bigger in the second and third generations (delayed vaginal opening, decreased percentage of regular estrous cycle, and decreased GnRH interpulse interval) than in the first. The reproductive phenotypes in rats are associated with alterations in both transcriptional and histone posttranslational modifications of hypothalamic genes involved in reproductive competence. Moreover, females of the first generation exposed in utero to these EDCs showed alteration of the thyroid and impaired maternal behavior, which could cause additional epigenetic change in subsequent generations [ 62].

EDCs can also act as obesogens and promote early adiposity rebound, changing metabolic or peripheral signals, and increases in adrenal androgen levels, inducing early pubertal development [ 63].

EDCs, and in particular EEDs, may produce epigenetic changes not only in puberty genes but also in other tissues and cells. In fact, these compounds could act as carcinogens and induce breast cancer. Clearly, EEDs are not the only factors responsible for these phenomena. Their effect is influenced by environmental carcinogens and unhealthy foods such as refined sugar, processed fats, and food additives, which promote molecular damage that increases the risk of breast cancer.

Puberty amplifies this process; young women today live in an obesogenic world and they are exposed to several contemporaneous risk factors, especially girls who live in disadvantaged conditions [ 64].

Moreover, the exposure to these compounds and the development of mammary glands expands the epigenetic changes that EEDs cause in breast tissue, promoting tumorigenesis and increasing the chance of damage that might prove to be significant.

In conclusion, exposure to EDCs during puberty increases the risk of developing breast cancer during the lifetime.

Although these results are still somewhat controversial, it is important for pediatricians to keep in mind the new risks for young girls during puberty, and to provide the right information to their families.

Author Contributions

Conceptualization, L.I.; methodology, L.I. and L.L. (Licia Lugli); resources, L.L. (Laura Lucaccioni), V.T.; writing-original draft preparation, L.L. (Laura Lucaccioni), V.T., L.M.; writing-review and editing, N.B., A.B., B.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare no conflicts of interest.

References

# data[].attributes.attachment_link.title
Endocrine-Disrupting Chemicals and Their Effects during Female Puberty: A Review of Current Evidence

# data[].attributes.attachment_link.description
Puberty is the process of physical changes between childhood and adulthood during which adolescents reach sexual maturity and become capable of reproduction. It is considered one of the main temporal windows of susceptibility for the influence of the endocrine-disrupting chemicals (EDCs).

# data[].attributes.headline
Carissa Perez: Exposure to PCBs, PBBs, and DDT (known EDCs) has been connected to females at a young age experiencing a menarche, while other studies have not found this connection.

# data[].attributes.subject
Carissa Perez: Exposure to PCBs, PBBs, and DDT (known EDCs) has been connected to females at a young age experiencing a menarche, while other studies have not found this connection.

# data[].attributes.attachment_link.readable_text
Elly Den Hond PhD, VITO, Department of Toxicology, Boeretang 200, B-2400 Mol, Belgium. E-mail: elly.denhond@vito.be

Summary

In this overview of the literature, epidemiological research studying the effect of endocrine disrupters on the onset of puberty is summarized. In girls, earlier age at menarche was reported after exposure to polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs), persistent pesticides [dichlorodiphenyltrichloroethane (DDT)] and phthalate esters. However, several other studies found no effect of these compounds on age at menarche or pubertal Tanner stages. One study reported a delaying effect of dioxin-like compounds on breast development. In boys, exposure to PCBs, PCDFs or the pesticide endosulfan was associated with delayed puberty or decreased penile length. Much of the results found in population studies are in accordance with experimental studies in animals. However, the mixture of different components with antagonistic effects (oestrogenic, anti-oestrogenic, anti-androgenic) and the limited knowledge about the most critical window for exposure (prenatal, peri-natal and pubertal) may hamper the interpretation of results.

Introduction

Observations in humans and wildlife over the past 40 years show a worrying trend of adverse effects on the development of the reproductive system. In birds, fish, reptiles and mammals, there is accumulating evidence of decreased fertility, reduced hatching success, and feminization as well as masculinization of different species in various areas (). In humans, decreased sperm counts, an increasing incidence of disorders such as cryptorchidism and hypospadias in children and a rising incidence of hormone-related cancers such as breast and testicular cancer have been observed (; ). Expert scientists have hypothesized that these trends may result from the effects of low levels of chemicals in the environment which interfere with or alter the body's endocrine system. These chemicals, known as endocrine disrupters, may act through different mechanisms. Certain endocrine disrupters mimic the effects of natural hormones by triggering their specific receptors, others bind to hormone receptors and block the action of the natural hormones. They can also interfere with the production, transport, metabolism or excretion of naturally occurring hormones (; ).

Puberty is a complex neuroendocrine process for which the primary mechanism is still unclear (). Adolescent growth spurt is driven by the continuing influence of growth hormones from the prepubertal period, with the additional influence of sex steroids in adolescence. During puberty, there is a rapid growth in height and muscular size, with increasing pulmonary volumes. Other important changes are the development of primary and secondary sexual characteristics such as growth of pubic hair, and an increase in breast and testis size ( Karlberg, 2002). In this short overview of the literature, we specifically looked at the effect of endocrine disrupters on the secondary sexual characteristics. We selected epidemiological studies that studied age at menarche in girls, testicular volume increase in boys and/or pubertal Tanner stages ( Marshall & Tanner, 1969, 1970) in either of the sexes.

Epidemiological research in girls

Table 1 gives an overview of the epidemiological studies in girls.

Reference

Study area

Pollutant

Exposure

Subjects

Findings associated with higher exposure

One rural and two urban villages in Belgium

PCBs Dioxin (Calux ®)

Pubertal

120 girls

High serum dioxin was associated with retarded breast development Dioxin did not effect menarche or pubic hair stage PCBs had no effect on menarche or pubertal stages

PBBs, polybrominated biphenyls; PCBs, polychlorinated biphenyls; DDE, dichlorodiphenyldichloroethylene (=metabolite of DDT); NHANES, National Health and Nutrition Examination Survey; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Accidental exposure to polybrominated biphenyl (PBB) in Michigan food cohort

Some of the epidemiological research on endocrine disrupters is performed in areas with accidental exposure to a specific chemical compound. In 1973, accidental contamination of the Michigan food chain with PBB led to the exposure of more than 4000 individuals (). In utero PBB exposure of babies born after the accident was estimated from maternal serum PBB measurements taken in the years after the pregnancy by means of a maternal decay rate model. In 1997, 26 years after the accident, mothers from the registry and their female offspring aged 5-24 years were contacted by mailing to provide information on the breast-feeding duration, on pubertal development (self-reporting of age at menarche and Tanner stage) and possible confounders. The mean PBB level was 17.3 ppb (range: 0-1142 ppb); the mean polychlorinated biphenyl (PCB) level was 5.6 ppb (range: 0-78 ppb). Breast-fed daughters of mothers with a high estimated serum PBB level (≥7 ppb) were found to have an earlier menarche (11.6 years, n = 16) than non-breastfed girls with low (<1 ppb) exposure (12.5 years, n  = 61). The hazard ratio was 3.4 (95% CI: 1.3-9.0). Further, a high peri-natal PBB exposure was associated with earlier pubic hair stage in breast-fed girls. Age at menarche and Tanner stage had little association with PCB exposure. The main congener in the Michigan accident (PBB153) has been described as oestrogenic, which may explain the early menarche related to high exposure. However, it is somewhat surprising that breast development was unaltered. Possibly, retrospective self-reporting is more reliable for age at menarche than it is for breast development. Pubic hair development, which is under control of the adrenal gland, was accelerated. There is limited evidence that the adrenal gland is a target of PBBs.

Accidental exposure to dioxin in Seveso

In 1976, as a result of a chemical explosion, residents of Seveso (Italy) experienced the highest levels of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), the most toxic congener of dioxin, in a human population. In a retrospective analysis (), 282 women who were pre-menarchal at the time of the explosion (mean age: 6.9 years, range: 0-17 years) were interviewed by a trained nurse to determine age at menarche retrospectively. TCDD was measured in archived sera. The median serum TCDD level was 140.3 ppt (range: 3.6-56 000 ppt). The hazard ratio associated with a 10-fold increase in TCDD was 0.95 (95% CI: 0.83-1.09), meaning that there was no significant change in the onset of menarche. The conclusions were similar if TCDD was considered as a categorical variable or if the analysis was restricted to women <8 years of age at the time of explosion. The authors hypothesized that the postnatal but prepubertal TCDD exposure in this cohort, although substantial in dose, likely missed the critical window for exposure effects.

The Michigan anglers cohort: exposure to PCBs and dichlorodiphenyltrichloroethane (DDT)

The Great Lakes (MI, USA) have been polluted with industrial wastes since the beginning of the 20th century, mainly by PCBs and DDT. These compounds have bioaccumulated in the marine food chain and therefore, anglers and fish eaters were considered to be at high risk for potential health effects. In 2004, results were reported from a group of 151 girls born between 1950 and 1980 from mothers belonging to the Michigan anglers cohort (). Maternal serum PCB and dichlorodiphenyldichloroethylene (DDE) concentrations at the time of the pregnancy were backward extrapolated from repeated maternal serum measurements between 1973 and 1991. The daughter's age at menarche and possible confounders were acquired by telephone interviews. Menarche was reached between the age of 9 and 17 years. In multivariate analysis, DDE ( p = 0.04) but not PCB ( p = 0.08) lowered age at menarche. An increase in the in utero DDE exposure of 15  μ g/L reduced age at menarche by 1 year. Possible mechanisms for the effect of DDE include androgen blocking, an oestrogen-mimicking effect or induction of aromatase, an enzyme that catalyses the conversion of C19 steroids (e.g. testosterone) to oestrogens.

'Environment and Health' Biomonitoring Study in Belgium: exposure to PCBs and dioxin

In 1999, 120 Belgian girls with a mean age of 17.4 years living either in a rural control area, an urban suburb hosting two waste incinerators, and another urban suburb in the vicinity of a primary non-ferrous smelter, were enrolled in a biomonitoring study (; ). Exposure to endocrine disrupters was estimated by the sum of PCB congeners 138, 153 and 180 in serum, and by the Calux ® (BioDetection System, Amsterdam, The Netherlands) assay, a bio-assay measuring the sum of all dioxin-like compounds in serum. Four trained school physicians recorded age at menarche and staged sexual maturation according to Tanner. Mean values of the Calux ® assay were significantly higher in both suburbs [0.17 (95% CI: 0.13-0.22) and 0.21 (0.17-0.26) ngTEQ/L] than in the rural area [0.11 (0.09-0.13) ngTEQ/L]. In one suburb near the two waste incinerators, significantly less girls (67%) had reached the adult stage of breast development compared with girls in the rural area (90%) or in the other urban area (79%). Moreover, at the individual level, breast development was inversely related to dioxin exposure: a doubling of the serum dioxin concentration increased the odds of not having reached the adult stage of breast development by 2.3 ( p = 0.02). Pubic hair growth and age at menarche were not related to PCB or dioxin exposure. The Calux ® assay is sensitive to a range of compounds activating the Aryl hydrocarbon (Ah) receptor, including dioxins and coplanar and mono-ortho-PCBs, which all may have anti-oestrogenic activity. It is therefore plausible that breast development in girls, which is an oestrogenic effect, may be retarded by dioxin-like compounds detected by the calux assay.

Immigrant study in Belgium: exposure to DDT

The patient study of Krstevska-Konstantinova et al. (2001) is conceptually different from epidemiological population studies that attempt to construct dose-effect relationships but the study equally investigates the role of endocrine disrupters in the development of puberty in girls. Based on patient files and adoption registries in Belgium, the prevalence of precocious puberty was found to be 80-fold higher in foreign children than in Belgian natives. This was without evidence of particular ethnic background or a history of delayed growth or development because of malnutrition or infectious diseases. In a group of patients with precocious puberty, serum DDE concentrations were significantly higher in foreign adopted (1.20 ng/mL; n = 15) or foreign non-adopted girls (1.04 ng/mL; n = 11) than in Belgian native girls (0.13 ng/mL; n  = 15). The authors hypothesized that migration may interrupt exposure to endocrine disrupters. Precocious puberty then could result indirectly from withdrawal of the negative feedback effect of the sex steroids and their environmental analogs and/or directly from accelerated hypothalamic maturation caused by sex steroids. However, as no native girls with precocious puberty served as controls, the associations found in the study remain speculative.

Case-control study of thelarche patients in Puerto Rico: exposure to pesticides and phthalates

Most of the epidemiological research studying the relationship between endocrine disrupting chemicals and pubertal development has focused on persistent compounds in the environment, i.e. PCBs, PBBs, dioxins, DDE and other persistent pesticides. However, recent attention is also focusing on indoor pollutants such as phthalates, which are present in food packaging materials and polyvinyl chloride plastics such as soft plastic toys. Since 1979, paediatric endocrinologists in Puerto Rico have detected an alarming increase in the number of patients with premature thelarche (). Therefore, in a case-control study including 41 thelarche patients and 35 controls, serum samples were screened for a number of endocrine-disrupting chemicals such as pesticides and their metabolites, and several compounds of the phthalate ester family. Detectable levels of phthalate esters were observed in 68% of the serum samples of thelarche patients compared with 14% of the controls. Moreover, the total concentrations of phthalates and of some individual phthalate esters such as DEHP were significantly higher in the patients' samples than in the control samples. According to the authors, these findings could be explained by the oestrogenic and anti-androgenic properties of phthalate esters that have been reported in vitro. However, the findings of were criticized (). As the phthalate levels in the Puerto Rican girls were high in comparison with other studies, the question was raised whether there may have been artificial contamination of the blood samples from vinyl laboratory equipment and tubing. Furthermore, in the human body, phthalate esters are rapidly metabolized to monoesters but these metabolites are not active under in vitro conditions.

National Health and Nutrition Examination Survey (NHANES III): environmental lead exposure

Environmental lead is generally not recognized as endocrine disrupter. Yet, in two recent studies, both using the same NHANES database, higher blood lead concentrations were associated with delays in self-assessed age at menarche and physician determined pubic hair and breast development (; ). The slight variations in the conclusions of the two studies could be explained by different selections of age and race classes. These results indicate that lead and possibly also other heavy metals such as cadmium, may confound the results of studies investigating the effect of pesticides, PCBs or dioxins, as they often occur in the same environment, e.g. in the neighbourhood of landfills, waste incinerators, non-ferro industry, etc.

Summary and remarks in girls

Epidemiological research on the effects of endocrine disrupters on sexual maturation is hampered by numerous pitfalls. One major problem is the mixture of different agents with potential oestrogenic, anti-oestrogenic and anti-androgenic activities in the environment. Another important problem is the limited knowledge about the time lag between exposure and effect. For most effects, the critical window of exposure has not yet been identified, so that it is not always clear whether to look at in utero, lactational, pubertal or life-long exposure. Additionally, epidemiological research in general may be influenced by many factors such as selection of the study area, sample size, adjustment for confounders, choice of the endpoints, etc.

The DDT, DDE, several PCB congeners, PBB congeners and phthalate esters have been described as oestrogenic or anti-androgenic agents in animal studies (; Hansen, 1998). Some of the findings reported in population studies are in line with this theory: earlier age at menarche was associated with higher prenatal PBB exposure in the Michigan food cohort (); with higher prenatal DDE exposure in offspring of the Michigan fish eaters (); with higher pubertal DDE exposure in immigrants moving to Belgium ( Krstevska-Konstantinova et al., 2001) and with higher pubertal phthalate esters in Puerto Rico (). However, age at menarche was not associated with DDE in the North Carolina Cohort () or with PCBs in any of the studies (; ; ). One of the reasons for the negative results on PCBs, is that often total PCB concentrations in serum are measured, while various PCB congeners may have different, sometimes antagonistic, effects ( Hansen, 1998). None of the studies that assessed breast development by Tanner stage, found a significant association with PBBs (), PCBs (; ; ) or DDE ().

Dioxins and dioxin-like PCBs have been described as anti-oestrogenic in animals (). The findings of retarded breast development in adolescents with higher dioxin-like compounds in serum () are in line with this theory. However, in the same population, no effect on age at menarche was observed. Postnatal but prepubertal TCDD exposure had no significant effect on age at menarche in the population of Seveso ().

Epidemiological research in boys

Table 2 summarizes the main results of epidemiological studies in boys.

Reference

Study area

Pollutant

Exposure

Subjects

Findings associated with higher exposure

One rural and two urban villages in Belgium

PCBs Dioxin (Calux ®)

Pubertal

80 boys (mean age 17.3 years)

Negative association between serum PCBs and pubertal stages (genital + pubic hair) No effect of dioxin on pubertal stages Testicular volume was lower in areas with higher PCB exposure but there was no association at the individual level

DDE, dichlorodiphenyldichloroethylene (=metabolite of DDT); PCBs, polychlorinated biphenyls; PCDFs, polychlorinated dibenzofurans.

'Environment and Health' Biomonitoring Study in Belgium: exposure to PCBs and dioxin

In the Flemish 'Environment and Health' study, 80 boys with a mean age of 17.3 years were included. Both genital development and pubic hair growth (determined by a physician) were at a lower stage in the area hosting two waste incinerators compared with the rural control area or the urban village near the lead smelter. A doubling of the serum concentration of PCB congener 138 increased the odds of not having matured into the adult stage of genital development by 3.5 ( p = 0.04); similarly for PCB congener 153 in relation to male pubic hair growth, the odds ratio was 3.5 ( p = 0.04). Respective odds ratios for the sum of marker PCB 138, 153 and 180 were 3.8 ( p = 0.06) and 2.7 ( p = 0.06). Testicular volume was significantly lower in boys of both polluted suburbs compared with boys of the control area, but was not related to pubertal exposure of PCBs or dioxin (measured by the Calux ® assay). Both PCB congeners 153 and 138 have been described as oestrogenic agents. Therefore, an inverse correlation with genital development and pubic hair growth, which both are androgenic effects, is in line with the expected outcome. It was speculated that testicular volume may be more closely associated with maternal exposure to endocrine disrupters during pregnancy, as testicular volume is determined by the number of Sertoli cell which are fixed prenatally and xeno-oestrogens may inhibit the proliferation of Sertoli cells.

Summary and remarks in boys

Compared with girls, the number of studies studying the effect of endocrine disrupters on pubertal development in boys are much more limited. Research in boys is more focused on semen quality, which is much more closely related to fertility. The findings on delayed puberty in boys exposed to PCBs, PCDFs, and endosulfan are in line with the general concept that these compounds work as xeno-oestrogens.

Summary

In this overview of the literature, epidemiological research studying the effect of endocrine disrupters on the onset of puberty is summarized. In girls, earlier age at menarche was reported after exposure to polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs), persistent pesticides [dichlorodiphenyltrichloroethane (DDT)] and phthalate esters. However, several other studies found no effect of these compounds on age at menarche or pubertal Tanner stages. One study reported a delaying effect of dioxin-like compounds on breast development. In boys, exposure to PCBs, PCDFs or the pesticide endosulfan was associated with delayed puberty or decreased penile length. Much of the results found in population studies are in accordance with experimental studies in animals. However, the mixture of different components with antagonistic effects (oestrogenic, anti-oestrogenic, anti-androgenic) and the limited knowledge about the most critical window for exposure (prenatal, peri-natal and pubertal) may hamper the interpretation of results.

Introduction

Observations in humans and wildlife over the past 40 years show a worrying trend of adverse effects on the development of the reproductive system. In birds, fish, reptiles and mammals, there is accumulating evidence of decreased fertility, reduced hatching success, and feminization as well as masculinization of different species in various areas (). In humans, decreased sperm counts, an increasing incidence of disorders such as cryptorchidism and hypospadias in children and a rising incidence of hormone-related cancers such as breast and testicular cancer have been observed (; ). Expert scientists have hypothesized that these trends may result from the effects of low levels of chemicals in the environment which interfere with or alter the body's endocrine system. These chemicals, known as endocrine disrupters, may act through different mechanisms. Certain endocrine disrupters mimic the effects of natural hormones by triggering their specific receptors, others bind to hormone receptors and block the action of the natural hormones. They can also interfere with the production, transport, metabolism or excretion of naturally occurring hormones (; ).

Puberty is a complex neuroendocrine process for which the primary mechanism is still unclear (). Adolescent growth spurt is driven by the continuing influence of growth hormones from the prepubertal period, with the additional influence of sex steroids in adolescence. During puberty, there is a rapid growth in height and muscular size, with increasing pulmonary volumes. Other important changes are the development of primary and secondary sexual characteristics such as growth of pubic hair, and an increase in breast and testis size ( Karlberg, 2002). In this short overview of the literature, we specifically looked at the effect of endocrine disrupters on the secondary sexual characteristics. We selected epidemiological studies that studied age at menarche in girls, testicular volume increase in boys and/or pubertal Tanner stages ( Marshall & Tanner, 1969, 1970) in either of the sexes.

Epidemiological research in girls

Table 1 gives an overview of the epidemiological studies in girls.

Reference

Study area

Pollutant

Exposure

Subjects

Findings associated with higher exposure

One rural and two urban villages in Belgium

PCBs Dioxin (Calux ®)

Pubertal

120 girls

High serum dioxin was associated with retarded breast development Dioxin did not effect menarche or pubic hair stage PCBs had no effect on menarche or pubertal stages

PBBs, polybrominated biphenyls; PCBs, polychlorinated biphenyls; DDE, dichlorodiphenyldichloroethylene (=metabolite of DDT); NHANES, National Health and Nutrition Examination Survey; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Accidental exposure to polybrominated biphenyl (PBB) in Michigan food cohort

Some of the epidemiological research on endocrine disrupters is performed in areas with accidental exposure to a specific chemical compound. In 1973, accidental contamination of the Michigan food chain with PBB led to the exposure of more than 4000 individuals (). In utero PBB exposure of babies born after the accident was estimated from maternal serum PBB measurements taken in the years after the pregnancy by means of a maternal decay rate model. In 1997, 26 years after the accident, mothers from the registry and their female offspring aged 5-24 years were contacted by mailing to provide information on the breast-feeding duration, on pubertal development (self-reporting of age at menarche and Tanner stage) and possible confounders. The mean PBB level was 17.3 ppb (range: 0-1142 ppb); the mean polychlorinated biphenyl (PCB) level was 5.6 ppb (range: 0-78 ppb). Breast-fed daughters of mothers with a high estimated serum PBB level (≥7 ppb) were found to have an earlier menarche (11.6 years, n = 16) than non-breastfed girls with low (<1 ppb) exposure (12.5 years, n  = 61). The hazard ratio was 3.4 (95% CI: 1.3-9.0). Further, a high peri-natal PBB exposure was associated with earlier pubic hair stage in breast-fed girls. Age at menarche and Tanner stage had little association with PCB exposure. The main congener in the Michigan accident (PBB153) has been described as oestrogenic, which may explain the early menarche related to high exposure. However, it is somewhat surprising that breast development was unaltered. Possibly, retrospective self-reporting is more reliable for age at menarche than it is for breast development. Pubic hair development, which is under control of the adrenal gland, was accelerated. There is limited evidence that the adrenal gland is a target of PBBs.

Accidental exposure to dioxin in Seveso

In 1976, as a result of a chemical explosion, residents of Seveso (Italy) experienced the highest levels of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), the most toxic congener of dioxin, in a human population. In a retrospective analysis (), 282 women who were pre-menarchal at the time of the explosion (mean age: 6.9 years, range: 0-17 years) were interviewed by a trained nurse to determine age at menarche retrospectively. TCDD was measured in archived sera. The median serum TCDD level was 140.3 ppt (range: 3.6-56 000 ppt). The hazard ratio associated with a 10-fold increase in TCDD was 0.95 (95% CI: 0.83-1.09), meaning that there was no significant change in the onset of menarche. The conclusions were similar if TCDD was considered as a categorical variable or if the analysis was restricted to women <8 years of age at the time of explosion. The authors hypothesized that the postnatal but prepubertal TCDD exposure in this cohort, although substantial in dose, likely missed the critical window for exposure effects.

The Michigan anglers cohort: exposure to PCBs and dichlorodiphenyltrichloroethane (DDT)

The Great Lakes (MI, USA) have been polluted with industrial wastes since the beginning of the 20th century, mainly by PCBs and DDT. These compounds have bioaccumulated in the marine food chain and therefore, anglers and fish eaters were considered to be at high risk for potential health effects. In 2004, results were reported from a group of 151 girls born between 1950 and 1980 from mothers belonging to the Michigan anglers cohort (). Maternal serum PCB and dichlorodiphenyldichloroethylene (DDE) concentrations at the time of the pregnancy were backward extrapolated from repeated maternal serum measurements between 1973 and 1991. The daughter's age at menarche and possible confounders were acquired by telephone interviews. Menarche was reached between the age of 9 and 17 years. In multivariate analysis, DDE ( p = 0.04) but not PCB ( p = 0.08) lowered age at menarche. An increase in the in utero DDE exposure of 15  μ g/L reduced age at menarche by 1 year. Possible mechanisms for the effect of DDE include androgen blocking, an oestrogen-mimicking effect or induction of aromatase, an enzyme that catalyses the conversion of C19 steroids (e.g. testosterone) to oestrogens.

'Environment and Health' Biomonitoring Study in Belgium: exposure to PCBs and dioxin

In 1999, 120 Belgian girls with a mean age of 17.4 years living either in a rural control area, an urban suburb hosting two waste incinerators, and another urban suburb in the vicinity of a primary non-ferrous smelter, were enrolled in a biomonitoring study (; ). Exposure to endocrine disrupters was estimated by the sum of PCB congeners 138, 153 and 180 in serum, and by the Calux ® (BioDetection System, Amsterdam, The Netherlands) assay, a bio-assay measuring the sum of all dioxin-like compounds in serum. Four trained school physicians recorded age at menarche and staged sexual maturation according to Tanner. Mean values of the Calux ® assay were significantly higher in both suburbs [0.17 (95% CI: 0.13-0.22) and 0.21 (0.17-0.26) ngTEQ/L] than in the rural area [0.11 (0.09-0.13) ngTEQ/L]. In one suburb near the two waste incinerators, significantly less girls (67%) had reached the adult stage of breast development compared with girls in the rural area (90%) or in the other urban area (79%). Moreover, at the individual level, breast development was inversely related to dioxin exposure: a doubling of the serum dioxin concentration increased the odds of not having reached the adult stage of breast development by 2.3 ( p = 0.02). Pubic hair growth and age at menarche were not related to PCB or dioxin exposure. The Calux ® assay is sensitive to a range of compounds activating the Aryl hydrocarbon (Ah) receptor, including dioxins and coplanar and mono-ortho-PCBs, which all may have anti-oestrogenic activity. It is therefore plausible that breast development in girls, which is an oestrogenic effect, may be retarded by dioxin-like compounds detected by the calux assay.

Immigrant study in Belgium: exposure to DDT

The patient study of Krstevska-Konstantinova et al. (2001) is conceptually different from epidemiological population studies that attempt to construct dose-effect relationships but the study equally investigates the role of endocrine disrupters in the development of puberty in girls. Based on patient files and adoption registries in Belgium, the prevalence of precocious puberty was found to be 80-fold higher in foreign children than in Belgian natives. This was without evidence of particular ethnic background or a history of delayed growth or development because of malnutrition or infectious diseases. In a group of patients with precocious puberty, serum DDE concentrations were significantly higher in foreign adopted (1.20 ng/mL; n = 15) or foreign non-adopted girls (1.04 ng/mL; n = 11) than in Belgian native girls (0.13 ng/mL; n  = 15). The authors hypothesized that migration may interrupt exposure to endocrine disrupters. Precocious puberty then could result indirectly from withdrawal of the negative feedback effect of the sex steroids and their environmental analogs and/or directly from accelerated hypothalamic maturation caused by sex steroids. However, as no native girls with precocious puberty served as controls, the associations found in the study remain speculative.

Case-control study of thelarche patients in Puerto Rico: exposure to pesticides and phthalates

Most of the epidemiological research studying the relationship between endocrine disrupting chemicals and pubertal development has focused on persistent compounds in the environment, i.e. PCBs, PBBs, dioxins, DDE and other persistent pesticides. However, recent attention is also focusing on indoor pollutants such as phthalates, which are present in food packaging materials and polyvinyl chloride plastics such as soft plastic toys. Since 1979, paediatric endocrinologists in Puerto Rico have detected an alarming increase in the number of patients with premature thelarche (). Therefore, in a case-control study including 41 thelarche patients and 35 controls, serum samples were screened for a number of endocrine-disrupting chemicals such as pesticides and their metabolites, and several compounds of the phthalate ester family. Detectable levels of phthalate esters were observed in 68% of the serum samples of thelarche patients compared with 14% of the controls. Moreover, the total concentrations of phthalates and of some individual phthalate esters such as DEHP were significantly higher in the patients' samples than in the control samples. According to the authors, these findings could be explained by the oestrogenic and anti-androgenic properties of phthalate esters that have been reported in vitro. However, the findings of were criticized (). As the phthalate levels in the Puerto Rican girls were high in comparison with other studies, the question was raised whether there may have been artificial contamination of the blood samples from vinyl laboratory equipment and tubing. Furthermore, in the human body, phthalate esters are rapidly metabolized to monoesters but these metabolites are not active under in vitro conditions.

National Health and Nutrition Examination Survey (NHANES III): environmental lead exposure

Environmental lead is generally not recognized as endocrine disrupter. Yet, in two recent studies, both using the same NHANES database, higher blood lead concentrations were associated with delays in self-assessed age at menarche and physician determined pubic hair and breast development (; ). The slight variations in the conclusions of the two studies could be explained by different selections of age and race classes. These results indicate that lead and possibly also other heavy metals such as cadmium, may confound the results of studies investigating the effect of pesticides, PCBs or dioxins, as they often occur in the same environment, e.g. in the neighbourhood of landfills, waste incinerators, non-ferro industry, etc.

Summary and remarks in girls

Epidemiological research on the effects of endocrine disrupters on sexual maturation is hampered by numerous pitfalls. One major problem is the mixture of different agents with potential oestrogenic, anti-oestrogenic and anti-androgenic activities in the environment. Another important problem is the limited knowledge about the time lag between exposure and effect. For most effects, the critical window of exposure has not yet been identified, so that it is not always clear whether to look at in utero, lactational, pubertal or life-long exposure. Additionally, epidemiological research in general may be influenced by many factors such as selection of the study area, sample size, adjustment for confounders, choice of the endpoints, etc.

The DDT, DDE, several PCB congeners, PBB congeners and phthalate esters have been described as oestrogenic or anti-androgenic agents in animal studies (; Hansen, 1998). Some of the findings reported in population studies are in line with this theory: earlier age at menarche was associated with higher prenatal PBB exposure in the Michigan food cohort (); with higher prenatal DDE exposure in offspring of the Michigan fish eaters (); with higher pubertal DDE exposure in immigrants moving to Belgium ( Krstevska-Konstantinova et al., 2001) and with higher pubertal phthalate esters in Puerto Rico (). However, age at menarche was not associated with DDE in the North Carolina Cohort () or with PCBs in any of the studies (; ; ). One of the reasons for the negative results on PCBs, is that often total PCB concentrations in serum are measured, while various PCB congeners may have different, sometimes antagonistic, effects ( Hansen, 1998). None of the studies that assessed breast development by Tanner stage, found a significant association with PBBs (), PCBs (; ; ) or DDE ().

Dioxins and dioxin-like PCBs have been described as anti-oestrogenic in animals (). The findings of retarded breast development in adolescents with higher dioxin-like compounds in serum () are in line with this theory. However, in the same population, no effect on age at menarche was observed. Postnatal but prepubertal TCDD exposure had no significant effect on age at menarche in the population of Seveso ().

Epidemiological research in boys

Table 2 summarizes the main results of epidemiological studies in boys.

Reference

Study area

Pollutant

Exposure

Subjects

Findings associated with higher exposure

One rural and two urban villages in Belgium

PCBs Dioxin (Calux ®)

Pubertal

80 boys (mean age 17.3 years)

Negative association between serum PCBs and pubertal stages (genital + pubic hair) No effect of dioxin on pubertal stages Testicular volume was lower in areas with higher PCB exposure but there was no association at the individual level

DDE, dichlorodiphenyldichloroethylene (=metabolite of DDT); PCBs, polychlorinated biphenyls; PCDFs, polychlorinated dibenzofurans.

'Environment and Health' Biomonitoring Study in Belgium: exposure to PCBs and dioxin

In the Flemish 'Environment and Health' study, 80 boys with a mean age of 17.3 years were included. Both genital development and pubic hair growth (determined by a physician) were at a lower stage in the area hosting two waste incinerators compared with the rural control area or the urban village near the lead smelter. A doubling of the serum concentration of PCB congener 138 increased the odds of not having matured into the adult stage of genital development by 3.5 ( p = 0.04); similarly for PCB congener 153 in relation to male pubic hair growth, the odds ratio was 3.5 ( p = 0.04). Respective odds ratios for the sum of marker PCB 138, 153 and 180 were 3.8 ( p = 0.06) and 2.7 ( p = 0.06). Testicular volume was significantly lower in boys of both polluted suburbs compared with boys of the control area, but was not related to pubertal exposure of PCBs or dioxin (measured by the Calux ® assay). Both PCB congeners 153 and 138 have been described as oestrogenic agents. Therefore, an inverse correlation with genital development and pubic hair growth, which both are androgenic effects, is in line with the expected outcome. It was speculated that testicular volume may be more closely associated with maternal exposure to endocrine disrupters during pregnancy, as testicular volume is determined by the number of Sertoli cell which are fixed prenatally and xeno-oestrogens may inhibit the proliferation of Sertoli cells.

Summary and remarks in boys

Compared with girls, the number of studies studying the effect of endocrine disrupters on pubertal development in boys are much more limited. Research in boys is more focused on semen quality, which is much more closely related to fertility. The findings on delayed puberty in boys exposed to PCBs, PCDFs, and endosulfan are in line with the general concept that these compounds work as xeno-oestrogens.

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Elly Den Hond PhD, VITO, Department of Toxicology, Boeretang 200, B-2400 Mol, Belgium. E-mail: elly.denhond@vito.be In this overview of the literature, epidemiological research studying the effect of endocrine disrupters on the onset of puberty is summarized. In girls, earlier age at menarche was reported after exposure to polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs), persistent pesticides [dichlorodiphenyltrichloroethane (DDT)] and phthalate esters.

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Rania Kraus: Early exposure to a variety of EDCs results in the early onset of puberty in girls, and often linked with increased chances of cancer later in life.

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Rania Kraus: Early exposure to a variety of EDCs results in the early onset of puberty in girls, and often linked with increased chances of cancer later in life.

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3065309/

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INTRODUCTION

Recent studies have demonstrated a progressive decrease in age of onset of puberty in children around the world. Although the exact reason for this shift is not completely understood, it is generally accepted to be the outcome of a complex interaction between genetic, endocrine and environmental factors. It is also known that owing to the acceleration of industrialization throughout the world, a gradual but significant increase has occurred in the number and amount of environmental pollutants. Some of these environmental pollutants are natural or synthetic chemicals with considerable effects on the endocrine system. The chemicals that have negative effects on the endocrine system are called endocrine disruptors (EDs). EDs exert their effects through different mechanisms: by binding to the relevant hormone receptors; by direct action on cell signaling pathways or on the central nervous system and the neuroendocrine system; by suppression of hormone synthesis; or through their toxic effects on the relevant organs. Several EDs such as phytoestrogens, topical and natural estrogens, pesticides, industrial chemicals and phthalates have been identified as possible agents affecting pubertal development in humans. The potential of EDs to cause precocious puberty has first been noticed in the early 1990s. Afterwards, the effects of EDs on the onset and course of puberty have been demonstrated in numerous animal and human studies and, subsequently, the use of some of these chemicals has even been prohibited. This paper aims to review the present information regarding environmental EDs that may negatively affect pubertal development.
The Effects of Endocrine Disruptors on Puberty
Recent studies have demonstrated that age of onset of puberty has shifted to younger ages by about 1 or 2 years, while no change has been noted in the age of menarche. The triggering mechanism for the earlier onset of puberty is not clearly understood. It is thought to occur as a result of a complex interaction between genetic, hormonal and environmental factors. Recently, ED chemicals have been intensively accused of being potential hazardous environmental factors ( 1, 2, 3, 4, 5, 6).

EDs are environmental chemicals, which may either be natural or synthetic. Some of the major EDs are presented in . EDs accumulate in the environment in the long term and are introduced into the human body through water, air, foodstuffs, or through equipments used in the office and home. Additionally, it has been demonstrated that EDs can be transferred from the mother to the fetus via placenta or to the baby via breast milk ( 1, 2, 3, 4, 5).

Due to their hormone-like characteristics, EDs mostly affect the endocrine system in an agonist- or antagonist-specific manner and can be classified according to their mechanisms of action. They influence puberty through their estrogenic, antiestrogenic, androgenic, antiandrogenic effects or through their direct effects on the gonadotropin-releasing hormone (GnRH). These chemicals may exert their estrogenic effects either directly by binding to estrogen receptors, increasing aromatase activity and increasing estrogen sensitivity or indirectly by their effect on GnRH, leading to an increase in endogenous estrogen production. All of these effects may result in precocious puberty. EDs produce antiestrogenic and androgenic effects through inhibition of aromatase enzyme activity and steroidogenic enzyme production. They display antiandrogenic effects via suppression of testicular steroidogenesis and androgen-receptor blockade. Thus, depending on their mechanism of action, EDs may lead to precocious puberty, to delayed puberty, or to sexual differentiation disorders ( 1, 4, 5, 6, 7, 8, 9, 10, 11). Classification of some of the EDs according to their mechanism of action is presented in .
Natural Endocrine Disruptors
The best known chemicals in this group are phytoestrogens, which are relatively weak compared to endogenous estrogen. They are found in several nutrients that are frequently consumed in daily life (i.e. carrots, garlic, apple, coffee, cherry, parsley, legumes). Phytoestrogens have estrogenic effects when consumed in huge amounts and antiestrogenic effects at low concentrations ( 4, 7, 8).
Synthetic Endocrine Disruptors
Diethylstilbestrol (DES) is the best known ED with strong estrogenic activity. It was first synthesized in 1938 and has since been widely used worldwide for medical zindications including pregnancy toxemia and preterm labor. However, a twofold increase in breast cancer incidence was observed in mothers exposed to DES. It was also shown that the incidence of cervical cancer, ovarian germ cell cancer, cervical or vaginal dysplasia, and vaginal clear-cell adenocarcinoma was increased in female infants born to mothers exposed to DES ( 12). The production or marketing of this chemical is prohibited since 1997.

Many chemicals including pesticides, fungicides, herbicides h ₂o used in agriculture, cleaning substances used in daily life, contents of cosmetic products, dyes, plastic substances and solvents are likely to be EDs. As neutralization or inactivation is difficult and most of these substances often accumulate in fat tissue, they may persist in the body for long periods of time and cause harmful effects ( 4, 5, 6, 7, 8, 9, 10, 11). Time of exposure to EDs is important in terms of their detrimental effects. Male rodents exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in intrauterine life were found to experience problems in masculinization of internal and external genitalia, descent of testicles, androgen production and in spermatogenesis. On the other hand, it was observed that postnatal exposure to TCDD was associated only with impairment of spermatogenesis and somatic and genital growth. The dose and duration of exposure to EDs is also important in terms of potential negative consequences. The negative effects may become more serious as the duration of exposure and dose increase ( 1, 11, 13).

EDs do not necessarily lead to similar effects in all situations. For example, phytoestrogens have estrogenic effects in high doses, while they exert antiestrogenic effects in low doses ( 1, 7).
Studies on Role of EDs in Precocious Puberty
Several animal and human studies have been conducted investigating the role of EDs in precocious puberty. EDs may cause early puberty via their estrogenic or antiandrogenic effects and also by increasing GnRH production ( 1, 4, 5, 6, 7, 8, 9, 10, 11).
Animal Studies
EDs have first been noted in experimental studies and gained significance through evidence from animal experience. Following the accidental spill of high amounts of dichlorodiphenyltrichloroethane (DDT) and dicofol from a chemical company into Lake Apopka in Florida in 1980s, it has been noted that male alligators living in the lake had smaller phalluses, reduced serum testosterone levels and abnormal gonadal structures concurrently with high serum DDT levels ( 14). It was observed that administration of methoxychlor, a pesticide with potential estrogenic effects, in female rats during pregnancy and lactation periods, led to vaginal opening and appearance of puberty at a very early age in their female offspring ( 15). Similarly, accelerated and early vaginal opening was noted in female rats following subcutaneous injections of bisphenol A (BPA), genistein, resvetarol, zearalenone or DES in the prenatal period, findings confirming the estrogenic effects of these chemicals ( 16, 17). Administration of methoxychlor or 17 beta-estradiol after the cessation of breastfeeding also resulted in early vaginal opening in female rats ( 15, 17). In addition, Markey et al ( 18) showed that prenatal exposure to BPA caused early growth and proliferation of breast tissue as well as increased susceptibility to endogenous estrogen in female rats. Methoxychlor, an antiandrogenic and estrogenic agent, and also vinclozolin, an antiandrogenic agent, were shown to lead to delayed puberty in male rats and precocious puberty in female rats ( 19).

Dioxins are environmentally toxic chemicals. TCDD is a type of dioxin, which exerts its estrogenic or antiandrogenic effects by causing alterations in gene expression via binding to aryl hydrocarbon receptors. In utero, exposure of rodents to even low doses of this substance was found to result in reproductive system abnormalities and precocious puberty findings in the females and delayed puberty in the males ( 7, 8, 9, 10, 11, 20, 21, 22). Other chemicals that can produce the same effects by binding to aryl hydrocarbon receptors include industrial products such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ether and the pesticide methoprene ( 1, 7, 8, 9, 10, 11).

Some of the environmental chemicals may impair neuroendocrine functions through their effect on the central nervous system and the hypothalamic-hypophyseal-gonadal (HHG) axis. These include pesticides such as thiram, molinate, metam sodium, chlordimeform, amitraz, triazoles, dichloroacetic acid, atrazine, propazine, simazine, methanol and linuron ( 1). It has been shown in a study on rats that atrazine causes delayed puberty by suppressing luteinizing hormone (LH) and prolactin levels ( 23, 24, 25). In rats prenatally exposed to BPA, an increase in estrogen feedback as well as development of precocious puberty via inhibition of tyrosine hydroxylase activity in rostral preoptic periventricular neurons have been observed ( 26). Howdeshell et al ( 27) also noted precocious puberty in rats prenatally exposed to BPA.

Some of the EDs affect puberty by inhibiting the synthesis of endogenous hormones such as testosterone, 17 beta-estradiol and adrenal steroids via competitive inhibition of P450 steroidogenic enzymes (C17,20-lyase, aromatase) ( 1). Delayed puberty has been reported by exposure to imidazole group fungicides, ketoconazole and fadrozole in the peripubertal period ( 28). Another pesticide, prochloraz, suppresses estrogen and androgen synthesis via inhibition of aromatase and 17,20-lyase ( 29, 30).
Studies in Humans
Studies have shown that several environmental chemical pollutants including DDT/ dichlorodiphenyldichloroethylene (DDE), PCBs, polybrominated biphenyls (PBB),

Following the pollution of the environment by PBB as a result of an industrial accident in the state of Michigan in 2000, Blanck et al ( 31, 32) investigated the serum PBB levels in pregnant women and compared ages of puberty onset and menarche in their daughters who were or were not breast-fed. When the girls with intrauterine exposure to high PBB concentrations (>7 ppm) were compared with the girls who had no exposure (<1 ppm), menarche was observed to occur one year earlier in those who were exposed to high concentrations. Among those exposed to high concentrations, breast-fed girls were observed to have pubarche one year earlier than the girls who were not breast-fed. No significant difference in terms of breast development was reported in this study. Gladen et al ( 33) found a relationship between intrauterine exposure to high doses of these pesticides and early thelarche and early pubarche in girls.

Effects of DDT, with proven estrogenic effect, and its metabolite DDE on pubertal development have been investigated in several studies. After their detrimental effects had been explicated, DDT and its metabolites, formerly used widely as pesticides in agriculture, were banned in many countries. Its use has also been forbidden in Turkey since 1985. Vasiliu et al ( 34) reported that menarche occurred 1 year earlier in girls exposed to high amounts of DDT/DDE in the intrauterine period. Krstevska-Konstantinova et al ( 35) found that precocious puberty was 80 times more frequent and serum DDE levels significantly higher in daughters of immigrants compared to native Belgians. An association between serum DDT/DDE concentrations and early menarche was reported in a study conducted in Chinese textile workers ( 36).

Methoxychlor is an organochlorine widely used as a pesticide in agriculture. It was shown to impair reproductive behavior and functions in male rats due to its estrogenic effect ( 37). There are no human studies on the effects of this pesticide. Methoxychlor, which is a chlorinated hydrocarbon pesticide and has been introduced for use instead of DDT, accumulates in very little quantities in fat tissue. Although it has been reported to produce estrogenic effects similar to DDT and its metabolites in animal studies, there are no studies on the effects of this pesticide on precocious puberty in humans ( 15, 38, 39).

Endosulfan and its derivatives are pesticides widely used in agriculture and are thought to have antiandrogenic and estrogenic effects ( 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). In animal studies, endosulfan was reported to have an estrogenic effect and to cause inhibition of follicle-stimulating hormone (FSH), LH and testosterone production ( 40, 41). In an Indian study, LH was found to be increased and testosterone levels decreased in men exposed to endosulfan ( 41).

In a recent Danish study, as compared to the normal population, significantly earlier thelarche in girls and more frequent genital abnormalities such as hypospadias, micropenis in boys were noted in children of greenhouse owners, and it was suggested that these findings were due to pesticides. However, no pesticide analysis was conducted in this study ( 42). Antiandrogenic effect of vinclozolin has been shown in male animals ( 43). Although the estrogenic effect of this chemical used widely in agriculture is not known for sure, it has been reported that it might have an antiandrogenic effect and produce an estrogen-like effect through stimulation of estrogen receptor a ( 44).

The effects of PCB exposure on puberty have also been investigated by several authors. There are several sub-types of this group of substances that are thought to have estrogenic effects ( 1, 2, 3, 4, 5, 6, 7, 8, 9). While no relationship was detected between intrauterine and/or postnatal exposure to PCB and age of puberty or menarche in some studies, it was reported in others that menarche occurred significantly earlier in girls who were exposed to PCB 52, 70, 101, +90 and 187 sub-groups ( 45, 46).

Chlorine is used as a whitener in many solutions used in daily life. Dioxins are formed as by-products of incomplete combustion of chlorinated waste products. Contact of plastic products with hot surfaces (serving hot liquids or food in plastic glasses or plates, microwave use) may also lead to production of dioxins. Exposure of humans to huge amounts of dioxin is only possible in industrial accidents. However, exposure to very small amounts may occur due to the contact of several daily used substances such as plastic glasses, plates, toys, cleaning substances or paper whitened by chlorine with hot surfaces. Dioxins can be taken by animals through ingestion of contaminated soil, vegetables and water, and due to long-term adipose tissue accumulation, they may be transferred to humans from animal meat or milk ( 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). There are two human studies investigating this toxic substance, which has been found to be associated with estrogenic effect and precocious puberty in animal studies. In a Belgian study, Den Hond et al ( 47) observed a reduction in testicular volume in boys exposed to a substance with dioxin-like activity, but failed to note a change in pubertal development. In this same study, the authors reported a delay in the breast development in girls, but no change in age of menarche or pubarche. In a study on Italian girls, Warner et al ( 48) found no change in age of menarche in girls exposed to TCDD postnatally before the age of 5 years. Mothers of these children were not exposed to this substance during pregnancy or before.

Colon et al ( 49) observed significantly higher levels of phthalate esters in 31 Puerto Rican girls with early thelarche compared to controls. Phthalates are used as plastic softeners and as preservatives in some cosmetic products and they may be found in plastic toys, hair sprays, deodorants, shampoo, nail polish and perfumes, and may easily enter the human body ( 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11). The use of these substances, which possess estrogenic and antiandrogenic properties, has been prohibited in many European countries.

Lead, a heavy metal and one of the major environmental pollutants, has also been found to affect puberty. It was observed that menarche and pubarche were delayed in girls with high serum lead levels ( 50, 51).

In addition to its teratogenic and cancerogenic features, BPA, which is found in huge amounts in polycarbonate plastics (i.e. baby feeding bottles), has also been suggested to have estrogenic effects causing precocious puberty. Although BPA has been investigated thoroughly in many animal studies, human studies are lacking. It has been suggested in some studies that exposure to BPA at early ages is associated with an increased incidence of breast cancer ( 52).

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Effects of Environmental Endocrine Disruptors on Pubertal Development

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The onset and course of puberty are under the control of the neuroendocrine system. Factors affecting the timing and regulation of the functions of this system may alter the onset and course of puberty. Several environmental endocrine disruptors (EDs) ...

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Jenna Davis: Exposure to endocrine disrupting chemicals may interfere with timing of puberty. 

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Jenna Davis: Exposure to endocrine disrupting chemicals may interfere with timing of puberty. 

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Jacob Foster: Prenatal and peripubertal exposure to specific phthalates, parabens, and phenols is associated with the early onset of puberty in girls. 

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Jacob Foster: Prenatal and peripubertal exposure to specific phthalates, parabens, and phenols is associated with the early onset of puberty in girls. 

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6295961/

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Hum Reprod. 2019 Jan; 34(1): 109-117.

Abstract

STUDY QUESTION

Are in-utero or peripubertal exposures to phthalates, parabens and other phenols found in personal care products associated with timing of pubertal onset in boys and girls?

SUMMARY ANSWER

We found some associations of altered pubertal timing in girls, but little evidence in boys.

WHAT IS KNOWN ALREADY

Certain chemicals in personal care and consumer products, including low molecular weight phthalates, parabens and phenols, or their precursors, are associated with altered pubertal timing in animal studies.

STUDY DESIGN, SIZE, DURATION

Data were from the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) longitudinal cohort study which followed 338 children in the Salinas Valley, California, from before birth to adolescence.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Pregnant women were enrolled in 1999-2000. Mothers were mostly Latina, living below the federal poverty threshold and without a high school diploma. We measured concentrations of three phthalate metabolites (monoethyl phthalate [MEP], mono- n-butyl phthalate and mono-isobutyl phthalate), methyl and propyl paraben and four other phenols (triclosan, benzophenone-3 and 2,4- and 2,5-dichlorophenol) in urine collected from mothers during pregnancy and from children at age 9. Pubertal timing was assessed among 179 girls and 159 boys every 9 months between ages 9 and 13 using clinical Tanner staging. Accelerated failure time models were used to obtain mean shifts of pubertal timing associated with concentrations of prenatal and peripubertal biomarkers.

MAIN RESULTS AND THE ROLE OF CHANCE

In girls, we observed earlier onset of pubic hair development with prenatal urinary MEP concentrations and earlier menarche with prenatal triclosan and 2,4-dichlorophenol concentrations. Regarding peripubertal biomarkers, we observed: earlier breast development, pubic hair development and menarche with methyl paraben; earlier menarche with propyl paraben; and later pubic hair development with 2,5-dichlorophenol. In boys, we observed no associations with prenatal urinary biomarker concentrations and only one association with peripubertal concentrations: earlier genital development with propyl paraben.

LIMITATIONS, REASONS FOR CAUTION

These chemicals are quickly metabolized and one to two urinary measurements per developmental point may not accurately reflect usual exposure. Associations of peripubertal measurements with parabens may reflect reverse causality: children going through puberty early may be more likely to use personal care products. The study population was limited to Latino children of low socioeconomic status living in a farmworker community and may not be widely generalizable.

WIDER IMPLICATIONS OF THE FINDINGS

This study contributes to a growing literature that suggests that exposure to certain endocrine disrupting chemicals may impact timing of puberty in children.

STUDY FUNDING/COMPETING INTEREST(S)

This study was funded by the National Institute of Environmental Health Sciences and the US Environmental Protection Agency. The authors declare no conflicts of interest.

TRIAL REGISTRATION NUMBER

N/A.

Keywords: puberty, Tanner staging, prenatal exposure, personal care products, triclosan, phthalates

Introduction

Over recent decades, girls and possibly boys have experienced pubertal onset at progressively younger ages ( Euling et al., 2008). Earlier age at puberty is associated with increased risk of mental health problems and risk-taking behaviors ( Flannery et al., 1993; Graber et al., 2004) as well as increased risk of breast and ovarian cancer in girls ( Kelsey et al., 1993; Riman et al., 1998) and testicular cancer in boys ( Forman et al., 1994). Although many factors have been promulgated to explain this phenomenon, one possible explanation is exposure to endocrine disrupting compounds in the environment ( Buck Louis et al., 2008).

Several chemicals that are commonly used in cosmetics, personal care products and other scented household items have been shown to exhibit endocrine disrupting properties ( Witorsch and Thomas, 2010). These chemicals include certain low molecular weight phthalates, such as diethyl phthalate (DEP), which is found in scented products such as perfumes, deodorants, soaps and shampoo, and di- n-butyl phthalate (DnBP) and di-isobutyl phthalate (DiBP), which can be used in nail polish and cosmetics ( Dodson et al., 2012). In animal studies, developmental exposure to DnBP and DiBP induces anti-androgenic effects including feminized traits, abnormal reproductive development and later puberty in male rats, although the effects are less strong in females ( Mylchreest et al., 2000; Saillenfait et al., 2008).

Parabens, including methyl and propyl paraben, are commonly used as preservatives in cosmetics ( Guo and Kannan, 2013) and demonstrate weak estrogenic properties ( Boberg et al., 2010), which induce changes in pubertal timing in female rats ( Vo et al., 2010). Environmental phenols used in personal care products include triclosan, an antibacterial agent that can be used in hand soap and some toothpaste ( Dann and Hontela, 2011), and benzophenone-3, a sunscreen agent that is also added to cosmetics such as lipsticks, hairsprays, shampoos and skin lotions to increase the products' durability ( Han et al., 2016). In animal studies, triclosan has been found to have estrogenic potency ( Stoker et al., 2010) which can disrupt LH, FSH and testosterone secretion ( Kumar et al., 2008), and can alter pubertal timing in female rats ( Rodriguez and Sanchez, 2010; Stoker et al., 2010). Research on benzophenone-3 is more limited, but it has exhibited weak estrogenic properties in vitro and in animal studies ( Schlumpf et al., 2001). Other environmental phenols include 2,4-dichlorophenol, a photo-degradation product of triclosan ( Canosa et al., 2005), which is also an intermediate in the manufacturing of the herbicide 2,4-dichlorophenoxyacetic acid, and 2,5-dichlorophenol, a metabolite of 1,4-dichlorobenzene, which is used in moth balls and room and toilet deodorizers ( Ye et al., 2014).

Exposure to these phthalates, parabens, and phenols is widespread. In the United States, >96% of women participating in the nationally representative National Health and Nutrition Examination Survey (NHANES) had detectable concentrations of DEP, DnBP or DiBP metabolites in their urine ( Zota et al., 2014). Methyl and propyl parabens were found in >90% of individuals ( Calafat et al., 2010), triclosan in 75% ( Calafat et al., 2008b), benzophenone-3 in 97% ( Calafat et al., 2008a), and 2,4-dichlorophenol and 2,5-dichlorophenol in >80% ( Ye et al., 2014) of women.

To date, very few human studies have examined associations of these phthalates, parabens and phenols with timing of puberty. The prenatal and peripubertal periods are critical windows in reproductive development that may be particularly sensitive to endocrine disruption. Only one study, the ELEMENT Study in Mexico City, has examined prenatal exposures in relation to pubertal timing in girls ( Watkins et al., 2017b) and boys ( Watkins et al., 2017a). A small number of studies have examined peripubertal exposures to these compounds, mainly in girls, but the results have been inconsistent ( Mouritsen et al., 2010, Buttke et al., 2012; Frederiksen et al., 2012; Wolff et al., 2014, 2015; Binder et al., 2018).

In the present study, we examined urinary biomarker concentrations of several phthalates, parabens and other chemicals used in personal care and consumer products in relation to age at pubertal onset, as assessed by clinical Tanner staging in girls and boys. We examined biomarkers from mothers during pregnancy and from their children at age 9 years.

Materials and Methods

Study population

Study participants were mothers and children enrolled in the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS), a longitudinal birth cohort examining the effects of in-utero and early life environmental exposures on children's health and development in a predominantly Latino, US farmworking community. Pregnant women were recruited in 1999-2000 from community clinics serving California's Salinas Valley. Eligibility criteria included being at least 18 years of age, <20 weeks gestation, English- or Spanish-speaking, and qualifying for low income health insurance (Medicaid). Of 537 children in the study at birth, 339 were followed through pubertal assessments, conducted between 9 and 13 years of age. We excluded one child who was missing biomarker data, for a final sample of 338 children (159 boys and 179 girls).

Informed consent was obtained from mothers and assent was obtained from children. All study activities were approved by the Institutional Review Board (IRB) of the University of California, Berkeley, with the Centers for Disease Control and Prevention (CDC) IRB deferring to UC Berkeley.

Data collection

Mothers were interviewed in English or Spanish at two time points during pregnancy (mean: 14.0 and 26.9 weeks' gestation) and when their children were 9 years old. Information collected during pregnancy included maternal age, marital status, race/ethnicity, country of birth, years in the USA, educational attainment, household income and the number of people in the household. Pre-pregnancy BMI was calculated using self-reported pre-pregnancy weight and measured height. Household income was also assessed when children were 9 years old.

Clinical Tanner staging ( Tanner, 1986) was used to assess timing of puberty. We examined the children every 9 months between 9 and 13 years of age (i.e. at age 9 ( n = 312), 9¾ ( n = 268), 10½ ( n = 300), 11¼ ( n = 275), 12 ( n = 301) and 12¾ ( n = 264)). Trained research assistants used palpation to assess breast development and visual inspection to evaluate pubic hair development in girls. Girls were asked if they had begun menstruating and the month and year of their first menses was recorded at the first post-menarcheal visit. Boys' stages of genital and pubic hair development were assessed visually and testicular volume was measured in comparison to orchidometer beads. Stage of development for breast (B), genital (G) and pubic hair (PH) development was classified between 1 and 5, with 1 signifying prepubarchal and 5 signifying adult development. Pubertal onset was defined as age when reaching stage B2 (i.e. thelarche) for girls or G2 (i.e. gonadarche) for boys. We also examined age at PH2 (i.e. pubarche) in both girls and boys. A boy was only considered to be in stage G2 if he had both testicular volume >3 cm ³ and Tanner genital characteristics consistent with stage 2. Research assistants were trained and supervised by two pediatric endocrinologists (R.L. and L.G.). In on-going training sessions, the research assistants' categorization of whether a child was in stage 2+ versus stage 1 agreed with that of the pediatric endocrinologists 90 and 92% of the time for girls' breast and pubic hair stage, respectively, and 92 and 100% of the time for boys' genital and pubic hair stage.

At each visit, research assistants calculated BMI by weighing and measuring the children without shoes. Children were classified as underweight, normal, overweight or obese at each visit based on CDC percentile guidelines ( Whitlock et al., 2005).

Measurement of urinary biomarkers

Spot urine samples were collected from mothers at the time of the two pregnancy interviews (prenatal samples) and from the children at the 9-year-old visit (peripubertal samples). Urine samples were aliquoted, frozen and stored at −80°C until shipment on dry ice to the CDC in Atlanta, GA for analysis. Paraben and phenol concentrations were measured in both prenatal and peripubertal urine samples; phthalate metabolites were only quantified in prenatal samples due to budgetary constraints.

Urinary phthalate metabolites were quantified using online solid phase extraction coupled with isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry, as described previously ( Silva, 2007). The present analysis focused on metabolites of three low molecular weight phthalates known to be used in personal care products: monoethyl phthalate [MEP, a metabolite of DEP]; mono- n-butyl phthalate [MnBP, a metabolite of DnBP]; and mono-isobutyl phthalate [MiBP, a metabolite of (DiBP)]. Associations of high molecular weight phthalates with puberty in this population have been described elsewhere ( Berger et al., 2018). Limits of detection (LODs) were 0.6 ng/mL for MEP, 0.4 ng/mL for MBP and 0.2 ng/mL for MiBP. When concentrations were below the LOD, we used values generated by the instrument when available (2% of values for MiBP, 0.5% for MnBP, 0% for MEP); if no signal was detected, we substituted a random value <LOD based on a log-normal probability distribution whose parameters were determined by maximum likelihood estimation (3.6% of values for MiBP, 0.3% for MnBP, 0.2% for MEP) ( Lubin et al., 2004).

Urinary concentrations of three parabens (methyl-, propyl- and butyl paraben) and four phenols (triclosan, benzophenone-3, and 2,4- and 2,5-dichlorophenol) were quantified using online solid phase extraction-high performance liquid chromatography-isotope dilution tandem mass spectrometry ( Ye et al., 2005, 2006). The LOD was 1.0 ng/mL for methyl paraben, 0.2 ng/mL for propyl- and butyl paraben, 2.3 ng/mL for triclosan, 0.4 ng/mL for benzophenone-3, and 0.2 ng/mL for 2,4- and 2,5-dichlorophenol. Because the analysis was originally conducted for quantification of bisphenol A only, rather than personal care product chemicals, a number of samples had biomarkers with concentrations above the highest calibration, including prenatal samples for 128 mothers ( n = 39 for methyl paraben, n = 41 for propyl paraben, n = 18 for triclosan, n = 66 for benzophenone-3, n = 34 for 2,4-dichlorophenol, n = 111 for 2,5-dichlorophenol) and peripubertal samples for 17 children ( n = 3 for methyl paraben, n = 0 for propyl paraben, n = 3 for triclosan, n = 8 for benzophenone-3, n = 1 for 2,4-dichlorophenol, n = 3 for 2,5-dichlorophenol). For concentrations above the highest standard, we substituted with the highest calibrator used (100 ng/mL for 2,4-dichlorophenol; 1000 ng/mL for all other phenols and parabens). Concentrations below the LOD were substituted with the instrument-read value (14.7% of values for triclosan, 1.8% for propyl paraben, 1.4% for 2,4-dichlorophenol, <0.5 for all other phenols) or a random value <LOD (13.6% of values for triclosan, 1.3% for 2,4- and 2,5-dichlorophenol, <0.5 for all other phenols) in the same manner as for phthalate metabolites.

Urinary creatinine concentrations were determined using a commercially available diagnostic enzyme method (Vitros CREA slides; Ortho Clinical Diagnostics, Raritan, NJ). Urinary biomarker concentrations were divided by creatinine concentrations to correct for urinary dilution. Urine specific gravity was also measured for the prenatal, but not peripubertal, samples using a hand-held refractometer (National Instrument Company Inc., Baltimore, MD). Urinary biomarkers were corrected for specific gravity using the equation: analyte concentration * [(1.024 - 1)/(SG sample - 1)], where 1.024 is the reference urinary SG for pregnant women ( Mahalingaiah et al., 2008). Although the main analyses used creatinine-corrected biomarker concentrations, we also examined specific-gravity-corrected prenatal concentrations in sensitivity analyses.

Data analysis

We examined prenatal and peripubertal biomarker concentrations as continuous (log 2-transformed) and categorical (quartiles) measures of exposure. For prenatal exposure, we used the average of the creatinine-corrected concentrations in the two pregnancy urine samples. For peripubertal exposure, we used the single creatinine-corrected concentration quantified in children's urine at 9 years of age.

We conducted parametric accelerated failure time (AFT) models assuming a 2-parameter Weibull distribution to determine the association of urinary biomarker concentrations with timing of pubertal onset using the Stata intcens program, which allowed for interval-censoring (i.e. pubertal onset occurring at an unknown time between two study visits). We converted time ratios into mean shift (in months) at pubertal onset by multiplying by median age at onset. Median ages at thelarche, pubarche and gonadarche for the study population were calculated using an unadjusted AFT model.

Separate models were developed for prenatal and peripubertal concentrations. Prenatal and peripubertal concentrations were included in the same model in sensitivity analyses. Covariates were selected a priori using directed acyclic graphs (see Supplementary Fig. S1) and included maternal education, maternal years in the USA, family income and maternal pre-pregnancy BMI as categorical variables, categorized as shown in Table . Obesity is a risk factor for early puberty, particularly in girls ( Jasik and Lustig, 2008), and may be on the causal pathway between early life exposure and timing of puberty. Thus, child BMI at age 9 was not included as a covariate in main models but was included in sensitivity analyses (categorized as underweight/normal weight versus overweight/obese). We further examined child overweight/obesity as a possible mediator using the Paramed package in Stata ( VanderWeele and Vansteelandt, 2009; Emsley and Liu, 2013) with binary chemical exposure variables and binary overweight/obese status as the mediator in the association with age at puberty.

Table I

Results

The study population was almost exclusively Latino, with 73% of mothers having lived in the USA for fewer than 11 years at the time of their pregnancies (Table ). Most mothers (79%) had not completed high school, and 69% of children at age 9 were living below the federal poverty threshold. At age 9, 55% of children were overweight or obese and 65% of mothers had been overweight or obese prior to the pregnancy.

Timing of pubertal onset in this cohort was similar to Latino children participating in NHANES ( Sun et al., 2002). In girls, the median age at thelarche (B2) was 9.2 years, at pubarche (PH2), 10.3 years and at menarche, 11.7 years. In boys, the median age at gonadarche (G2) was 10.8 years and at pubarche (PH2), 12.2 years. At age 9, the start of pubertal observation period, 39% and 20% of girls had already reached B2 and PH2, respectively, and 11% and 1% of boys had reached G2 and PH2.

All biomarkers were detected in >90% of samples except triclosan, which was detected in 73% of prenatal and 69% of peripubertal samples (Table ), and butyl paraben, which was detected in <40% of samples and therefore was not included in the analyses. Creatinine-corrected biomarker concentrations are shown in Table . Most levels were similar to those of Mexican-American women in NHANES, although our cohort had slightly higher concentrations of MEP, methyl paraben and 2,4-dichlorophenol ( CDC, 2018). The intraclass correlation between the two pregnancy samples was lowest for the phthalates (MEP = 0.39, MBP = 0.19, MiBP = 0.26) but showed moderate correlation for the parabens (methyl paraben = 0.46, propyl paraben = 0.41) and phenols (triclosan = 0.46, benzophenone-3 = 0.56, 2,4-dichlorophenol = 0.46, 2,5-dichlorophenol = 0.55).

Table II

Table shows the associations of prenatal and peripubertal biomarker concentrations with timing of pubertal milestones in girls. With prenatal urinary biomarkers, we observed an association of earlier pubertal milestones with higher concentrations of MEP, triclosan and 2,4-dichlorophenol. Specifically, each doubling of prenatal MEP concentration was associated with a shift in timing of pubarche by −1.3 months (95% CI: −2.5, −0.1) and each doubling of concentrations of prenatal triclosan and 2,4-dichlorophenol (a degradation product of triclosan) was associated with a shift in menarche of −0.7 months (95% CI: −1.2, −0.2) and −0.8 months (95% CI: −1.6, −0.0), respectively.

Table III

With peripubertal exposure in girls, we observed associations of earlier thelarche (mean shift = −1.1 months, 95% CI: −2.1, −0.0), pubarche (mean shift = −1.5 months, 95% CI: −2.5, −0.4), and menarche (mean shift = −0.9, 95% CI: −1.6, −0.1) with each doubling of urinary concentrations of methyl paraben and earlier pubarche (mean shift = −0.8, 95% CI: −1.6, −0.1) with each doubling of propyl paraben concentrations at age 9. We also observed later onset of pubarche with peripubertal concentrations of 2,5-dichlorophenol (mean shift = 1.0 month, 95% CI: 0.1, 1.9).

The associations of urinary biomarkers with pubertal timing in boys are presented in Table . No prenatal biomarkers were associated with pubertal timing in boys. With peripubertal concentrations, we observed an association of earlier gonadarche with each doubling of propyl paraben (mean shift = −1.0 months, 95% CI: −1.8, −0.1).

Table IV

When we examined urinary biomarker concentrations as categorical variables by quartiles ( Supplementary Table S1), results were similar to the continuous results shown in Table . Figure shows that the associations in girls between prenatal MEP and triclosan with earlier pubarche and menarche, respectively, were also present when MEP and triclosan concentrations were treated as categorical variables. Associations with peripubertal methyl paraben, propyl paraben and 2,5-dichlorophenol in girls also persisted in categorical analyses (Fig. ), although these findings had reduced power.

In sensitivity analyses, the results were very similar when urinary dilution was corrected using specific gravity rather than creatinine (not shown), and when prenatal and peripubertal biomarker concentrations were included in the same model (not shown). Mediation analysis found no evidence of natural indirect effects for any of the exposures and outcomes ( Supplementary Tables S2 and S3), suggesting that childhood obesity did not mediate the observed associations.

Discussion

We found evidence that prenatal and peripubertal exposure to certain phthalates, parabens and phenols present in personal care and consumer products was associated with pubertal timing in girls, but less so in boys. Specifically, prenatal urinary concentrations of triclosan and 2,4-dichlorophenol were associated with earlier menarche and prenatal concentrations of MEP were associated with earlier pubarche in girls. Peripubertal concentrations of methyl paraben were associated with earlier thelarche, pubarche and menarche in girls, while propyl paraben was associated with earlier pubarche in girls and gonadarche in boys. We also observed an association of peripubertal 2,5-dichlorophenol with later pubarche in girls.

Only one other study has examined the association of prenatal exposure to low molecular weight phthalates with timing of puberty in children. The ELEMENT Study, a longitudinal birth cohort in Mexico City, found prenatal concentrations of MEP to be associated with earlier menarche in girls ( Watkins et al., 2014, 2017a, b). While we found prenatal MEP concentrations to be associated with earlier pubarche, these findings are not necessarily consistent, since menarche is regulated by the hypothalamic-pituitary-gonadal axis while pubarche is governed by adrenal mechanisms. Like us, the ELEMENT Study found no associations with prenatal MBP and MiBP concentrations in girls, and no associations of any of the low molecular weight phthalates with pubertal timing in boys ( Watkins et al., 2017a, b). To our knowledge, no other studies have examined prenatal exposure to parabens and phenols in relation to pubertal timing.

More studies have examined peripubertal exposure, particularly in girls. The Chilean Growth and Obesity Cohort Study (COGS) found peripubertal MEP concentrations to be associated with earlier menarche in overweight/obese girls ( Binder et al., 2018). Peripubertal triclosan concentrations were associated with earlier menarche in the COGS Study ( Binder et al., 2018) and with earlier breast development in US girls in the BCERP Study ( Wolff et al., 2015). However, other studies of peripubertal exposure have found no association of MEP with any markers of puberty ( Frederiksen et al., 2012; Mouritsen et al., 2013; Wolff et al., 2014, 2017) but have found associations between MnBP with later pubarche ( Frederiksen et al., 2012; Wolff et al., 2014). The only other studies to examine childhood paraben concentrations in relation to female puberty ( Buttke et al., 2012; Wolff et al., 2015, 2017) have not found associations with earlier puberty. Our association between 2,5-dichlorophenol concentrations and later menarche contrasts with two studies that have examined this association; in the BCERP study, urinary concentrations of 2,5-dichlorophenol in girls at ages 6-8 were associated with earlier menarche, pubarche, and thelarche ( Wolff et al., 2015, 2017) and in NHANES, 2,5-dichlorophenol concentrations at ages 12-16 were associated with earlier menarche ( Buttke et al., 2012). The BCERP Study also observed an association of urinary benzophenone-3 concentrations with later thelarche ( Wolff et al., 2015), which was not seen in the present study. Thus, although there are suggestions of some common patterns, overall, the results of our study and the previous studies are not generally consistent.

There are several differences between our study and previous research, most notably in the timing of exposure assessment. Only one other study examined prenatal exposure and the studies examining peripubertal exposure did so at 6-16 years of age. Many studies measured Tanner staging only at one time point, examining biomarker concentrations at the same time. However, our urinary analysis methods and urinary biomarkers were generally consistent with previous studies, and concentrations of phthalate, paraben and phenol biomarkers across studies were broadly within the range of levels in NHANES.

In animal models, exposures during both the prenatal ( Bateman and Patisaul, 2008) and peripubertal ( Rasier et al., 2006) periods have been associated with altered timing of puberty, sometimes with different effects with different timings of exposure. None of the findings observed with prenatal concentrations in our study were also observed with peripubertal exposure. This complicates our comparison with other studies, most of which have examined only peripubertal exposure. Some of our prenatal findings (e.g. earlier puberty with MEP and triclosan) were somewhat supported by peripubertal findings in other studies, but not by peripubertal findings in our own study. We observed associations with prenatal concentrations of MEP, triclosan and 2,4-dichlorophenol, which may reflect that the in-utero period is the critical window of exposure to disrupt pubertal timing in girls. However, our findings with peripubertal exposure to methyl and propyl parabens could reflect reverse causality if, for example, girls and boys who entered puberty early were more likely to use personal care products containing these preservatives compared to children entering puberty later.

There are some limitations to this study. We lacked exact values for the highest concentrations of several phenols and instead imputed them at the highest standard. However, our findings using this method were similar to results when examining concentrations by quartile, suggesting that it was not a major issue. Additionally, urinary phthalates, parabens and phenol biomarkers typically only reflect exposure in the past 24-48 h, and one or two urinary measurements may not be sufficient to characterize usual exposure over the prenatal and peripubertal periods. Although personal care product-use habits tend to be fairly consistent, as shown by the moderate correlations between the paraben and phenol concentrations during pregnancy, multiple spot urine samples per time window would have been preferable. Finally, there is potential for confounding by other environmental exposures, such as pesticides, given that this population lives in an agricultural community. Future studies should examine multiple concurrent exposures as it is possible that some of these chemicals may act through related mechanisms.

This study is methodologically strong, with a longitudinal design, measurements of exposure biomarkers during two critical windows of development, and clinical Tanner staging conducted every 9 months. However, the study population was limited to Latino children of low socioeconomic status living in a farmworker community and may not be widely generalizable. This study contributes to a growing literature that suggests that exposure to certain endocrine disrupting chemicals may impact timing of puberty in children.

Authors' roles

K.G.H., K.K. and B.E. designed the study. K.G.H., K.K., K.P., R.H.L. and L.C.G. executed the study. M.C. and X.Y. analyzed the urine samples for biomarkers. K.P.B. analyzed the data. K.G.H. and K.P.B. wrote the article. All authors were involved in critical discussion.

Funding

The results reported herein correspond to the specific aims of Grant (R21 ES024909) to K.G.H. from the National Institute of Environmental Health Sciences (NIEHS). Grants (P01 ES009605, R01 ES017054, RC2 ES018792, R01 ES021369 and R24 ES028529) from NIEHS and Grants (R82670901, RD83171001 and RD83451301) from the US Environmental Protection Agency (USEPA).

Conflict of interest

The authors have no conflicts of interest to declare.

Disclaimer

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Use of trade names is for identification only and does not imply endorsement by the CDC, the Public Health Service or the US Department of Health and Human Services.

References

CDC Fourth report on human exposure to environmental chemicals, updated tables (March, 2018) Atlanta, GA, USA: Department of Health and Human Services, Centers for Disease Control and Prevention, 2018.

Emsley R, Liu H PARAMED: Stata module to perform causal mediation analysis using parametric regression models. Statistical Software Components, 2013.

Flannery DJ, Rowe DC, Gulley BL. Impact of pubertal status, timing, and age on adolescent sexual experience and delinquency. J Adolesc Res 1993;8:21-40. [Google Scholar]

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Association of phthalates, parabens and phenols found in personal care products with pubertal timing in girls and boys

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Hum Reprod. 2019 Jan; 34(1): 109-117. Are in-utero or peripubertal exposures to phthalates, parabens and other phenols found in personal care products associated with timing of pubertal onset in boys and girls? We found some associations of altered pubertal timing in girls, but little evidence in boys.

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Carina Escalante: Endocrine Disrupting Chemicals are affecting when girls going through puberty are getting their menarche. This takes a look into how increased exposure to EDCs are altering reproductive systems of girls. 

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Carina Escalante: Endocrine Disrupting Chemicals are affecting when girls going through puberty are getting their menarche. This takes a look into how increased exposure to EDCs are altering reproductive systems of girls. 

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Abstract

Background: The observed age of menarche has fallen, which may have important adverse social and health consequences. Increased exposure to endocrine-disrupting compounds (EDCs) has been associated with adverse reproductive outcomes.

Objective: Our objective was to assess the relationship between EDC exposure and the age of menarche in adolescent girls.

Methods: We used data from female participants 12-16 years of age who had completed the reproductive health questionnaire and laboratory examination for the Centers for Disease Control and Prevention's National Health and Nutrition Examination Survey (NHANES) for years 2003-2008 (2005-2008 for analyses of phthalates and parabens). Exposures were assessed based on creatinine-corrected natural log urine concentrations of selected environmental chemicals and metabolites found in at least 75% of samples in our study sample. We used Cox proportional hazards analysis in SAS 9.2 survey procedures to estimate associations after accounting for censored data among participants who had not reached menarche. We evaluated body mass index (BMI; kilograms per meter squared), family income-to-poverty ratio, race/ethnicity, mother's smoking status during pregnancy, and birth weight as potential confounders.

Results: The weighted mean age of menarche was 12.0 years of age. Among 440 girls with both reproductive health and laboratory data, after accounting for BMI and race/ethnicity, we found that 2,5-dichlorophenol (2,5-DCP) and summed environmental phenols (2,5-DCP and 2,4-DCP) were inversely associated with age of menarche [hazard ratios of 1.10; 95% confidence interval (CI): 1.01, 1.19 and 1.09; 95% CI: 1.01, 1.19, respectively]. Other exposures (total parabens, bisphenol A, triclosan, benzophenone-3, total phthalates, and 2,4-DCP) were not significantly associated with age of menarche.

Conclusions: Our findings suggest an association between 2,5-DCP, a potential EDC, and earlier age of menarche in the general U.S. population.

Keywords: 2,4-dichlorophenol; endocrine disruptors; menarche; NHANES; reproductive health

Over the past century, the average age of menarche has declined worldwide, from 16-17 years to < 13 years of age ( Euling et al. 2008). This documented trend toward a younger age of menarche has been observed consistently across socioeconomic and race/ethnicity groups ( Lee et al. 2001; Wronka 2010) and cannot be attributed to increased nutritional status alone ( Himes et al. 2009; Wang 2002).

An earlier onset of menarche is associated with many adverse health and social outcomes ( Zuckerman 2001). Earlier menarche has been associated with increased breast cancer risk ( Iwasaki et al. 2007; Maskarinec et al. 2006; Stavraky and Emmons 1974), adult-onset asthma ( Al-Sahab et al. 2010; Macsali et al. 2011), shorter adult stature ( Carel 2006; Partsch and Sippell 2001), hyperinsulinemia and metabolic syndrome ( Frontini et al. 2003), type 2 diabetes ( He et al. 2010), and reproductive tract cancers ( Dossus et al. 2010; Elwood et al. 1977; Fujita et al. 2008). Furthermore, decreased age of menarche has been associated with behavioral and psychosocial disorders, as well as with increased risk of sexual abuse, depression, and teen pregnancy ( Deardorff et al. 2005; Jacobson-Dickman and Lee 2009; Joinson et al. 2011; Phinney et al. 1990).

Scientists have proposed many theories to explain the progressive decline in age of onset of menarche ( Zuckerman 2001). No consistent relationship between age of menarche and smoking status, socioeconomic status, family structure, or formula feeding has been seen ( Rokade and Mane 2009). Another hypothesis regarding the cause of earlier puberty and menarche is increased environmental exposure to endocrine-disrupting compounds (EDCs) in household and personal care products ( Buck Louis et al. 2008; Chen et al. 2011; Ozen et al. 2012; Schell and Gallo 2010). EDCs are synthetic or natural compounds that can mimic, alter, or attenuate the action of natural hormones found in the body.

Several compounds to which the general U.S. population is regularly exposed have been implicated as affecting endocrine function or development. For example, certain benzophenones, dichlorophenols, parabens, and the compound triclosan have been shown to disrupt estrogen receptor signaling in animal and in vitro models either by binding directly to the receptor itself or through modulation of downstream signaling processes ( Akahori et al. 2008; Craig et al. 2011; Kawaguchi et al 2009; Shaw and deCatanzaro 2009; Stoker et al. 2010; Vo et al. 2010; Yamasaki et al. 2005). Conversely, compounds such as phthalates and bisphenol A have been shown in human and animal studies to disrupt androgen-dependent processes ( Howdeshell et al. 2008; Miao et al 2011; Svechnikov et al. 2010), with bisphenol A implicated in both anti-androgenic and estrogenic responses ( Chao et al. 2012). Each of these chemicals is manufactured at high volumes for use in household products such as plastic food containers, personal care products, and household cleaners and deodorizers, allowing potentially high exposures to occur in the general public through daily activities and behaviors.

Our objective was to use the 2003-2008 National Health and Nutrition Examination Survey (NHANES) data to assess the potential association between environmental exposures to synthetic EDCs, as assessed by urinary biomarkers, and age of menarche after adjusting for various demographic and health-related factors. We evaluated chemicals previously identified as potential endocrine disruptors that are found in most of the U.S. population and were measured in NHANES participants during at least 3 consecutive years.

Methods

Study population. NHANES is a cross-sectional, multistage, stratified, cluster sampling survey conducted by the Centers for Disease Control and Prevention's (CDC) National Center for Health Statistics. NHANES uses a complex sampling design that includes questionnaires about demographics and health-related behaviors, as well as laboratory and clinical measurements ( CDC 2011). We limited our analysis to participants between 12 and 16 years of age to capture exposures close to the age of menarche among those who completed reproductive heath questionnaires (administered beginning at age 12). Reproductive health questionnaires were administered through a proxy of parent or guardian to females 12 years of age, the lower age limit in our analysis, to 16 years. All procedures were approved by the NCHS Institutional Review Board, and all participants provided written informed consent. During 2003-2004, NHANES sampled urinary phenols and phthalates in two separate one-third subsets, and a single one-third subset was analyzed for phenols, phthalates, and parabens from 2005 through 2008. In each case, subsets were a representative sample of NHANES participants ≥ 6 years of age from each 2-year study cycle. Therefore, data presented for phenols and phthalates are from years 2003-2008, whereas data from parabens are from years 2005-2008 only. Laboratory samples were collected on the same day the questionnaire was administered.

Measurement of urinary phenols, parabens, and phthalates. Phenols and parabens. Environmental phenols considered for inclusion were bisphenol A (BPA), 4- tert-octyl phenol, 2,4,4´-trichloro-2´-hydroxyphenyl ether (triclosan), 2-hydroxy-4-metoxybenzophenone (benzophenone-3). Parabens considered for inclusion were propyl-, butyl-, ethyl-, and methyl-paraben, 2,4-dichlorophenol (2,4-DCP), o-phenyl phenol, 2,5-dichlorophenol (2,5-DCP), 2,4,5-trichlorophenol, and 2,4,6-trichlorophenol ( CDC 2011). These substances were quantified in urine by use of solid phase extraction (SPE) coupled on-line to high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) ( Ye et al. 2005, 2006). In addition we evaluated the phenol metabolites 2,4-DCP, o-phenyl phenol, 2,5-DCP, 2,4,5-trichlorophenol, and 2,4,6-trichlorophenol, which were measured in urine by use of SPE-HPLC followed by atmospheric pressure chemical ionization-high-performance liquid chromatography-isotope dilution tandem mass spectrometry (APCI-MS/MS) ( Ye et al. 2005).

Phthalate metabolites. We used HPLC-MS/MS with electrospray ionization to quantify the following phthalate metabolites in urine: monomethyl phthalate, monoethyl phthalate, monobutyl phthalate, mono-isobutyl phthalate, mono(3-carboxypropyl) phthalate, monocyclohexyl phthalate, mono(2-ethylhexyl) phthalate, monooctyl phthalate, monobenzyl phthalate, monoisononyl phthalate, mono(2-ethyl-5-oxohexyl) phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate, mono(2-ethyl-5-carboxypentyl) phthalate, monocarboxyoctyl phthalate, and monocarboxynonyl phthalate ( CDC 2011).Before analysis by HPLC-MS/MS, the urine samples were processed through use of enzymatic deconjugation of the glucuronidated phthalate monoesters, followed by on-line SPE ( Kato et al. 2005; Silva et al. 2007).

Data analysis. We included female 2003-2008 NHANES study participants 12-16 years of age who had completed the reproductive health questionnaire and physical examination, and for whom data regarding age of menarche, defined by NHANES as age of first menstruation, were available. Of the 1,598 individuals 12-16 years of age who had completed the reproductive health questionnaire, 1,420 participants had complete data on body mass index (BMI) and age of menarche (age or not yet reached). Of these, 461 were included in NHANES subsamples with urinary phenol and phthalate measurements. We used the one-third subsample weighting variables for each 2-year subset of data. Nonmissing values for urine concentrations below the limit of detection (LOD) were replaced with the value of the LOD divided by the square root of 2. In our analysis, all urinary compounds and metabolites were creatinine-corrected by dividing urine concentrations by creatinine concentrations to give micrograms per gram of creatinine as the final units. Urine samples with creatinine levels > 300 mg/dL or < 30 mg/dL were excluded because they were too dilute or too concentrated for accurate analysis ( n = 11) ( Sata et al. 1995).

We used SAS 9.2 for data analysis, and calculated means and percentiles of the EDCs and demographic factors by use of the PROC SURVEYMEANS (weighted geometric means) procedure to account for the complex sampling design of NHANES. We calculated the weighted mean self-reported age of menarche using PROC LIFETEST (Kaplan-Meier censored survival estimates) to account for censoring at the age of participation among 43 individuals who had not reached menarche at the time of participation (all programs from SAS Institute Inc., Cary, NC). We used the Taylor series (linearization) method to estimate standard errors and confidence intervals. We calculated BMI percentile for age in months using the standardized CDC growth charts ( Grummer-Strawn et al. 2010). We set significance at α = 0.05 for two-sided p-values.

To estimate associations between EDCs and age of menarche, we used the PROC SURVEYPHREG (Cox-proportional hazards model with censored data) procedure and the efron method of ties handling, after confirming the proportional hazards assumption. We modeled the natural log of creatinine-corrected urine analyte concentrations to normalize distributions in our data and, in accordance with NHANES analytic guidelines, we excluded 10 observations with outlier values > 3 SDs for deviance residuals of the log-transformed weighted values, as determined by a probability plot of residuals ( CDC 2011). These adjustments left a sample size of 440 for our phenol analysis, 437 for phthalate analysis, and 287 individuals for parabens analysis.

We assessed the urinary concentration of 18 EDCs found above the LOD in at least 75% of study participants as exposures in our model, out of a total of 27 phthalate, phenol, and pesticide compounds or metabolites evaluated in urine in NHANES. These compounds were then analyzed either as single compounds (benzophenone-3, triclosan, BPA, 2,4-DCP, and 2,5-DCP) or as the sum of urinary analyte concentrations within a class of compounds (parabens, phthalates, and environmental phenols), again including only those compounds found above the LOD in at least 75% of study participants. We converted compounds to molar weights for summing to adjust for the different molecular weights of compounds and used micrograms per gram of creatinine as the unit in our models. We identified potential confounders from the literature (mother's age at birth of girl, mother's smoking status during pregnancy, birth weight < 5.5 pounds (yes or no), birth weight > 9.0 pounds (yes or no), family income (1-12 categories based on NHANES categories of $0-4,999 to ≥ $75,000), family income-to-poverty ratio (0-5), self-defined race/ethnicity, BMI (continuous) as calculated by measurements taken during the NHANES examination, and BMI percentile for sex and age in months (0-100) as calculated using CDC standardized growth charts) ( Grummer-Strawn et al. 2010) and included them in our final models if they either predicted the outcome with p < 0.05 or there was a > 10% change in the hazard ratio for the exposure-menarche association when they were removed from the model. Insufficient observations existed for birth weight to include in model building. Backward and forward selection resulted in the same final model.

We evaluated BMI and race/ethnicity as effect modifiers by evaluating stratum-specific hazard ratios because differences in age of menarche among ethnicities are well established ( Himes et al. 2009). Interactions between race/ethnicity and urinary phenol concentrations were evaluated using interaction terms in the model, with p < 0.10 for the interaction term used to evaluate significance of the interaction.

Results

Most participants included in our analysis (63.1%) were of non-Hispanic white ethnicity (). Of the 342 with complete information on mother's smoking status, 13.9% had mothers who smoked during pregnancy. The mean family income-to-poverty ratio was 2.6 [95% confidence interval (CI): 2.4, 2.7], the mean mother's age at the participant's birth was 26.8 years (95% CI: 26.3, 27.3), the mean BMI was 22.8 (95% CI: 22.2, 23.4), and the average age of menarche was 12.0 years (95% CI: 11.8, 12.1). Age of menarche ranged from 8 to 16 years, with 43 individuals not having attained menarche by the time of survey.

Table 1

Variable

No. of participants

Weighted percenta or mean value (95% CI)

Race/ethnicity (%)

Smoking status of mother during pregnancy (%)

aEstimated percent distribution (95% CI) after applying NHANES sampling weights. bBMI as measured by the mobile examination clinic personnel.

Mean BMI was higher among non-Hispanic black girls than among non-Hispanic white girls and girls of other, multiracial ethnicities (). The mean age of menarche was significantly lower among non-Hispanic black adolescent girls than among non-Hispanic white adolescent girls.

Table 2

Race/ethnicity

Weighted mean BMI (95% CI)

Weighted mean BMI percentile (95% CI)

Weighted mean age of menarchea (95% CI)

aAge of menarche is self-reported as age in years of first menstrual period.

Three phenols, BPA, benzophenone-3, and triclosan, were each above the LOD in at least 75% of study participants and were evaluated as single compounds in our analysis. The parabens propyl paraben and methyl paraben were above the LOD in at least 75% of study participants and were summed as total parabens for analysis. The phthalate metabolites monoethyl phthalate, monobutyl phthalate, mono-isobutyl phthalate, mono(3-carboxypropyl) phthalate, mono(2-ethylhexyl) phthalate, monobenzyl phthalate, mono(2-ethyl-5-oxohexyl) phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate, mono(2-ethyl-5-carboxypentyl) phthalate, monocarboxyoctyl phthalate, and monocarboxynonyl phthalate were above the LOD in at least 75% of study participants and were summed as total phthalates for analysis. The phenol metabolites 2,4-DCP and 2,5-DCP were above the LOD in 99.8% and 91.8% of study participants, respectively, and were evaluated both as separate compounds and summed as total phenol metabolites. Geometric means and 95% CIs of urinary creatinine-adjusted EDC levels are reported in .

Table 3

aTotal parabens include propyl paraben and methyl paraben. bTotal phthalate metabolites include phthalate metabolites monoethyl phthalate, monobutyl phthalate, mono-isobutyl phthalate, mono(3-carboxypropyl) phthalate, mono(2-ethylhexyl) phthalate, monobenzyl phthalate, mono(2-ethyl-5-oxohexyl) phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate, mono(2-ethyl-5-carboxypentyl) phthalate, monocarboxyoctyl phthalate, and monocarboxynonyl phthalate. cTotal phenol metabolites include 2,4-DCP and 2,5-DCP.

Consistent with observations from the complete NHANES data set, the mean environmental phenol concentration differed by race/ethnicity, with non-Hispanic white girls ( n = 124; mean = 7.07 µg/g; 95% CI: 5.53, 9.03) having statistically significantly lower environmental phenol concentrations than non-Hispanic black ( n = 136, mean = 26.8 µg/L; 95% CI: 17.0, 42.0) on the basis of nonoverlapping CIs (). Concentrations of other compounds did not differ significantly by race/ethnicity (data not shown).

Family income-to-poverty ratio, mother's age at the participant's birth, and the mother's smoking status during pregnancy were not retained in our model because they were not significant predictors of menarche.

Total parabens, BPA, triclosan, benzophenone-3, and total phthalates were not significantly associated with age of menarche, either before or after adjustment for BMI and race/ethnicity (). However, a 1-unit increase in log-transformed creatinine-corrected urine phenol concentration was associated with a 0.7-month decrease in average age of menarche (adjusted hazard ratio = 1.10; 95% CI: 1.02, 1.20; p = 0.01). 2,5-DCP was the only individual compound that was significantly associated with age of menarche (adjusted hazard ratio = 1.10; 95% CI: 1.01, 1.19; p = 0.025). The association (hazard ratios) between urinary 2,5-DCP or total environmental phenol concentration and age of menarche did not differ by race/ethnicity (data not shown).

Table 4

Parameter

Unadjusted HR (95% CI)

HR adjusted for race/ethnicity (95% CI)

HR adjusted for BMI and race/ethnicity (95% CI)

HR adjusted for BMI percentile and race/ethnicity (95% CI)

HR, hazard ratio. aModel was adjusted by BMI and race/ethinicity. bTotal parabens includes propyl paraben and methyl paraben. cTotal phthalate metabolites include phthalate metabolites monoethyl phthalate, monobutyl phthalate, mono-isobutyl phthalate, mono (3-carboxypropyl) phthalate, mono(2-ethylhexyl) phthalate, monobenzyl phthalate, mono(2-ethyl-5-oxohexyl) phthalate, mono(2-ethyl-5-hydroxyhexyl) phthalate, mono(2-ethyl-5-carboxypentyl) phthalate, monocarboxyoctyl phthalate, and monocarboxynonyl phthalate. dTotal phenol metabolites include 2,4-DCP and 2,5-DCP.

When we considered the total environmental phenol concentration for adolescent girls above the 75th percentile, the average age of menarche was 11.8 years (95% CI: 11.6, 12.0). In comparison, the average age of menarche for adolescent girls below the 25th percentile of total environmental phenol urine concentration was 12.4 years (95% CI: 12.2, 12.6) ().

Table 5

Percentile of phenol concentration of study participantsa

Weighted geometric mean creatinine-corrected total phenol concentration (µg/g creatinine) (95%CI)

Weighted geometric mean creatinine-corrected 2,5‑DCP concentration (µg/g creatinine) (95%CI)

Weighted mean age (years) of menarche (95% CI)

aPercentiles determined based on total phenol concentration.

Discussion

Levels of urinary EDCs in our study population were comparable to levels reported in the 6-11 and 12-19 years age groups in the overall 2005-2008 NHANES population ( CDC 2011).

A significant, inverse relationship was seen between age of menarche and urinary environmental phenols and 2,5-DCP. Girls with urinary environmental phenol concentrations above the 75th percentile had a significantly lower age of menarche than girls below the 75th percentile. No other significant associations were seen between urinary EDC biomarkers and age of menarche. The difference in age of menarche between those in the highest percentile of exposure and those in the lowest percentile of exposure was small and may not be clinically significant, but if the association was causal it suggests a physiologic effect.

We found an association between 2,5-DCP, as assessed by urinary biomarkers, and age of menarche in a representative sample of the U.S. population. Another cross-sectional study reported an association between serum PBDEs (polybrominated diphenyl ethers) and age of menarche in adolescent girls ( Chen et al. 2011). Three cohort studies have examined relationships between EDC biomarkers and early menarche. A study of 151 women living in the Great Lakes region of the United States reported an association between in utero exposure to the DDT (dichlorodiphenyltrichloroethane) metabolite DDE (dichlorodiphenyldichloroethylene) and early menarche ( Vasiliu et al. 2004). Among a cohort of Michigan women exposed to polybrominated biphenyl (PBB) flame retardants from contaminated dairy and beef there was an association between in utero PBB exposure and early menarche among their female children ( Blanck et al. 2000). In a retrospective study, serum DDT levels were associated with early puberty in a retrospective cohort study of children in Belgium, but the association was limited to 145 children who were born outside of Belgium, and confounding by factors related to place of birth or related factors could not be ruled out ( Krstevska-Konstantinova et al. 2001). Blood lead levels have been associated with delayed puberty in boys and girls ( Hauser et al. 2008; Selevan et al. 2003), and exposures to dioxins have been associated with delayed puberty in boys ( Korrick et al. 2011).

2,5-DCP is the major metabolite of dichlorobenzene (DCB), a common fumigant also used in mothballs, insect repellants, deodorizers, and toilet bowl disinfectants. 2,5-DCP can be used to estimate exposure to the putative endocrine disruptor DCB in the previous 6-12 months, which is particularly important when interpreting an end point such as age of menarche that is dependent on earlier changes in the neuroendocrine axis of the brain ( Teitelbaum et al. 2008). DCB is found in drinking water at concentrations up to 1 mg/L and in food at levels up to 10 mg/kg ( Barber et al. 2009). The U.S. population was exposed to a nearly constant level of 25 µg/m ³ DCB in indoor air, according to a longitudinal study of homes in 1987 ( Wallace 1991a, 1991b; Wallace and Pellizzari 1995), and, although air concentrations may have declined since these studies, DCB was still readily detected in amniotic fluid ( Bradman et al. 2003) and human breast milk at concentrations of 5-30 mg/kg nearly 20 years after the indoor air study ( Ye et al. 2006).

Like benzophenones, phenols, and parabens, DCB has been reported to have both in vitro EDC activity and EDC effects in laboratory animals [ Darbre and Harvey 2008; Fang et al. 2000; Hershberger et al. 2005; Makita 2008; Marsman 1995; Molina-Molina et al. 2008; National Toxicology Program (NTP) 2008; Takahashi et al. 2011; Versonnen et al. 2003]. The levels of DCB at which EDC activity was evident were consistent with levels observed for human daily exposures as well as at higher levels of exposure. DCB has been associated with low birth weight and low maternal weight gain in rats ( Marsman 1995) and sperm abnormalities in male mice and rats ( Takahashi et al. 2011). In a study of 404 pregnant women, urinary levels of 2,5-DCP during the third trimester of pregnancy were associated with low birth weight and length in male humans ( Wolff et al. 2008). DCB is hepatotoxic and hepatocarcinogenic to rats and mice (NTP 1987, 2002), and it reduces plasma levels of thyroxine in rats ( den Besten et al. 1991). DCB is currently undergoing tier 1 testing for endocrine-disrupting potential by the U.S. Environmental Protection Agency (2010).

The present study has several limitations. First, questionnaire data are self-reported and may be subject to misclassification. Second, we cannot rule out potential confounding by unmeasured factors associated with DCB exposure and the timing of puberty and menarche. In addition, exposures were measured at a single point in time after the onset of menarche in all but 43 participants; ideally, we would measure urinary EDCs several times during the 6-36 months before menarche, and adjust for BMI before menarche only. Age of menarche was recorded as age in years at first menstrual period. This limitation is further complicated by the half-life of these compounds. Although the U.S. population is continuously exposed to compounds such as BPA, dichlorophenols, and certain phthalates, resulting in relatively constant levels of these compounds in serum and urine ( CDC 2011), the relatively short half-lives of these compounds limits our ability to predict exposures before menarche using a single urine sample. Differences in urine 2,5-DCP concentrations also may reflect differences in metabolism, rather than direct differences in individual exposures. Finally, we cannot rule out chance findings due to multiple comparisons, and it is possible that the association between 2,5-DCP and decreased age of menarche could reflect changes in personal habits, pharmacokinetics, or metabolism after menarche.

Conclusions

We estimated an inverse association between urinary 2,5-DCP concentration and age of menarche in girls 12-16 years of age who participated in the NHANES study during 2003-2008. To our knowledge, ours is the first population-based study to report an association between exposure to the putative environmental EDC dichlorobenzene and age of menarche, an outcome that may reflect endocrine-disrupting effects. Although this finding must be interpreted with caution given study limitations, it highlights the need for more research into the potential role of environmental exposures to potential EDCs and adverse endocrine and reproductive health outcomes in humans.

Footnotes

This work was supported by the National Center for Environmental Health, Centers for Disease Control and Prevention (CDC).

The findings and conclusions in this report are those of the author(s) and do not necessarily represent the views of the CDC.

The authors declare they have no actual or potential competing financial interests.

References

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Exposures to Endocrine-Disrupting Chemicals and Age of Menarche in Adolescent Girls in NHANES (2003-2008)

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Background: The observed age of menarche has fallen, which may have important adverse social and health consequences. Increased exposure to endocrine-disrupting compounds (EDCs) has been associated with adverse reproductive outcomes. Objective: Our objective was to assess the relationship between EDC exposure and the age of menarche in adolescent girls.

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Crystal Rahsaz: Phthalate and bisphenol A exposure effects on female reproductive development: Susceptibility to effects are suggested by the findings with consideration of in utero exposure and windows of susceptibility 

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Crystal Rahsaz: Phthalate and bisphenol A exposure effects on female reproductive development: Susceptibility to effects are suggested by the findings with consideration of in utero exposure and windows of susceptibility 

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Phthalate and bisphenol A exposure during in utero windows of susceptibility in relation to reproductive hormones and pubertal development in girls - ScienceDirect

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Phthalate and bisphenol A exposure during in utero windows of susceptibility in relation to reproductive hormones and pubertal development in girls

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Investigated exposure during in utero windows of development and female puberty. * In utero phthalate exposure was associated with higher peripubertal testosterone. * 3rd trimester phthalate exposure was associated with higher peripubertal DHEA-S. * In utero MEHP was associated with lower odds of breast development.

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Ashlin Klocke: The effects of EDCs on puberty timing is sex specific, depends on exposure window, and affects particular pubertal milestones differently.

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Ashlin Klocke: The effects of EDCs on puberty timing is sex specific, depends on exposure window, and affects particular pubertal milestones differently.

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6009831/

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INTRODUCTION

Puberty is a fundamental process of maturational changes that mark the transition from sexual infancy to reproductive maturity. It is now recognized that the age of onset of breast development, exemplifying an early stage of puberty in girls, has declined in recent decades, although the age of menarche, an advanced pubertal milestone, has not changed significantly ( 1, 2, 3). The data on pubertal timing in boys is inconsistent, with some reporting an earlier onset and others finding no changes in the age of pubertal development in recent decades ( 4, 5). While genetic factors remain the predominant determinant of pubertal timing, the shift towards an earlier age of puberty in the past century coincided with improvements in public health and nutrition, and more recent changes have been attributed to obesity. It has also been speculated that endocrine disrupting compounds (EDCs) are potential contributors to the observed shifts in pubertal timing ( 1, 2, 5, 6). Here we review publications from 2016 and 2017 that examine EDC associations with pubertal milestones, focusing on possible environmental triggers of puberty in both males and females.

General principles of EDC actions

A number of different classes of exogenous compounds with endocrine active properties interfere with endogenous hormone action ( 7). These Endocrine Disrupting Compounds (EDCs) can bind hormone receptors and affect their synthesis, transport, metabolism, and/or elimination. Among the various types of EDCs are persistent organic compounds that bio-accumulate through the food chain and have half-lives of years to decades. These chemicals, pesticides (dichloro-diphenyl-trichloroethane [DDT], pyrethroids), dioxins, polychlorinated biphenyls (PCBs), and flame retardants (polybrominated diphenyl ethers [PBDEs]), are present ubiquitously across the ecosystem and detectable in humans worldwide (reviewed in ( 5, 7). Other EDCs, such as phthalates and phenols, have shorter biological half-lives and are measurable for only a short duration post-exposure, but may still exert long-term health effects. EDCs are not natural ligands, and therefore do not have the same binding specificity and affinity as endogenous hormones, and may elicit non-receptor mediated effects. They exert antagonistic and agonistic actions on hormonal axes and pathways, and may have divergent effects at low versus high concentrations- consistent with their non-monotonic dose response curves. Both acute and delayed (latent) effects of EDCs have been reported. The prenatal, infancy, and childhood periods are considered critical windows of susceptibility due to ongoing developmental processes that are vulnerable to EDCs. The specific health consequences are dependent on the life stage at the time of exposure, particular compound, the endogenous hormonal milieu, and which health outcomes are being examined.

Phenols and phthalates

Phthalates and phenols include a number of hormonally active chemicals with anti-androgenic and estrogenic activity that are present at relatively high concentrations compared to pesticides and other industrial chemicals.

Bisphenol A (BPA) is the phenol that has drawn the most public interest (reviewed in ( 8)). BPA and other phenols are plasticizers used in both medical products and consumer goods, such as medical tubing, plastic bottles and toys, dental sealants, and linings of metal container. BPA is now banned in the production of infant food and beverage containers such as baby bottles, and has been removed from many consumer products. At lower concentrations, BPA and other phenols have weak estrogenic properties but can also compete with endogenous estrogens for binding, and at higher concentrations, they have been shown to have anti-androgenic properties.

To investigate the associations of BPA exposures with puberty, a Thai study compared urinary BPA concentrations in 41 girls with precocious puberty to 47 age matched controls ( 9). The girls with precocious puberty had higher urinary BPA levels than controls, and among the group with early puberty, those who were obese or overweight had higher values than those of normal weight. One challenge with this study is that the investigators used a relatively advanced cut-off of 8-9 years to define early puberty. In addition, samples were collected at the time of the exam, reflecting contemporaneous exposures rather than exposure assessment prior to the outcome measure- the onset of puberty.

Miao and colleagues also conducted a cross-sectional study to investigate the association between BPA exposure and pubertal development in 655 girls aged 9-18 years in Shanghai, China ( 10). Urinary BPA levels were measured and categorized according to whether they were unmeasurable (<LOD) or measurable (>LOD). If they were measurable, they were grouped as "above" or "below" the median concentrations for the cohort. A physician assessed pubertal development by Tanner staging and collected self-reported age at menarche. After adjusting for potential confounders, girls with higher values of BPA were more likely to have delayed menarche compared to girls with undetectable urinary BPA levels. Girls aged 9-12 years with measurable urinary BPA levels were more likely to have reached pubic hair stage 2; while among girls aged >15 years, those with detectable urinary BPA were less likely to have reached pubic hair stage 5. These authors concluded that contemporaneous BPA exposures were associated with delayed menarche and with earlier onset of P2 but later attainment of P5. Again a limitation of this study was that the exposure assessment occurred concurrently with the outcome assessment, making it difficult to establish causality.

BPA and phthalate levels were compared in 42 Turkish girls with idiopathic central precocious puberty, 42 girls with peripheral precocious puberty, and 50 healthy non-obese age-matched girls as controls ( 11). The authors did not find any differences in mean values of urinary BPA, LH, FSH or estradiol among the three groups. However, they reported that plasma DEHP and MEHP, were higher in the group with central precocious puberty than in the other two groups, suggesting a potential central site of action for these phthalate metabolites.

A longitudinal US puberty cohort study based at three centers (the Bay area in northern California, Cincinnati, Ohio, and Manhattan, NY was established through the Breast Cancer Environmental Research Program (BCERP) to examine the role of EDCs on pubertal development ( 12). 1051 girls had urinary levels of phenols and phthalates measured at age 6-8 years and were followed annually ( 13). Higher 2,5-dichlorophenol was associated with a 7-month earlier onset of menarche, while mono-3-carboxypropyl phthalate (MCPP) was associated with a 4-5 month later age of both thelarche and menarche, that was greater in lean girls. The association of MCPP with delayed pubertal onset and menarche was a new and unanticipated finding. MCPP is a metabolite of several phthalates and exposures is through household products as well as diet. These authors also reported that triclosan, an anti-bacterial and anti-fungal agent in soaps and personal care products, and benzophenone-3, a ketone in cosmetics for UV protection were both associated with earlier thelarche but not menarche ( 13).

Wang and colleagues evaluated the association of urinary BPA with pubertal development in 671 Chinese boys aged 9 to 18 years ( 14). Urinary BPA levels were categorized as <LOD, LOD to the 75 th%ile and >75 th%ile levels. The boys were grouped according to sexual maturity staging as early-, mid-, and late puberty. Of note is that all 13 years olds recruited were already in early puberty and that none of the boys < 11 years of age had progressed to late puberty. Moderate levels of urinary BPA correlated with earlier onset of pubic hair and genital stage 2, and showed a trend towards testicular enlargement. Urinary BPA was associated with later attainment of mature milestones, suggesting an association of moderate levels of urinary BPA with slower pubertal progression. These authors concluded that BPA exposure was associated with earlier age of adrenarche and genital stage 2, but delayed attainment of genital stage 5. Adjusting for BMI did not alter these associations.

Flame retardants/polybrominated diphenyl ethers (PBDEs)

Another class of EDCs of particular interest are the flame retardants, or polybrominated diphenyl ethers (PBDEs) used in carpeting, clothing, and furniture. The Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS) was a birth cohort in California established to investigate the health effects of gestational and childhood exposures to agricultural pesticides and other environmental chemicals ( 15). This paper examined the association of flame retardants with pubertal timing in Latino low income mothers in this agricultural community. Four PBDEs were measured in maternal serum during pregnancy or at delivery as a measure of prenatal exposures and childhood exposures were assessed in PBDE measurements at age 9 years in the offspring. The children were seen every 9 months from 9-13 years of age for pubertal staging, and menarche was self-reported. In this cohort of 309 boys and 314 girls, prenatal PBDE exposure was associated with later menarche in girls and earlier pubarche in boys. Associations were stronger for normal weight girls. Childhood PBDE exposure showed a similar trend towards later menarche but it was not statistically significant, and no association was found at all for boys. There was no association between prenatal or childhood PBDE exposure and thelarche or pubarche in girls. These results are similar to those reported for the BCERP cohort, the only other longitudinal cohort study of girls' PBDE exposure ( 12). In the CHAMOCOS cohort, maternal PBDE levels were also associated with higher LH values in the boys at age 12 years ( 16).

A cross sectional case-control study from Italy compared serum PBDEs in children referred for idiopathic central precocious puberty (n=37) and premature thelarche (n=56) to 31 prepubertal control girls ( 17). Serum was collected for PBDE measurement at the time of physical exam. They found that girls with premature thelarche had higher PBDE levels than girls with precocious puberty and controls. This study had the same limitations as the other cross-sectional studies described above, ie they did not assess exposure prior to pubertal onset, and also used a relatively late age of 8 years to define precocious puberty onset. The authors conclude that their findings suggest that PBDE has a peripheral estrogenic action but no central action on the hypothalamic-pituitary-ovarian axis.

PCBs and Dioxins

Eskanazi and colleagues also reported the association of organochlorine chemicals with pubertal timing and reproductive hormones in the CHAMACOS boys, examined at ages 9 to 12.75 years for pubertal staging ( 16). Of the 312 boys who completed the 12 year visit, 234 provided a blood sample for hormone analysis. A 10-fold increase in maternal DDT levels was associated with an 18% decrease in boys' LH and testosterone values, while a 10-fold increase in DDE (a DTT metabolite) was associated only with a decrease in LH. Conversely, higher prenatal PCBs were associated with higher 12 year old FSH values in the boys. These associations between the boys' chemical levels at age 9 years and hormone values at 12 years was mediated by the boys' BMI.

The longitudinal Russian Boys Study cohort examines the associations of mixed childhood exposures with pubertal development ( 18). 473 boys had serum dioxin-like compounds (DLCs) and PCBs measured at 8-9 years of age. Genital staging and testicular volume determination was assessed at annual follow-up visits until age 18 years. The highest quartile of TEQs (a measure of dioxin potency) compared to the lowest was associated with a delay in pubertal onset and maturation by 11.6 months for testicular volume (TV >3 ml for onset; >20 ml for sexual maturity) and 10 months for genital staging (defined as G2 and G5) ( 19). Conversely, non-dioxin-like PCBs co-adjusted for TEQs, were associated with earlier pubertal onset by 8.3 months (TV) and 6.3 months (G2) and earlier sexual maturity by 6-7 months. These compounds were not associated with changes in timing of pubic hair development. In this cohort, higher quartiles of TCDD and PCDD TEQs were also associated with lower sperm concentration, total sperm count, and total motile sperm count (p-trends ≤ 0.05) ( 20), suggesting that childhood exposures to these organochlorines not only altered pubertal timing but were also associated with poorer semen parameters, raising concerns about long term effects of EDCs on reproductive function and fertility.

Dietary exposures

One area of particular interest to parents is whether dietary exposures through dairy, meat or soy intake can alter the timing of puberty. A longitudinal study of 515 Chilean girls recruited at ages 3-4 years investigated the relationship between dairy intake and pubertal changes ( 21). Breast DXA was performed in 290 girls and 324 girls reported age of menarche. Breast volume was highest in girls who drank more sweetened milk-based drinks and lowest in those who ate the most yogurt. Consumption of yogurt and low fat milk was associated with later menarche by 4.6 months. Another group looked at 367 mother-daughter dyads in the UK Avon Longitudinal Study of Parents and Children ( 22). Six phytoestrogens were measured in urine collected during pregnancy. Enterodiol (a lignan metabolite) was associated with lower risk of earlier menarche, defined as <11.5 years of age, while O-DMA (a daidzein metabolite) was associated with an increased risk of earlier menarche, The four other lignans studied, including enterolactone were not associated with age of menarche. This study controlled for timing of sample collection, race, maternal education, age at menarche delivery, BMI, diet and smoking history, infant feeding (maternal breast milk vs soy milk), childhood BMI and vegetarianism. These findings contrasted with the BCERP data that showed an association of enterolactone, a lignan metabolite, both a 4-5 month later age of menarche as well as later thelarche ( 13). This association was more marked in leaner girls and the authors speculate that the conflicting findings may be due to different exposure windows (gestational versus childhood) or sample size.

A prospective study of 456 girls in Bogota, Columbia, investigated the association of red meat intake at ages 5-12 and age at menarche via food-frequency questionnaires ( 23). They found earlier menarche with higher red meat consumption. Those who ate meat more than twice daily had significantly earlier menarche than those who ate it less than four times per week. The investigators also found that those who ate canned tuna/sardine intake more than once per week had later menarche than those who ate them less than once per month. Average age of menarche was 12.4 years in this study. Intake of dairy, poultry, eggs, and other fish were not significantly associated. The meat effect was attenuated when adjusted for BMI.

Pesticides

Another area of considerable interest is whether there is a difference between organic food and conventional food. A study examined 3-phenoxybenzoic acid (3-PBA), a nonspecific metabolite of 18 pyrethroids in 305 9-15 year old Chinese girls with self-reported pubertal staging and menarche status ( 24). These insecticides can be both estrogenic and anti-androgenic, and are used in homes and for agriculture. Exposure occurs via diet as well as through skin. Higher 3-PBA was associated with later pubertal development for breast development, pubic hair, and menarche. The Avon Longitudinal Study examined gestational exposures to weakly estrogenic organochlorines ( 24). Exposure occurs through fatty foods (meat, fish, dairy), as well as via the placenta and maternal breast milk. This nested case-control study measured maternal serum organochlorine concentrations at random times in pregnancy. Among 3682 girls who self-reported age at menarche; 218 girls were 'early' (menarche before 11.5 years), and 230 were "controls" (ages 11.5 years or older). There were no associations between in utero exposure to organochlorines and early menarche. The investigators controlled for race, mother's BMI, mother's age at menarche and at delivery, SES, birth order, child's BMI at age 7, duration of breastfeeding, child's birth weight, and trimester of sample collection.

The associations of insecticides measured as urinary metabolites (3-PBA) with self-reported pubertal staging and self-measured testicular volumes were examined in 463 Chinese boys, ages 9-16 years ( 24). 3-PBA was detected in 98.7% of all urine samples and was inversely correlated with the frequency of organic food intake. After controlling for confounders such as SEC, age, and other birth history factors, higher levels of 3-PBA were associated with increased odds of being at a more advanced stage of puberty and having higher urinary gonadotropins.

Air pollution

Lastly, the BCERP group published two studies on the effects of tobacco exposure and air pollution. Prenatal and contemporary second-hand tobacco exposure was assessed by questionnaire and urinary cotinine measurement at enrollment. Girls with higher prenatal and secondhand smoke exposure have earlier pubarche but not thelarche. Pubarche occurred 10 months earlier in the highest vs the lowest prenatally exposed groups ( 25). To examine the effects of exposure to the polycyclic aromatic hydrocarbons in air pollution, residential proximity to traffic metrics was used as a proxy measure ( 26). Proximity to major roads at age 6-8 years was associated with earlier PH across all ethnic groups, controlling for confounders. Higher exposed girls had pubarche 2-9 months earlier than lower exposed. There was no association with thelarche timing.

Contributor Information

Louise C. Greenspan, Pediatric Endocrinologist, Kaiser Permanente, San Francisco, Clinical Professor of Pediatrics, University of California, San Francisco School of Medicine, (415) 833-3533, fax (415) 833-4177.

Mary M. Lee, Chair and the Stoddard Professor of Pediatrics, Department of Pediatrics, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, 508-857-3199, 508-856-1540.

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Endocrine Disrupters and Pubertal Timing

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Puberty is a fundamental process of maturational changes that mark the transition from sexual infancy to reproductive maturity. It is now recognized that the age of onset of breast development, exemplifying an early stage of puberty in girls, has declined in recent decades, although the age of menarche, an advanced pubertal milestone, has not changed significantly ( 1, 2, 3).

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Chloe Mullins: Endocrine Disrupting Chemicals and Their Effects during Female Puberty: May increase the risk of breast cancer later in life

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Chloe Mullins: Endocrine Disrupting Chemicals and Their Effects during Female Puberty: May increase the risk of breast cancer later in life

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Abstract

Puberty is the process of physical changes between childhood and adulthood during which adolescents reach sexual maturity and become capable of reproduction. It is considered one of the main temporal windows of susceptibility for the influence of the endocrine-disrupting chemicals (EDCs). EDCs may act as single chemical agents or as chemical mixtures; they can be pubertal influencers, accelerating and anticipating the processing of maturation of secondary sexual characteristics. Moreover, recent studies have started to point out how exposure to EDCs during puberty may predispose to breast cancer later in life. In fact, the estrogen-mimicking endocrine disruptors (EEDs) may influence breast tissue development during puberty in two main ways: the first is the action on the proliferation of the breast stromal cells, the second concerns epigenetic mechanisms. The aim of this mini-review was to better highlight what is new and what is not completely known regarding the role of EDCs during puberty.

Keywords: puberty, Endocrine disrupting chemicals (EDCs), Window of susceptibility, Early Puberty, Estrogen-mimicking Endocrine Disruptors (EEDs), breast development, breast cancer

1. Background

Endocrine-disrupting chemicals (EDCs) influence one or more functions of the endocrine system and cause several adverse health effects in human beings. They are ubiquitous in nature, being found in industrial chemicals, drugs, plastics, and other materials common in everyday life [ 1].

EDCs may represent a risk for our health, working through many different mechanisms of action. In fact, they may take several routes of absorption (oral, dermal, etc.), affect different temporal windows (prenatal and postnatal life, puberty), and be made up of many combinations of chemicals (mixtures), leading to extremely difficulty in interpreting their influence on human wellbeing.

Moreover, their effect seems to be transgenerational, occurring over at least two or three generations. As the window of susceptibility, puberty is considered one of the hot spots in the lifetime when EDCs may exert their effects [ 2].

The main aims of this mini-review were (1) to provide an update of what we know about the relationships between EDCs and pubertal trends, and (2) to highlight the new role of EDCs as influencers of breast cancer predisposition later in life.

2. Puberty and Pubertal Onset

Puberty is the process of physical change that occurs between childhood and adulthood, during which adolescents reach sexual maturity and become capable of reproduction.

The main clinical manifestations of puberty are the growth spurt, progression of secondary sexual characteristics, development of gonads, and changes in body composition. These features lead to an increase in strength and endurance [ 3].

During pubertal development, changes in the brain activity are also detectable: the frontal lobes increase their volume and activity, leading to more complex psychological understanding [ 4]. The whole process involves years of development on both a mind and a body level.

In the last two centuries, pubertal timing has changed dramatically. Data show that the age of the menarche is experiencing a declining trend, from 17 years old at the beginning of nineteenth century to 13 years old by the 50s [ 5]. In the United States, the onset of puberty in girls is occurring earlier than previous studies have documented, with breast and pubic hair development appearing on average 1 year earlier in white girls and 2 years earlier in African-American girls. Beyond these data, it is also underlined how this early onset of puberty seems not to be correlated with a parallel anticipation of the menarche age. According to the most recent studies, the age of menarche has decreased by only few months [ 6].

As far as timing is concerned, in the 60s, Tanner et al. identified the normal range of pubertal onset as between 8 and 13 years in girls, and between 9 years and 6 months and 13 years and 8 months in boys [ 3, 4]. Therefore, the development of secondary sexual characteristics is considered precocious when it happens in girls under the age of 8 years old and in boys younger than 9 years old.

The timing of pubertal onset depends on genetic and environmental factors. The genes involved in this process are the ones that control GnRH hypothalamic secretion, pituitary development and its functioning, hormone synthesis and bioactivity, energy homeostasis and growth, and potential peripheral feedback from sex steroids [ 7].

Studies show that some populations are much more likely to develop precocious puberty: girls; African-Americans; individuals presenting other medical conditions (McCune-Albright syndrome, congenital adrenal hyperplasia) or experiencing or having experienced radiation therapy of the central nervous system; those exposed to sex hormones; children that may have been in contact with estrogen or testosterone cream or ointment, or with other substances such as adults' medications, dietary supplements, or food containing phytoestrogens.

The nutritional status of the pediatric population influences the age of pubertal onset. Adolescents with anorexia nervosa usually reach menarche later [ 8], highlighting a correlation between nutritional state and development. Girls who are overweight or obese have a higher probability of being diagnosed with central precocious puberty or early puberty compared to the general population [ 9]. Genomic studies and human epidemiological data suggest a correlation between early infancy weight gain, early pubertal onset, and later obesity in adulthood, which can be explained by the overlapping of genes involved in pubertal onset and obesity [ 10]. Moreover, obesity induces hyperinsulinemia and hyperleptinemia, which can affect linear growth and advanced puberty [ 11].

Both events, pubertal timing and obesity, are influenced by the environment, and there is a strong interaction between genes and environments during critical periods of development such as puberty. This interaction is due to substances, EDCs, which derail the normal hormonal process.

3. EDCs and Puberty

In 2015, the Endocrine Society defined an EDC as "an exogenous chemical or mixture of chemicals, which interferes with any aspect of hormone action". The EDCs affect the control of energy balance, reproduction, hormone-sensitive cancer, thyroid, neurodevelopment, and the neuroendocrine system. They can act at any level in the hypothalamic-pituitary-gonadal system or the peripheral tissue. During a lifespan, there are some critical points (windows of development) that are important because during them, cells are rapidly proliferating and epigenetic changes are more likely to occur. These periods are fetal life, neonatal life, and puberty [ 12].

Moreover, EDCs act at any level in the hypothalamic-pituitary-gonadal-peripheral tissue-endocrine axis. Indeed, they can act not only as agonists or antagonists of estrogen receptors, but can also target and act against androgen and progesterone receptors. They may also mimic the natural occurrence of estrogens and androgens. Furthermore, EDCs bind to cell receptors and block the functions of endogenous hormones, thus acting as anti-estrogens and anti-androgens [ 13].

The most important effect of EDCs is considered to be the anticipation of or, more generally, impact on the timing of female puberty [ 14]. Nevertheless, this effect is difficult to demonstrate since low-level exposures to a mixture of EDCs may mask the effects of some of them. Moreover, inter-individual variation in susceptibility might interfere with an objective analysis. Finally, the effects on humans to EDCs exposure may show up with a considerable delay.

We analyzed the most important and well-known EDCs that might interfere with natural pubertal timing ().

Table 1

Atrazine

Herbicide

<1 day

Dermal absorption, inhalation

Prenatal life

ER agonist

Early puberty

↑incidence of breast cancer

BPA

Polycarbonate plastics, epoxy resins, plastic toys and bottles, lining of food cans

4-5 h

Ingestion, dermal absorption, inhalation

Prenatal life
Puberty

ER agonist
Antiandrogen

Premature thelarche

Induce neoplastic transformation

DES

Nonsteroidal estrogen medication used in the past

3-6 h

Ingestion

Prenatal life
Puberty

ER agonist
Epigenetic silencing of mRNA

Not known

↑breast cancer

DDT/DDE

Contaminated water, soil crops, fish

6-10 years

Ingestion, dermal absorption, inhalation

Prenatal life and lactation period

ER agonist
Antiandrogen
Antiprogestin
Induction of aromatase

Precocious puberty
Anticipated menarche
Later onset of puberty

↑cells proliferation index and mammary tumor cells proliferation

Dioxin

By-product of chlorinated herbicide production, smelting, chlorine bleaching of paper

7-11 years

Ingestion, inhalation

Prenatal and lactation
Puberty

ER agonist
Antiandrogen
Interfere with sex-steroid synthesis

Delayed breast development

Induce epigenetic silencing of key tumor suppressor (TCDD)

Parabens

Cosmetics and pharmaceutical products, toothpaste and food preservatives.

<24 h

Dermal absorption, ingestion

Pre- post-natal life and puberty

ER agonist

Not known

↑incidence of breast cancer

PBDEs/PBB

Furniture, mattresses, carpet pads, automobile seats, flame-retardant textiles

2 days-3 months

Ingestion, dermal abruption, inhalation

Prenatal life, perinatal period, peripuberty

ER agonist/antagonist
Antiandrogen

Early pubic hair in boys
Premature thelarche
Early/late menarche
Early puberty

Not known

PCBs

Contaminated air and food, skin contact with old electrical equipment

12 days-16 years

Ingestion, dermal absorption, inhalation

Prenatal life

ER agonist/antagonist
Antiandrogen

Early onset of menarche
Delayed pubertal development

Accumulates in breast adipose tissue

PFOA

Carpeting, upholstery, floor wax, firefighting foam, sealants

3.8 years

Ingestion, inhalation

Prenatal life
Puberty

Induced estrogen

Delayed pubertal development

↑susceptibility to carcinogenesis in future generations

Phthalates

Contaminated food, PVC plastics and flooring, personal care, medical devices and tubing

12 h

Ingestion, dermal absorption, inhalation

Prenatal life

ER agonist/antagonist
Antiandrogen
Interfere with androgen synthesis

Early puberty
Premature thelarche
Delayed pubic hair development

↑cells'proliferation in the breast

Pyrethroids

Contaminated water, soil, food

10 h

Ingestion, dermal abruption, inhalation

Androgen agonist

Delayed puberty

Not known

Vinclozolin

Fungicide

1-3 months

Ingestion, dermal absorption, inhalation

Prenatal life
Puberty

ER agonist
Antiandrogen
Epigenetic alteration

Not known

Dysregulates the gland development
↑formation of mammary tumor

3.1. BPA

Bisphenol A (BPA) is a precursor of plastics, polycarbonates, and epoxy resins. BPA-based plastic is clear and tough, and it is used in a lot of common objects such as plastic bottles, food storage containers, baby bottles, and CDs. Epoxy resins are utilized to line water pipes, as coating on the insides of many food and beverage containers, and in making thermal paper. This chemical is almost ubiquitous, which means that people are at great risk for exposure and, even if the estrogen receptor agonist activity is weak, its potential should not be underestimated. BPA also acts as an anti-androgen.

Studies demonstrate how this EDC is associated with premature thelarche, precocious puberty and pubertal development [ 15, 16, 17].

3.2. DDT/DDE

Dichlorodiphenyltrichloroethane, commonly known as DDT, is an organochlorine. It was originally developed as an insecticide for use in agriculture and it is colorless, tasteless, and odorless. Because of these features, exposure to DDT is imperceptible, but being exposed during fetal life and lactation can affect sexual development. Even if DDT is banned from the market, it will remain present in the environment as a persistent organic pollutant (POP). Moreover, in some low-income countries, it is still widely used.

DDT and its metabolite dichlorodiphenyldichloroethane (DDE) are the most studied of the pesticides that have been identified as EDCs. Both have estrogenic, anti-androgenic, and antiprogestin effect and induce aromatase. According to Vasiliu et al. (2004), there is an association between exposure to these chemicals and precocious puberty, delayed puberty, and earlier age of menarche [ 18, 19].

3.3. Dioxins

Dioxins are byproducts of burning or various industrial processes such as chlorinated herbicide production, smelting, or chlorine bleaching of paper. They have an estrogenic and anti-androgenic effect, and they interfere with sex steroid synthesis. Exposure to dioxins during the prenatal and lactation period is associated with delayed breast development. On the other hand, exposure to these chemicals has not been correlated with age of menarche or pubic hair development [ 20].

3.4. PBDEs and PBB

Flame retardants are a group of chemicals added to manufactured materials (plastics, textiles, surface finishes, and coatings). They are intended to prevent or slow down the further development of ignition. Some examples of such chemicals are polybrominated diphenyl ethers (PBDEs) and polybrominated biphenyl (PBB). They act as estrogens and anti-androgens. Data show an association between PBDEs and premature thelarche, earlier menarche, and earlier pubertal development [ 21, 22]. Furthermore, prenatal exposure to PBDEs is associated with later menarche in girls and earlier development of pubic hair in boys [ 23].

Moreover, exposure during the peri-pubertal period seems to interfere with reproductive development [ 24].

3.5. PCBs

Polychlorinated biphenyl (PCBs) is a dioxin-like compound derived from biphenyl. This chemical is widely used as a dielectric and coolant fluid in electrical apparatuses, in carbonless copy papers, and in heat transfer fluids. Its mechanism of action is rather similar to that of dioxins, but compared to dioxins, there is evidence that exposure during the prenatal period leads to early onset of menarche and to delayed pubertal development [ 25].

3.6. Phthalates

Phthalates are esters of phthalic anhydride. They are used as liquid plasticizers in plastic, flooring, personal care products, medical devices, and tubing. They increase the flexibility, transparency, durability, and longevity of materials. Their most common use is to soften polyvinyl chloride (PVC).

Their endocrine-disrupting mechanism is not fully clarified, but they either act as estrogen receptor agonists and antagonists, or androgen receptor antagonists. Moreover, they can also disrupt androgen synthesis.

Different studies have demonstrated a significant association with premature thelarche and precocious or early puberty [ 26, 27].

Phthalates can be classified as low- or high-molecular-weight phthalates. Depending on the class and the timing of exposure, different outcome have been observed. High-molecular-weight phthalate levels several years before puberty are associated with later pubic hair development and younger age of menarche; in contrast, low-molecular-weight phthalate levels are related to advanced breast or pubic hair development [ 28, 29].

3.7. Pyrethroids

A pyrethroid is an organic compound similar to a natural pyrethrin, which comes from the pyrethrum flower. This chemical is found in most common commercial household insecticides. It has been reported both as a weak androgen and for its association with late onset of puberty in girls [ 30, 31].

4. Puberty and EDCs: A Temporal Window for Breast Cancer

In girls, the first physical sign of puberty is thelarche, with physical breast changes in both the stroma and the epithelium. The amount of fibrous and fatty tissue in the stroma increases (the adult non-lactating breast is more than 80% composed of stroma) [ 32]. The ducts first elongate, proliferate, and form lobular structures composed of the growth ductal tree; with the achievement of sexual maturity, they decrease in size, the proliferative phase reducing [ 33]. Thus, puberty is the time in which the breast tissue undergoes rapid changes regulated by small fluctuations of endogenous hormones [ 34]. These rapid structural and cellular modifications are thought to create a "window of susceptibility" [ 35].

Breast tissue during this period of morphogenesis and re-modeling is particularly sensitive to carcinogenic effects; studies have demonstrated that exposure to radiation, DDT, medications, and disease before and during puberty increases breast cancer risk in adulthood [ 36, 37, 38, 39, 40].

The role of estrogen in breast development and tumorigenesis is confirmed. Prolonged and uninterrupted exposure to endogenous estrogen and administration of exogenous estrogens to postmenopausal women increases the risk of cancer. In contrast, gonadectomy in premenopausal women reduces the risk of breast cancer, while pharmacological agents serve to treat the same condition, blocking estrogen biosynthesis or estrogen receptors.

It is not entirely clear how human exposure to specific EDCs can be linked to breast cancer. The EDCs of interest in this mechanism are those with estrogenic activity: they are called estrogen-mimicking endocrine disruptors (EEDs).

The main effects of EEDs are two-fold: the first is their action on the proliferation of the stromal cells, the second concerns epigenetic mechanisms. EEDs affect stromal cells, interfering with the estrogen signal pathway [ 40, 41].

Moreover, EEDs may modify the breast matrix composition. All these changes macroscopically create a different breast density, which is one of the strongest predictors of premenopausal breast cancer risk [ 42].

Breast cancer is also associated with epigenetic changes such as DNA methylation, histone modifications, and non-coding RNAs (ncRNAs). These mechanisms work together to determine whether the epigenome may expresses itself and how, facilitating gene expression control, X chromosome inactivation, and/or genomic imprinting [ 43].

The most important chemical EEDs and their activities to promote or facilitate breast cancer are analyzed below ().

4.1. Atrazine

Atrazine is an herbicide used to prevent pre- and post-emergence broadleaf weeds in crops (corn and sugarcane) and turf. It can induce aromatase activity, increasing the level of estrogen in the body. Atrazine is not classified as a carcinogen or mutagen because its link with breast cancer is not totally clear. On this issue, studies are contradictory. Some studies on human reaction to atrazine exposure have seemed to detect a weak association between atrazine and breast cancer [ 44].

4.2. BPA

BPA increases the methylation levels of key genes associated with tumor development (e.g., BRCA1, CDKN2A, etc.), altering the epigenome in order to promote proliferation, senescence, and tumor development [ 45].

3D cultures of breast cells have shown that BPA reduces the number of tubules, increases spherical masses, and causes more deformed acini, indicating an ability to induce neoplastic transformation [ 46]. This because BPA exposure increases HOXB9 expression in breast cells, both in vitro and in vivo, through a mechanism that involves increased recruitment of transcription and chromatin modification factors. HOXB9 is a homeobox-containing gene that plays a key role in mammary gland development, and is associated with breast cancer. BPA acts as a competitor for estrogen receptors (ERs), which leads to the activation of this gene through chromatin modifications (histone methylation and acetylation) [ 47].

4.3. DDT/DDE

The estrogenic property of these compounds and their prominent role in mRNA regulation increase epithelial cells' proliferation index and mammary tumor cell proliferation [ 48]. Moreover, a prospective study showed that girls exposed to high levels of DDT and DDE were more likely to develop breast cancer than those who were exposed to a lower dose. On top of that, the younger the age at which a person was exposed to these compounds, the higher the risk of developing breast cancer [ 38]. These data support the hypothesis that the age of exposure must be considered for risk assessment.

4.4. DES

Diethylstilbestrol (DES) is a nonsteroidal estrogen medication that was used in the past to support during pregnancy women with a history of recurrent miscarriage, as hormone therapy for menopausal symptoms and estrogen deficiency, and as a treatment for breast cancer.

The development of breast cancer in women exposed to DES is mediated by the proliferative effects of estrogens, and by a differential expression of ncRNAs. In particular, epigenetically silenced mRNA causes disordered regulation of the p53-mediated apoptotic pathway [ 49].

4.5. Parabens

Parabens are a widely used class of preservatives in cosmetic and pharmaceutical products. They are used for their bactericidal and fungicidal properties. They are commonly found in shampoos, commercial moisturizers, topical/parenteral pharmaceuticals, suntan products, make-up, and toothpaste, and are also used as food preservatives.

They have estrogen-like effects and they may be associated with breast cancer etiology. This is demonstrated by the association between the utilization of paraben-containing body care products in the Western world and the increased incidence of breast cancer [ 50].

4.6. PCBs

Studies have demonstrated that polychlorinated biphenyls accumulate in breast adipose tissue, but so far, an association has not been proven between PCB exposure and breast cancer [ 51]. This is probably because PCBs are mixtures of congeners with varied properties, and because breast cancers of different subtypes are etiologically distinct diseases. The absence of stratified subgroup analysis on individual PCB exposure and patients with specific biological subtypes of breast cancer may have resulted in an underestimation of the correlations between PCBs and breast cancer [ 52]. Moreover, PCBs' mechanisms of action are different. In fact, the natures of the biochemical and toxic effects of PCB congeners are largely determined by their structure, as well as different PCB congeners sometimes exerting conflicting actions. Some PCBs act as estrogen agonists both in vitro and in experimental animal systems; some others have been shown to be anti-estrogenic, while others are enzyme inducers of phenobarbital (PB)-type cytochrome P450 [ 53]. The main mechanism involved in breast cancer seems to be the estrogen agonist mechanism.

4.7. PFOA

Perfluorooctatonic acid (PFOA) serves as a surfactant in the emulsion polymerization of fluoropolymers and as a building block for polymeric materials. It is used for various industrial applications (carpeting, upholstery, floor wax, firefighting foam, sealants etc.). Data show how in CD-1 and C57B1/6 mice, early PFOA exposure alters the mammary gland without changing other pubertal endpoints [ 54]. This development may increase susceptibility to carcinogenesis in future generations.

4.8. Phthalates

One characteristic of phthalates is their lipophilicity, so they accumulate in breast tissue where they cause modification of the mammary gland [ 55]. Epigenetic changes due to exposure to phthalates increase the proliferation of cells in the breast [ 56].

4.9. TCDD

2,3,7,8-Tetracloridibenzo-p-dioxin, or TCDD, is a famous organochlorine mainly known as a contaminant in Agent Orange, an herbicide used in the Vietnam War. The scientific community is aware of its catastrophic effects, including birth defects, pregnancy loss, and increased rates of cancer. In 2012, a retrospective study showed a significantly higher prevalence of breast cancers in women working in a chemical pesticide establishment due to their exposure to high levels of TCDD [ 57].

TCDD has a significant effect on the epigenome in breast and other tissues. It is interesting to note that TCDD induces an epigenetic silencing of key tumor suppressors, which may have a role in breast cancer development [ 58].

4.10. Vinclozolin

Vincozolin is a common dicarboximide fungicide used to control diseases in vineyards and on fruits and vegetables. Exposure to this pesticide in female rats causes the formation of mammary tumors and dysregulates gland development [ 59]. It promotes an epigenetic alteration in the germ line that creates a transgenerational disease state [ 60].

5. Conclusions and Perspectives

Most of these EDCs are present in everyday objects and in various common solutions; it is almost impossible not to be exposed to these chemicals, even if laws were to ban the use of most of them and restrict the others. It is known that environmental factors, including EDCs, are the most important determinant in explaining the puberty secular trend and the timing of menarche. The anticipation of these two endpoints of development can be explained by the action of environment during the pubertal period, and how it may advance and activate the central axis.

According to Parent et al., this paradigm of EDCs could be revised; in fact, environmental factors can affect puberty and reproduction through central and peripheral mechanisms, taking advantage of the critical windows of development [ 61]. During these periods, EDCs can modify genetic or epigenetic pathways.

The epigenetic pathways are interesting because of their long-term action. Studies on animals have demonstrated the effects of a mixture of EDCs on female sexual development over three generations, and that the effects are even bigger in the second and third generations (delayed vaginal opening, decreased percentage of regular estrous cycle, and decreased GnRH interpulse interval) than in the first. The reproductive phenotypes in rats are associated with alterations in both transcriptional and histone posttranslational modifications of hypothalamic genes involved in reproductive competence. Moreover, females of the first generation exposed in utero to these EDCs showed alteration of the thyroid and impaired maternal behavior, which could cause additional epigenetic change in subsequent generations [ 62].

EDCs can also act as obesogens and promote early adiposity rebound, changing metabolic or peripheral signals, and increases in adrenal androgen levels, inducing early pubertal development [ 63].

EDCs, and in particular EEDs, may produce epigenetic changes not only in puberty genes but also in other tissues and cells. In fact, these compounds could act as carcinogens and induce breast cancer. Clearly, EEDs are not the only factors responsible for these phenomena. Their effect is influenced by environmental carcinogens and unhealthy foods such as refined sugar, processed fats, and food additives, which promote molecular damage that increases the risk of breast cancer.

Puberty amplifies this process; young women today live in an obesogenic world and they are exposed to several contemporaneous risk factors, especially girls who live in disadvantaged conditions [ 64].

Moreover, the exposure to these compounds and the development of mammary glands expands the epigenetic changes that EEDs cause in breast tissue, promoting tumorigenesis and increasing the chance of damage that might prove to be significant.

In conclusion, exposure to EDCs during puberty increases the risk of developing breast cancer during the lifetime.

Although these results are still somewhat controversial, it is important for pediatricians to keep in mind the new risks for young girls during puberty, and to provide the right information to their families.

Author Contributions

Conceptualization, L.I.; methodology, L.I. and L.L. (Licia Lugli); resources, L.L. (Laura Lucaccioni), V.T.; writing-original draft preparation, L.L. (Laura Lucaccioni), V.T., L.M.; writing-review and editing, N.B., A.B., B.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare no conflicts of interest.

References

# data[].attributes.attachment_link.title
Endocrine-Disrupting Chemicals and Their Effects during Female Puberty: A Review of Current Evidence

# data[].attributes.attachment_link.description
Puberty is the process of physical changes between childhood and adulthood during which adolescents reach sexual maturity and become capable of reproduction. It is considered one of the main temporal windows of susceptibility for the influence of the endocrine-disrupting chemicals (EDCs).

# data[].attributes.headline
Skylar McFadden: increased exposure to BPA, dichlorobenzene, and household chemicals has a correlation with precocious puberty. 

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Skylar McFadden: increased exposure to BPA, dichlorobenzene, and household chemicals has a correlation with precocious puberty. 

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EarthTalk®
E - The Environmental Magazine

Dear EarthTalk: Is it true that American kids are going through puberty earlier today than in previous generations, and are there any environmental causes for this? - Paul Chase, Troy, NY

Research indicates that indeed Americans girls and boys are going through puberty earlier than ever, though the reasons are unclear. Many believe our widespread exposure to synthetic chemicals is at least partly to blame, but it's hard to pinpoint exactly why our bodies react in certain ways to various environmental stimuli.

Researchers first noticed the earlier onset of puberty in the late 1990s, and recent studies confirm the mysterious public health trend. A 2012 analysis by the U.S. Centers for Disease Control and Prevention (CDC) found that American girls exposed to high levels of common household chemicals had their first periods seven months earlier than those with lower exposures. "This study adds to the growing body of scientific research that exposure to environmental chemicals may be associated with early puberty," says Danielle Buttke, a researcher at CDC and lead author on the study. Buttke found that the age when a girl has her first period (menarche) has fallen over the past century from an average of age 16-17 to age 12-13.

Earlier puberty isn't just for girls. In 2012 researchers from the American Academy of Pediatrics (AAP) surveyed data on 4,100 boys from 144 pediatric practices in 41 states and found a similar trend: American boys are reaching puberty six months to two years earlier than just a few decades ago. African-American boys are starting the earliest, at around age nine, while Caucasian and Hispanics start on average at age 10.

One culprit could be rising obesity rates. Researchers believe that puberty (at least for girls) may be triggered in part by the body building up sufficient reserves of fat tissue, signaling fitness for reproductive capabilities. Clinical pediatrician Robert Lustig of Benioff Children's Hospital in San Francisco reports that obese girls have higher levels of the hormone leptin which in and of itself can lead to early puberty while setting off a domino effect of more weight gain and faster overall physical maturation.

Some evidence suggests that "hormone disrupting" chemicals may also trigger changes prematurely. Public health advocates have been concerned, for example, about the omnipresence of Bisphenol A (BPA), a synthetic chemical in some plastics, because it is thought to "mimic" estrogen in the body and in some cases contribute to or cause health problems. BPA is being phased out of many consumer items, but hundreds of other potentially hormone disrupting chemicals are still in widespread use.

Dichlorobenzene, used in some mothballs and in solid blocks of toilet bowl and air deodorizers, is also a key suspect in triggering early puberty. It is already classified as a possible human carcinogen, and studies have linked prenatal exposure to it with low birth weight in boys. The U.S. Environmental Protection Agency (EPA) has recently made screening Dichlorobenzene for hormonal effects a priority.

Parents can take steps to reduce our kids' so-called "toxic burden": Buy organic produce, hormone- and antibiotic-free meat and dairy and all-natural household cleaners. And keep the dialogue going about healthy food and lifestyle habits so kids learn how to make responsible, healthy choices for themselves.

CONTACTS: CDC, www.cdc.gov; AAP, www.aap.org.

EarthTalk® is written and edited by Roddy Scheer and Doug Moss and is a registered trademark of E - The Environmental Magazine (www.emagazine.com). Send questions to: earthtalk@emagazine.com. Subscribe: www.emagazine.com/subscribe. Free Trial Issue: www.emagazine.com/trial.

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centers for disease control and prevention

# data[].attributes.attachment_link.title
Rises in Early Puberty May Have Environmental Roots

# data[].attributes.attachment_link.description
EarthTalk® E - The Environmental Magazine Dear EarthTalk : Is it true that American kids are going through puberty earlier today than in previous generations, and are there any environmental causes for this? - Paul Chase, Troy, NY Research indicates that indeed Americans girls and boys are going through puberty earlier than ever, though the reasons are unclear.

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Olivia Kelly: A review done on several of the most common EDCs and their effects on puberty in biological females, most of which result in an increased risk of breast cancer.

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Olivia Kelly: A review done on several of the most common EDCs and their effects on puberty in biological females, most of which result in an increased risk of breast cancer.

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Neonatal Intensive Care Unit, Department of Medical and Surgical Sciences of the Mothers, Children and Adults, University of Modena and Reggio Emilia, via del Pozzo 71, 41124 Modena, Italy

Post Graduate School of Paediatrics, Department of Medical and Surgical Sciences of the Mothers, Children and Adults, University of Modena and Reggio Emilia, via del Pozzo 71, 41124 Modena, Italy

Pediatric Unit, Department of Medical and Surgical Sciences of the Mothers, Children and Adults, University of Modena and Reggio Emilia, via del Pozzo 71, 41124 Modena, Italy

Author to whom correspondence should be addressed.

Received: 3 March 2020 / Revised: 12 March 2020 / Accepted: 14 March 2020 / Published: 18 March 2020

Puberty is the process of physical changes between childhood and adulthood during which adolescents reach sexual maturity and become capable of reproduction. It is considered one of the main temporal windows of susceptibility for the influence of the endocrine-disrupting chemicals (EDCs). EDCs may act as single chemical agents or as chemical mixtures; they can be pubertal influencers, accelerating and anticipating the processing of maturation of secondary sexual characteristics. Moreover, recent studies have started to point out how exposure to EDCs during puberty may predispose to breast cancer later in life. In fact, the estrogen-mimicking endocrine disruptors (EEDs) may influence breast tissue development during puberty in two main ways: the first is the action on the proliferation of the breast stromal cells, the second concerns epigenetic mechanisms. The aim of this mini-review was to better highlight what is new and what is not completely known regarding the role of EDCs during puberty. View Full-Text

Lucaccioni, L.; Trevisani, V.; Marrozzini, L.; Bertoncelli, N.; Predieri, B.; Lugli, L.; Berardi, A.; Iughetti, L. Endocrine-Disrupting Chemicals and Their Effects during Female Puberty: A Review of Current Evidence. Int. J. Mol. Sci. 2020, 21, 2078. https://doi.org/10.3390/ijms21062078

AMA Style

Lucaccioni L, Trevisani V, Marrozzini L, Bertoncelli N, Predieri B, Lugli L, Berardi A, Iughetti L. Endocrine-Disrupting Chemicals and Their Effects during Female Puberty: A Review of Current Evidence. International Journal of Molecular Sciences. 2020; 21(6):2078. https://doi.org/10.3390/ijms21062078

Chicago/Turabian Style

Lucaccioni, Laura, Viola Trevisani, Lucia Marrozzini, Natascia Bertoncelli, Barbara Predieri, Licia Lugli, Alberto Berardi, and Lorenzo Iughetti. 2020. "Endocrine-Disrupting Chemicals and Their Effects during Female Puberty: A Review of Current Evidence" International Journal of Molecular Sciences 21, no. 6: 2078. https://doi.org/10.3390/ijms21062078

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# data[].attributes.attachment_link.title
Endocrine-Disrupting Chemicals and Their Effects during Female Puberty: A Review of Current Evidence

# data[].attributes.attachment_link.description
Puberty is the process of physical changes between childhood and adulthood during which adolescents reach sexual maturity and become capable of reproduction. It is considered one of the main temporal windows of susceptibility for the influence of the endocrine-disrupting chemicals (EDCs).

# data[].attributes.headline
Haylie Dillard: Being exposed to EDC earlier or later in life for girls can lead to different outcomes through mechanism and may be linked to environmental factors 

# data[].attributes.subject
Haylie Dillard: Being exposed to EDC earlier or later in life for girls can lead to different outcomes through mechanism and may be linked to environmental factors 

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6603611/

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Abstract

Over the last decades, the onset of puberty in girls has occurred earlier, but the tempo of pubertal progression has been relatively slower, resulting in a younger age at puberty onset without a change in age at menarche. Sufficient energy availability and adiposity contribute to early pubertal development, and environmental factors, such as endocrine-disrupting chemicals (EDCs), may affect not only the control of energy balance, but also puberty and reproduction. EDCs are hormonally active substances that can perturb puberty by acting both peripherally on target organs, such as adipose tissue or adrenal glands, and/or centrally on the hypothalamic-pituitary-gonadal (HPG) axis. Depending on whether the exposure takes place earlier during fetal and neonatal life or later during early childhood, EDCs can lead to different outcomes through different mechanisms. Evidence of associations between exposures to EDCs and altered pubertal timing makes it reasonable to support their relationship. However, human epidemiologic data are limited or inconsistent and cannot provide sufficient evidence for a causal relationship between EDC exposure and changes in pubertal timing. Further investigation is warranted to determine the overall or different effects of EDCs exposure during prenatal or childhood windows on pubertal milestones and to reveal the underlying mechanisms, including epigenetic marks, whereby early-life exposure to EDCs affect the HPG-peripheral tissue axis.

Keywords: Puberty, Endocrine disrupting chemicals, Secular trend

Introduction

Puberty begins with the release of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator from central nervous system inhibition after a quiescent period during childhood [ 1]. The age of menarche has definitely decreased from 16 years in the 1800s to 13 years in the 1960s, after which this downward trend seems to have slowed or even stopped [ 2]. The trend for earlier age at menarche has also been reported in Korea, from 16.6 years for females born between 1925 and 1929 to 12.6 years for females born between 1990 and 1994 [ 3, 4]. While the onset of puberty has been occurring earlier, the overall tempo of pubertal progression is reported to be relatively slower, resulting in skewing towards younger ages for the onset of puberty and towards older ages for the completion of puberty [ 2]. Such increased duration of the pubertal transition may result from gonadotropin-independent estrogenic action at the level of the breast which may originate from peripheral tissues [ 5].

Although genetic factors remain the major determinant of pubertal timing [ 6], the secular trend for earlier onset of puberty has coincided with improved public health and nutrition [ 7- 9]. Sufficient energy availability and early adiposity rebound contribute to early pubertal development. Endocrine-disrupting chemicals (EDCs) may affect not only the control of energy balance and adiposity, but also puberty and reproduction [ 2]. EDCs can act at any level in the 'hypothalamic-pituitary-gonadal (HPG)-peripheral tissues' axis [ 10]. Their effects can manifest right before puberty as well as much earlier, during fetal and neonatal life [ 2]. We reviewed the genetic and environmental factors that have been implicated in pubertal timing. We present the current evidence for the roles of EDCs on pubertal timing in girls based on human epidemiologic data and introduce potential mechanisms depending on the timing of exposure.

Factors implicated in pubertal timing by affecting the hypothalamic-pituitary-gonadal-peripheral tissues axis

Pubertal onset appears to be a highly heritable trait, but earlier sexual maturation in girls may be linked to environmental factors. Some genes implicated in precocious puberty ( DLK1, MKRN3, and KISS1) and in delayed puberty ( TACR3) are associated with variations in pubertal onset. The childhood GnRH pulse generator may be inhibited, in part, by MKRN3. Disinhibition of the HPG axis leads to the progressive amplification of pulsatile GnRH secretion which may be mediated by leptin levels and increased expression of neuropeptides, such as neurokinin B and kisspeptin, and their receptors [ 1]. In addition to central signals, peripheral metabolic and nutritional signals, such as leptin and insulin, play a permissive role in the proper function of the HPG axis [ 11].

Genes involved in pubertal timing or menarche reported by Elks et al. [ 12] and Perry et al. [ 13] include those related to hypothalamic GnRH secretion, pituitary development and function, hormone synthesis and bioactivity, energy homeostasis and growth, and potential peripheral feedback from sex steroids [ 13]. The overlap of genes involved in pubertal onset and obesity from genome wide association studies and human epidemiological data suggests a common pathway connecting early infancy weight gain, earlier pubertal onset, and later obesity in adulthood, although mechanisms whereby early-life changes in adiposity affect growth acceleration and early pubertal onset remain to be determined [ 14, 15].

Obese girls are at risk for early pubertal development. Girls maturing at earlier ages have higher body mass index (BMI) z-scores with advanced Tanner stages [ 16]. Obesity-induced hyperinsulinemia and hyperleptinemia can affect linear growth and advance puberty [ 17]. Since adrenarche starts earlier as childhood BMI and insulin levels gradually increase, the increased adrenal androgen levels in obese children may also be responsible for accelerated growth before puberty [ 17]. In addition, hyperinsulinemia can increase sex steroid bioavailability via stimulation of ovarian growth and steroidogenesis, reduction of sex hormone binding globulin, and increased conversion of androgens to estrogens by stimulating aromatase activity in adipose tissues [ 2]. Breast development can occur from the gonadotropin-independent release of estrogen by peripheral tissues, such as the adrenal glands and adipose tissue. Finally, increased sex steroid levels in obese girls can induce gonadotropin-independent or dependent precocious puberty.

Gene-to-environment interactions during critical periods of development could alter gene expression in the HPG-peripheral tissue axis through epigenetic mechanisms [ 18]. While recent data have started to reveal the epigenetic regulation of KISS1 expression [ 18, 19], epigenetic mechanisms that affect the HPG-peripheral tissue axis remain to be discovered. The intrauterine environment, birth size, nutrition, and (potentially) EDCs can affect adiposity and the timing of puberty. EDCs and adiposity may be the most concerning contributors to changes in puberty and reproduction. While sufficient energy availability provides clues to the mechanism of early pubertal development, changes in the control of both energy balance and reproduction may vary under the influence of common determinants, such as EDCs. These effects can manifest right before puberty as well as much earlier during fetal and neonatal life [ 20, 21]. Although the link between EDCs and pubertal timing does not prove causality, their association is highly suggestive [ 22].

Possible mechanisms for the effect of EDCs on pubertal timing in girls

EDCs are defined as "an exogenous chemical, or mixture of chemicals, that interferes with any aspect of hormone action" by the Endocrine Society's second scientific statement published in 2015 [ 10]. Endocrine disruptors can be naturally occurring or synthetic. This section reviews the effects of synthetic EDCs on pubertal timing in girls.

EDCs can act through several mechanisms at any level in the 'HPG-peripheral tissues' endocrine axis. Directly, EDCs may affect genes or HPG pathways unique to puberty. Indirectly, EDCs can act as obesogens and change metabolic programming during fetal and early childhood development, resulting in alterations in the metabolic and peripheral hormones associated with the onset of puberty [ 15]. A variety of chemical toxins that are ubiquitously present in our environment exert antagonistic and agonistic actions on hormonal axes and pathways at low versus high concentrations, and they demonstrate nonmonotonic dose-response curves [ 10]. Since many EDCs can act as agonists of estrogen receptors (ERs) or antagonists of androgen receptors (ARs), EDCs may mimic naturally occurring estrogens and androgens. In addition, EDCs might bind to a receptor within a cell and block the functions of endogenous hormones, thus acting as antiestrogens and antiandrogens [ 10, 23].

Low-level exposures to a mixture of EDCs may mask the effect of an individual compound. Moreover, interindividual and interspecies variation in susceptibility may be present due to differences in pharmacokinetics and/or genetic polymorphisms in key genes, complicating our ability to translate data from animal studies to human health [ 20]. In humans, the long interval from exposure to endpoints (latent effects) and the setting of mixed exposures over a lifetime make it difficult to analyze the health outcomes of EDCs [ 20]. Furthermore, it is difficult to distinguish the influence of nutrition and adiposity on pubertal outcomes from that of EDCs. Since the prenatal, neonatal, infancy, early childhood and peripubertal periods are critical windows of development, EDCs may have different effects depending on the time of exposure [ 2]. Though the mechanisms whereby fetal or neonatal life changes pubertal timing have not been fully elucidated, EDCs can alter the genes involved in the modification of epigenetic marks [ 2, 15]. Although researching the effect of EDCs on pubertal timing is difficult for the various aforementioned reasons, further investigation is warranted to determine which EDCs can affect pubertal timing, which periods of exposure are critical, and how different mechanisms are involved depending on the timing of exposure in humans [ 24].

Human epidemiologic data on EDCs in regards to pubertal timing in girls

The Endocrine Society's Scientific Statements present a review of the literature on human studies that assessed the associations between EDCs exposure and pubertal timing [ 10]. There have been extensive reviews about the contribution of EDCs on shifts in pubertal timing [ 2, 10, 24, 25] as well as a recent review of publications from 2016 to 2017 that examined EDC associations with pubertal milestones [ 26]. The EDCs most extensively studied in regards to puberty include bisphenol A (BPA), phthalate esters, pesticides (dichlorodiphenyl trichloroethane [DDT], pyrethroids), dioxins and polychlorinated biphenyls (PCBs), and flame retardants (polybrominated biphenyls [PBB], polybrominated diphenyl ethers [PBDEs]) (). This section reviews the human epidemiologic studies regarding the relationship of individual EDCs with pubertal development or menarche in girls.

Table 1.

Phthalates

Phthalates are used as liquid plasticizers in plastics and flooring, personal care products, medical devices and tubing with half-lives of 12 hours. Although the mechanism remains to be further elucidated, phthalates might act as ER agonists or AR antagonists and may also interfere with androgen synthesis [ 53]. Phthalates may be classified into low-molecular-weight phthalates (ester side-chain lengths, 1 to 4 carbons) and high-molecular-weight-phthalates (ester side-chain lengths, 5 or more carbons). Di(2-ethylhexyl) phthalate (DEHP) metabolites (mono(2-ethyl-5-oxohexyl) phthalate and mono(2-ethyl-5-hydroxyhexyl) phthalate), which are classified into high-molecular-weight-metabolites, were the phthalates most commonly studied.

The results from studies regarding the relationship between phthalate and puberty in girls conflict depending on the timing of exposure and/or different phthalate metabolites () [ 31, 33, 34, 36, 42, 45, 47- 49, 54- 65]. Different studies have shown significant associations with premature thelarche [ 54, 56] and precocious or early puberty [ 31, 36, 42, 55, 58, 61, 62, 64, 65], while no relationship with puberty [ 34, 57, 59] or an association with later onset of puberty [ 45, 47- 49, 60] have also been reported. Among several studies on phthalate exposure, nine studies had a longitudinal cohort design [ 33, 45, 47- 49, 59- 61, 63]. Prenatal exposure to high-molecular-weight phthalate metabolites was associated with later onset of puberty [ 48]. Early childhood exposure to phthalate metabolites was not associated to puberty [ 59] or associated with earlier [ 61] or later [ 45, 47, 49, 60] pubertal progression. Meanwhile, differences in pubertal development or age at menarche has been reported depending on the timing of exposure or classes of exposures (high or low-molecular-weight-phthalates) [ 33, 63]. Whereas high-molecular-weight phthalate levels were associated with later pubic hair development, low-molecular-weight phthalate levels were related to advanced breast or pubic hair development [ 33]. While prepubertal exposure to DEHP was associated with later menarche, pubertal exposure to monomethyl phthalate (low-molecular-weight) was related to earlier menarche only among overweight or obese girls and not among those that were of normal weight [ 63].

Revised paradigm of EDCs on pubertal timing and future directions

Parent et al. [ 2] suggested a revised paradigm of EDCs on pubertal timing. Since improvements in public health and nutrition have paralleled a downward trend in pubertal timing and menarche, environmental factors are the predominant determinants in pubertal timing. Classically, environmental factors, including EDCs, might advance pubertal timing via central mechanisms, particularly during the prepubertal period. According to the revised paradigm [ 2], environmental factors can influence puberty and reproduction through not only central but also peripheral mechanisms, depending on whether they take place early during fetal and neonatal life or late during the prepubertal period. Environmental factors can affect energy availability during the fetal and neonatal period or in infancy and early childhood. They can also act as obesogens and promote early adiposity rebound, leading to changes in metabolic or peripheral signals and increases in adrenal androgen levels, with subsequent early pubertal development. Environmental factors, including EDCs, might affect genetic or epigenetic pathways during critical windows of development. Further investigation is warranted in order to reveal the underlying mechanisms whereby early-life exposure to EDCs affects the HPG-peripheral tissue axis.

Conclusion

The onset of puberty in girls is occurring earlier, without changes in the timing of completion of puberty. Physiological variability and multiple other factors affect the initiation of puberty. Exposure to a broad mixture of EDCs is ubiquitous, and the shifts in pubertal timing may be mediated by exposures to EDCs at critical developmental windows. EDCs are hormonally active substances that can act via several mechanisms to perturb puberty either peripherally on target organs (adipose tissue or adrenal glands) or centrally via the HPG axis. EDCs can influence pubertal timing through different mechanisms depending on when the exposure occurs during early-life. Evidence for associations between exposures of EDCs and altered pubertal timing makes it reasonable to support the hypothesis that a relationship between EDCs and pubertal timing exists. However, the current data is insufficient and conflicting to provide sufficient evidence for a causal relationship between EDCs exposure and changes in pubertal timing in humans. Further human epidemiologic studies of prospective and longitudinal design are needed to determine the combined effect of EDC exposure on puberty and reproduction during critical periods. In addition, the underlying mechanisms whereby early-life exposures to EDCs influence puberty, including epigenetic marks, need to be explored in further studies.

Acknowledgments

This study was supported by grants from the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2018R1D1A1B07049806).

Footnotes

Conflict of interest No potential conflict of interest relevant to this article was reported.

References

29. Han EJ, Yim OK, Chung J, Baek SY, Kim Y. The study of relationship between the concentrations of Bisphenol A and DEHP in human plasma and precocious puberty. Anal Sci Technol. 2008;21:375-82. [ Google Scholar]

31. Lee SW, Lee WJ, Chae HW, Lee EB, Kim JH, Kim DH, et al. Determination of serum Di-(2-ethylhexyl) phthalate and bisphenol A level in children with idiopathic central precocious puberty. J Korean Soc Pediatr Endocrinol. 2009;14:154-62. [ Google Scholar]

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Early-life exposure to endocrine-disrupting chemicals and pubertal development in girls

# data[].attributes.attachment_link.description
Over the last decades, the onset of puberty in girls has occurred earlier, but the tempo of pubertal progression has been relatively slower, resulting in a younger age at puberty onset without a change in age at menarche. Sufficient energy availability ...

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Dr. Crespi: BPA exposure effects on puberty: the jury is still out--some studies make connections with BPA and early puberty, others do not

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Dr. Crespi: BPA exposure effects on puberty: the jury is still out--some studies make connections with BPA and early puberty, others do not

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5615581/

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Abstract

Many scientific studies have revealed a trend towards an earlier onset of puberty and have disclosed an increasing number of children that display precocious puberty. As an explanation, some authors have considered the global socio-economic improvement across different populations, and other authors have considered the action of endocrine disrupting chemicals (EDCs). Among these, bisphenol A (BPA), an aromatic compound largely used worldwide as a precursor of some plastics and chemical additives, is well known for its molecular oestrogen-like and obesogenic actions. We reviewed the medical literature of the previous 20 years that examined associations between BPA exposure and the age of puberty in humans, considering only those referring to clinical or epidemiological data. Of 19 studies, only 7 showed a correlation between BPA and puberty. In particular, the possible disruptive role of BPA on puberty may be seen in those with central precocious puberty or isolated premature breast development aged 2 months to 4 years old, even if the mechanism is undefined. Some studies also found a close relationship between urinary BPA, body weight, and early puberty, which can be explained by the obesogenic effect of BPA itself. The currently available data do not allow establishment of a clear role for BPA in pubertal development because of the conflicting results among all clinical and epidemiological studies examined. Further research is needed to fully understand the potential role of exposure to EDCs and their adverse endocrine health outcomes.

Keywords: bisphenol A, menarche, puberty, pubertal development, premature thelarche

1. Introduction

Endocrine-disrupting chemicals (EDCs) are a group of hormonally active agents to which we are directly or indirectly exposed on a daily basis and that have been widely used in industry for almost a century [ 1]. They interfere with natural hormones, stimulating or inhibiting their action by binding their receptors or by affecting their synthesis, transport, metabolism, and elimination [ 2]. As a result, they may cause different kinds of diseases. Concerning EDCs' toxic effects on health, many studies have confirmed that foetuses, infants, and children are more vulnerable than adults, primarily because of the important role of hormonal balance in both growth and development [ 3, 4]. Moreover, they receive more exposure to these substances than adults, as demonstrated by the higher quantities of EDCs found in urine and blood [ 5, 6].

Bisphenol A (BPA), one of the most studied EDCs, is an aromatic compound largely used worldwide as the precursor of plastics and chemical additives [ 7]. It is widely used in the production of polycarbonate plastics (very common for transparency, heat resistance, and mechanical properties) and epoxy resins for coating of cans for food and beverages. Humans are exposed to these products mainly through their diet [ 8] but also dermal absorption and inhalation [ 9]. Among these, we find EDCs in food and drink packaging, baby feeding bottles, recycling containers, polyvinyl chloride, toys, and dental sealants [ 10]. EDCs can also be transferred to the baby via the placenta or breast milk [ 11, 12]. Although many countries including Europe (EU Directive 2011/8/EU) have recently drawn up laws restricting the use of BPA in the production of basic necessities [ 13], this substance is still used on a large scale [ 14], with more than 3.5 million tons being produced every year [ 7]. The United States of America is the world's largest producer, with more than a 1 million tons of BPA produced per year (almost 25% of the global production) [ 15].

Despite the seriousness of prenatal exposure to EDCs' toxic effects, the pathological role of BPA becomes more evident during the pubertal period, when neuroendocrine mechanisms, during the development of reproductive organs and the appearance of secondary sexual characteristics, become more vulnerable to environmental stimulation [ 16, 17]. The aim of our review was to find all studies examining the association between BPA exposure and age of puberty in humans. A PubMed search via MEDLINE was undertaken to identify studies matching the following keywords: "bisphenol A" and puberty", "precocious puberty", "female puberty", "male puberty", "premature thelarche", "menarche", and "pubertal development". The same research was conducted with these keywords replacing the word "bisphenol A" with the most widely used acronym "BPA". We reviewed all papers published in the last 20 years, and the date of our last search was March 2017. We retrieved 111 studies, excluding animal studies, in vitro studies, BPA chemical analysis studies, and BPA pharmacology studies, and considering only articles that referred to clinical and/or epidemiological data. From the initial collection, we selected 17 scientific papers based on the above-listed criteria; we later added 2 other papers not found in the PubMed database but that were tracked through the bibliographical references. In the end, we split all works into three subgroups: BPA and female puberty, BPA and isolated premature thelarche, and BPA and male puberty.

2. Puberty Onset

Puberty is a period of transition between adolescence and adulthood. During this period, full reproductive maturity is reached through the maturation of gonads, permanent genital development, and appearance of secondary sexual characteristics [ 18]. The harmonic evolution of the phenomena characterizing puberty is closely linked to the activation of the hypothalamic-pituitary gonadotropic cells-gonadal axis [ 19]. Clinical and experimental evidence has indicated that the hypothalamic-pituitary-gonadal system is in a condition of repression during childhood.

Mechanisms responsible for the reactivation of this axis are currently unknown. Recently, a neuropeptide known as kisspeptin, encoded by the gene KISS-1, and its receptor GPR54 have been revealed as major regulators of the beginning of the reproductive pubertal axis, acting as potent stimulators of pulsatile secretion of the hypothalamic gonadotropin-releasing hormone (GnRH) [ 20, 21]. GnRH neurons have sex steroid receptors through which oestrogen and progesterone can modulate the activity of kisspeptin [ 22]. Leptin, a hormone produced by adipocytes and an important regulator of both fat mass and gonadal function, is among the factors leading to increased kisspeptin expression during puberty [ 23]. Other factors involved in controlling the levels of the GnRH network are the transcriptional regulator enhanced at puberty 1 (EAP1) and Ying Yang 1 [ 24], which operate as inhibitory regulators, and thyroid transcription factor 1 (TTF1) [ 25], which operates as a stimulator within the GnRH neuronal network.

Other possible puberty regulators may be RFamide-related peptide-3 (RFRP3) neurons, which seem to play an inhibitory action on the GnRH pathway [ 26, 27]: they were first identified in birds and have recently also been found in humans [ 28, 29]. Some authors have suggested that kisspeptin behaves as an accelerator of GnRH, while RFRP3 acts as a brake; through their modulation, the timing of pubertal development is scanned [ 30].

We know that, at the level of the tonic hypothalamic centre, sensitivity to the inhibition of gonadal hormones is greater in childhood and decreases at the onset of puberty. In fact, in childhood, the very small amount of hormones produced by gonads is able to inhibit the release of GnRH and the secretion of gonadotropins [ 31]. When puberty approaches, there is a progressive reduction of sensitivity of the tonic centre receptor to the sex steroids' actions. Then, a critical time coinciding with the onset of puberty arrives, during which gonadal hormones are no longer able to inhibit the hypothalamus neurosecretory activity. There is a massively elevated release of GnRH and LH-FSH gonadotropins, and the consequent activation of the gonads leads to an increased circulating level of androgens and oestrogens [ 32, 33].

Oestrogen plays a very important role in sexual differentiation, which occurs at the pubertal stage and is closely linked to several changes in the child's phenotype: breast growth [ 34], effect on pubertal growth spurt for both sexes (stimulation of linear growth at low concentrations and closure of the epiphyseal plates and cessation of linear growth at higher concentrations) [ 35], maturation of sexual characteristics [ 36], beginning of menses [ 37], and mutual influence on the hypothalamic-pituitary axis [ 38]. The last data explain why, in girls with early pubertal signs, elevated levels of oestradiol can be found in blood, even if the mechanism remains unclear [ 39]. Three different receptors through which oestrogen carries out its functions have been identified: two are nuclear receptors (αER and βER ⁰) [ 40, 41], while another one, the most recently discovered, works as a membrane receptor or a cytoplasmic receptor [ 42].

Androgens play a crucial role in male sexual development, and they have two origins: adrenal or gonadal. Adrenal androgens (DHEA, DHEAS, and androstenedione) increase in their secretion at 6-8 years. This moment corresponds with adrenarche, a process that precedes gonadarche by approximately 2 years, which instead represents the true onset of puberty in males. Gonadarche is regulated by increased production of testosterone by the testicular Leydig cells, which in turn are stimulated by activation of the pituitary gland via GnRH pulsatile secretion. Adrenal androgens are responsible for the initial stages of linear growth and pubic hair, changes of tone, appearance of acne, and appearance of hair in the androgen-sensitive areas. However, the most important role in puberty is played by testosterone in collaboration with pituitary hormones, which are implicated in linear growth, increase in testicular volume, appearance of male secondary sex characteristics, and increase in lean body mass [ 43].

During pre-pubertal age, the levels of endogenous sex steroids are very low, although receptors for oestrogens and androgens are expressed since early childhood [ 44]. This explains how a small change in levels, even from the outside, may cause a major change in balance of child hormones, such as several pubertal changes [ 45]. Diseases related to puberty include early and delayed puberty. Precocious puberty is defined as the onset of puberty characteristics in females under the age of 8 years and in males under the age of 9 years [ 46, 47]. The main clinical manifestations of suspicion for precocious puberty in girls are breast development and acceleration of growth rate; other signs include pubic or underarm hair development, acne, and puberty sweating. In boys, an increase in testicular volume (>4 mL) may occur. Late signs may include modifications of penis and scrotum in appearance and size, acne, pubic hair, underarm or facial hair development, and acceleration of linear growth [ 48]. Pubertal status is conventionally evaluated using Tanner's staging; for both boys and girls, early puberty is conventionally summed up in that a Tanner stage 2 or higher may occur earlier than expected [ 49, 50].

Precocious puberty is more prevalent in girls (almost 0.2%) than boys (almost 0.05%) [ 51]. This condition can be caused by early activation of the pituitary gonadostat (central precocious puberty, CPP) and ectopic production of gonadotropins or sex steroids (peripheral precocious puberty, PPP); it may have different origins, such as idiopathic, sporadic, familial, or neurogenic, which is secondary to central nervous system abnormalities [ 52]. Idiopathic central precocious puberty (ICPP) is the most common cause in females, being characterized by anticipated activation of the hypothalamic-pituitary-ovarian axis [ 53]. PPP is a rare condition and is usually characterized by the overproduction of sex hormones (oestrogen and testosterone) [ 54].

A careful remote and family medical history allows for suspicion of forms of early secondary and familiar precocious puberty, respectively. The clinical data listed above are important in association with other information dealing with signs of pubertal development at the pelvic ultrasonography in females (increase in uterine volume, longitudinal diameter >35 mm, uterine body/cervix ratio >1, thickening of endometrial rhyme, or increase in the diameter of the ovaries, longitudinal diameter >3 cm or increase in size of follicles, diameter >9 mm); with detection of testosterone in males (>50 ng/dL) and oestradiol in females (>20 pg/mL, even if for females low estradiol concentrations do not rule out the diagnosis of precocious puberty); or assessment of bone age at the left hand X-ray film (resulting higher than at least 1 year in comparison with the chronological age).

A definite diagnosis is based on the GnRH test: CPP is present when there is an LH blood peak >5 mUI/mL and LH/FSH ratio >0.66 after stimulation with the hypothalamic hormone analogue [ 55, 56, 57, 58, 59]. Magnetic resonance imaging allows discrimination between forms caused by neurologic diseases and those not (including idiopathic forms) [ 60]. On the other hand, PPP can be diagnosed by the same criteria except the detection of high oestradiol and low gonadotropin blood levels, LH peak <5 mUI/mL, absence of LH/FSH inversion after GnRH stimulation test, and negative cerebral magnetic resonance imaging [ 61, 62]. Pathological effects related to early puberty include insulin resistance, metabolic syndrome, psychosocial disorders, cardiovascular diseases, adult-onset asthma, and breast or reproductive system tumours [ 63, 64, 65, 66, 67].

3. Age of Puberty

Many studies have reported a current counter-trend of age for puberty onset in girls compared to the past. The mean onset of thelarche, pubarche, and menarche has decreased over the last decades [ 49, 68]. This trend has been observed both in America [ 69] and Europe [ 70]. A study by Biro et al. showed that 16% of girls (with a peak of 23.4% in black non-Hispanic girls) began pubertal development at the age of 7 years, while 30% (with a peak of 42.9% in black non-Hispanic girls) began at the age of 8, well below the previous average [ 71]. Askglaede highlighted the different average age for onset of secondary sexual characteristics between the beginning of the 1990s and the early years of this decade (in a time interval of approximately 15 years) in Denmark. If the average was 10.8 years old during the 1990s, it dropped 1.02 years between 2006 and 2008 (to 9.86 years old). Girls get their first periods on average 0.29 years earlier (13.13 years) [ 71]. Genetics, with the A2 polymorphism of the CYP17 gene [ 72], Oct-2 gene [ 73], and GPR54/KiSS-1 at the neuroendocrine level [ 74], and the environment play a role in the definition of puberty onset; in this respect, the general improvement of socio-economic and nutritional conditions of the population only partially justifies this anticipation [ 75]. Weight-gain, insulin resistance, and metabolic syndrome are well-known factors linked to this condition. Glucose and leptin have been shown to be involved in the control of GnRH secretion, but their role in the timing of puberty remains controversial [ 75]. What is certain is that obese girls are more likely to have early menarche [ 76]. Studies have shown that further decreases in the age of onset of pubertal activation signs (breast development in girls and increased testicular volume in boys) have occurred in recent years, despite the maintenance of substantially unchanged standards of life [ 70]. For this reason, industrialization and daily exposure to environmental pollutants are considered among the causes of this trend because of their ability to interfere with normal bodily function [ 77, 78]. It also explains why the number of studies dealing with EDC and their effects on pubertal development is increasing [ 37, 79]. One of those studies was conducted by Massart et al. in Italy, where in a limited area of northern Tuscany, they have reported a higher proportion of early puberty: the study has raised the suspicion that this growing incidence might be correlated with environmental factors [ 80].

Sun et al., comparing previous studies, found an anticipation of puberty also in males, but only for white and Hispanic males (not for non-Hispanic black males) [ 81], although other studies investigating the male pubertal timing trend have presented less reliable data [ 82].

4. Bisphenol A (BPA) and Its Oestrogen-Like Role

As an EDC, BPA is able to alter reproductive, nervous, cardiovascular, and immune systems and interferes with thyroid and pancreatic functions [ 82]. According to the pharmacological studies, the maximum BPA value to which we can daily expose ourselves as recommended by the European Food Safety Authority is almost 4 mcg/kg/day [ 83]. However, some studies recommend lower thresholds for exposure without toxicological relevance [ 84].

Although most of the molecular mechanisms of its effects on the human body are still unknown, BPA is widely studied for its oestrogen-like action. Because of its structural similarity with 17β-oestradiol, many studies have shown that BPA may act as a leading actor in the pathogenesis of several genital and reproductive disorders, such as male and female reproductive tract alterations, female and male infertility, precocious puberty, externalizing behaviour in girls, breast and prostate cancer, menstrual irregularities, and polycystic ovary syndrome (PCOS) [ 85, 86, 87, 88, 89, 90]. It is a xenooestrogen and competes with natural oestradiol for oestrogen receptors (αER, βER, and the membrane receptor) [ 91, 92, 93]. Furthermore, it may act antagonistically in the presence of oestrogens, such as 17β-oestradiol, and can interfere with oestrogen nuclear receptors [ 82, 94]. The possibility of simultaneously causing early puberty and having a negative role on fertility seems to confirm its dual aspect. This means that it behaves as an agonist or antagonist through the classical or alternative endocrine receptor-dependent signalling pathways [ 95]. Moreover, its interference with steroid hormones seems to be expressed by antagonizing the action of androgens and thyroid hormones [ 96, 97].

The pathologic mechanism of precocious puberty caused by BPA exposure is related to its oestrogen-like activity, which triggers a positive feedback process for the activity of the GnRH pulse generator, with increased LH and FSH central secretion despite the weak effect as a xenooestrogen [ 98]. Animal studies have shown the effects of BPA on the hypothalamic-pituitary-gonadal axis through alterations in brain sexual differentiation, higher GnRH secretion, and alterations in the oestradiol-induced LH surge [ 86]. In a recent study, Mueller et al. showed that BPA is able to induce precocious puberty by stimulating factors implicated in pubertal activation, such as KiSS1, and repressing factors inhibiting the onset of puberty, such as EAP1 and YY1. Epigenetic changes may also enable this mechanism. The same study demonstrated the stimulatory effect of BPA on Dnmt3b, a DNA methylase, with a possible effect of altered methylation of the genes belonging to the neuronal network of GnRH [ 99].

Another relevant issue is the correlation between BPA and obesity. This correlation was first studied in rats; when exposed prenatally to BPA, they develop dyslipidaemia, hyperleptinaemia, hyperglycaemia, and insulin resistance [ 100]. In a second step, the obesogen action of BPA in human beings was evaluated and confirmed [ 101, 102]. An interesting study by Trasande et al. showed high levels of urinary BPA in obese children and adolescents [ 103]. A more recent study by Li et al. found that, in Shangai, girls aged between 9 and 12 years with high levels of urinary BPA were linked to a more than 2-fold risk of obesity [ 104]. As obesity has an important role in puberty induction, it is not difficult to imagine a possible indirect action of BPA mediated by its interference with glucose and lipid metabolism [ 105]. Although several hypotheses have been raised, many uncertainties still remain.

5. Bisphenol A (BPA) and Female Puberty

One of the largest and longest clinical studies on pubertal development in girls and exposure to environmental phenols was conducted by Wolff et al. [ 106]. In 2008, they investigated the exposure to EDCs and pubertal status in a multiethnic group of 192 healthy 9-year-old girls living in New York City. In addition to phytooestrogens, 1,1′-dichloro-2,2′-bis(4-chlorophenyl)ethylene (DDE) and polychlorinated biphenyls (PCBs), they also measured the urinary BPA in this group of subjects. The choice of selecting 9-year-old patients stemmed from the opportunity of having a relatively equal distribution between subjects with puberty signs (53% had breast development predominantly at Tanner stage 2, and 31% had pubic hair development) or subjects with no signs of sexual development. Despite taking into account BMI as a possible modifier of hormonal exposure, they revealed no correlation between the values of urinary BPA and pubertal development [ 106].

Later, in 2010, Wolff and other colleagues expanded the study by taking into account 1151 American girls among those enrolled in the Breast Cancer and Environment Research Centers (BCERC) [ 107]. The last group was established by the National Institute of Environmental Health Sciences and the National Cancer Institute that studied environmental chemicals' influences on the timing of pubertal development in girls. Between 2004 and 2007, they recruited girls aged between 6 and 8 years and followed them throughout puberty. As in the previous study, the authors compared the pubertal stage of these girls and exposure to 3 classes of chemical contaminants: phenols (including BPA), phthalates, and phytooestrogens. The proportion of girls with breast or pubic hair development (30% and 22%, respectively) was lower 1 year after the recruitment compared to the previous study, given the lower age considered. A sample of urine taken at enrolment and another taken 1 year later was also analysed, but no association was found between the level of urinary BPA and pubertal stage [ 107].

In 2015, the same group evaluated 1239 girls taken from BCERC, all of whom were 6-8 years old at enrolment, in order to detect age at first puberty appearance in a 7-year follow-up and correlated this moment with urinary values of 10 different phenols, including BPA [ 108]. This study estimated a relative risk of early or late puberty linked to phenol exposure. In approximately 85% of girls, signs of pubertal activation (growth of breasts and pubic hair) appeared in the extended follow-up time, but no statistical correlation was found between levels of BPA and early pubertal development. According to this study, there was no correlation between BMI and levels of urinary BPA [ 108].

A similar study with a smaller group was conducted in Europe by Frederiksen et al., who investigated pubertal status in relationship with urinary levels of 8 hormonally active phenols in 129 Danish children and adolescents aged between 6 and 21, showing the absence of correlation between urinary BPA and pubertal development [ 109].

Other works have considered the correlation between BPA exposure and age at menarche. McGuinn et al. evaluated this association in girls through the study of the National Health and Nutrition Examination Survey (NHANES) in the period 2003-2010 [ 110]. All girls analysed were aged 12-19 years, and it was therefore possible to consider the effect of BPA exposure close to the age of menarche (indeed, 72.2% had reached menarche). Age, ethnicity, country of birth, annual household income, level of guardian graduation, and body max index were also considered as potential confounders. In this case, the index of exposure to BPA was represented by urinary BPA. Among the 987 girls that were studied, those with moderate levels of urinary BPA had delayed menarche compared to those with the lowest values. The girls layering for potential confounders did not reach statistical significance, although a common result with other studies was the finding of elevated values of urinary BPA in overweight girls (BM ≥ 85th percentile) who were more likely to have early onset menarche [ 111].

Another important American study was conducted by Buttke, Sircar, and Martin in 2012 [ 112]. They considered the link between exposure to certain EDCs and age of menarche in a group of 461 girls aged between 12 and 16 years who had been recruited from Centers for Disease Control and Prevention's National Health and Nutrition Examination Survey (NHANES)) between 2003 and 2008. In that survey, the study group was given a questionnaire on demographics and related health behaviours, and each subject underwent clinical and laboratory evaluations. Among the 461 girls, it was possible to analyse the levels of urinary BPA in 440 subjects. To obtain the most reliable results, the interaction between urinary phenols and BMI and ethnicity were also considered, as these factors affect the age of menarche. However, regardless of the adjustments made, urinary BPA levels were not significantly associated with age at menarche [ 112].

While these studies did not show a significant correlation between BPA and puberty, different results have been obtained from studies that took into account a population of girls with early puberty. Most of these works were carried out in Asian populations. Durmaz et al. tested the difference in urinary BPA between 28 non-obese Turkish girls affected by idiopathic central precocious puberty and 25 healthy age- and ethnicity-matched control girls [ 113]. Both were aged 4-8 years. In the control group, the girls considered had no signs of pubertal activation at the time of the study. The level of urinary BPA in the affected group of 26 girls was significantly ( p = 0.001) higher than that in the control group. The study also took into account the absence of correlation between urinary BPA levels and serum LH, FSH, and oestradiol levels. They did not observe any significant differences between early puberty and use of feeding bottles or nipples.

Another more recent study by Supornsilchai et al. considered the values of urinary BPA and pubertal stage in 88 Thai girls [ 114]. The girls were divided into two groups: 41 patients affected by precocious puberty and 47 healthy age-matched controls. Those with signs of early puberty had higher levels of BPA in the urine compared to the control group. In addition, obese or overweight girls with signs of pubertal activation had higher values of urinary BPA compared to normal weight girls and normal pubertal overweight or obese girls. As in other studies, BPA levels were not associated with levels of FSH, LH, or oestradiol [ 114].

To determine if there was an association between the onset of precocious puberty and levels of EDCs in the plasma, Kwon performed a study in 2011 that confirmed the association between BPA and early puberty [ 115]. In that case, the determination of exposure to BPA was obtained by dosing the concentration of BPA in serum associated with that of kisspeptin. Both the levels of serum kisspeptin and BPA were significantly higher in the study group, consisting of 31 girls who were diagnosed with central precocious puberty, compared to the control group of 30 healthy age-matched girls. Instead, no correlation was found between serum kisspeptin, BPA, LH, and FSH peak values [ 115].

In 2010, Qiao et al. compared the plasma levels of 3 different phenols (bisphenol A, octylphenol, and 4-nonylphenol) in 110 girls affected by precocious puberty and 100 normal girls [ 116]. Those values were compared with the volume of the uterus and ovaries and the value of oestradiol. BPA was identified in the blood of 40.9% of girls with precocious puberty compared to 2% of healthy controls. This confirmed that higher values of BPA are found in subjects with early onset of puberty. In such subjects, exposure to this contaminant positively influenced the volume of ovaries and the uterus.

Yum et al. did not agree with the results of previous trials conducted on populations of children with early puberty. In fact, they recruited 150 female patients affected by precocious puberty and 90 control subjects in the Seoul area, measuring plasma values of 10 different EDCs, including BPA [ 117]. In that study, the levels of BPA of precocious pubertal girls were lower than in healthy children, unlike other compounds, such as monobutyl phthalate (MBP), that was 1.3 times higher in the affected group compared to controls.

Similar results may be extrapolated from previous works conducted on the Korean population by Lee et al. in 2009 and Han et al. in 2008. In Lee's study, 30 patients (29 girls and 1 boy) with idiopathic CPP (diagnosed on the basis of clinical and hormonal tests) were included [ 118]. Levels of serum BPA in this group were not significantly different than those obtained from 30 normal control children. Han et al. came to the same conclusion one year earlier in his study evaluating serum BPA in 100 subjects (50 with precocious puberty and 50 without) [ 119].

In 2016, Buluş et al. were the first to evaluate the BPA and peripheral precocious puberty ratio [ 120]. Among the Turkish population, they selected 42 patients with idiopathic central precocious puberty (ICPP), 42 patients with peripheral precocious puberty (PPP), and 50 healthy non-obese age-matched girls as controls. The authors did not identify any statistically significant dissimilarity between the mean values of urinary BPA in the three groups. Moreover, they obtained additional data through a questionnaire investigating the possible source of BPA exposure in daily life; the duration of breastfeeding, playing time with plastic toys, and use of creams or cosmetics did not influence the development of early puberty. As a retrospective questionnaire, the answers might be biased. In contrast, both the groups with ICPP and PPP had a higher presence of polyvinyl chloride windows in their houses compared to the control group. In addition, LH, FSH, and oestradiol serum levels were detected in all three groups, but the results were affected by the underlying disease [ 120].

A more recent study that correlated peripheral precocious puberty and urinary levels of BPA was carried out by Lee et al. [ 121]. They selected 42 patients suffering from central precocious puberty and 40 patients with peripheral precocious puberty, comparing them with a group of 37 age- and sex-matched healthy subjects through the dosage of 84 different sex steroid hormones. The study was born from the idea that the biological changes involved in early puberty may cause changes in the metabolism of human steroids. Furthermore, there may be exposure to EDCs, in particular to bisphenol A. They found that the urinary levels of BPA were slightly higher but not statistically significant in both PPP and CPP groups than in the control group. They also showed that testosterone, androstenedione, androstenediol, 16-alpha-hydroxy-dehydroepiandrosterone, and 5-alpha-androstenedione were higher in both groups with precocious puberty compared to controls. Moreover, the levels of 17-beta-estradiol were higher in subjects with CPP than in the other two groups, and many other oestrogens were slightly increased in both precocious puberty groups. Furthermore, the levels of testosterone, 17-beta-estradiol, and pregnenolone were significantly higher in patients with elevated levels of urinary BPA [ 121].

describes the main epidemiological studies investigating the relationship between BPA and precocious puberty in girls.

Table 1

Reference

Study Area

Type of Study

Study Group

Subjects' Age

Exposition Measure

Correlation with PP

Other Remarks

BPA exposition and CPP

31 girls with CPP + 30 healthy girls

6-8 years old

Serum BPA

YES

-neither correlation between levels of serum kisspeptin and BPA; nor between BPA and LH or FSH

6. Bisphenol A (BPA) and Isolated Premature Thelarche

As isolated premature thelarche may anticipate a framework of central precocious puberty and result from exposure to xenooestrogens, it is appropriate to consider studies referring to this phenomenon [ 122, 123]. It is well known that physiological breast development accompanies an upsurge in steroid hormones, chiefly oestrogens [ 124]. Limited clinical or epidemiological studies have evaluated the relationship between the exposure to EDCs and premature thelarche [ 125]. One of the latest studies investigating BPA was conducted in 2014 on 251 girls aged 4 months to 2 years [ 126]. Chen et al. selected only patients with at least stage 2 thelarche per the criteria by Tanner and without other signs of pubertal development. The results obtained showed a serum concentration of BPA significantly higher in premature thelarche girls compared to 33 healthy age- and sex-matched controls. Moreover, within the study group, there was a different distribution of BPA values based on the age of girls: the youngest girls had much higher values than the 4-year-old girls. This highlights the possible harmful effect of higher exposure to BPA in the early stages of life [ 126].

A more recent study contrasts with this result. In 2015, Ozgen et al. measured the levels of urinary BPA in 28 girls with central precocious puberty, 28 girls with premature thelarche, and 22 pre-pubertal children as controls, without identifying any significant differences between the three groups [ 127]. In their study, they also measured the serum levels of kisspeptin and found higher concentrations in the CPP group but no correlation between the values of BPA and oestradiol. The contrast with Chen's study may derive from the different ages of patients selected (girls with isolated premature thelarche aged between 5.41 and 8.12 years were analysed); in fact, the previous study found higher values of urinary BPA in the younger girls.

summarizes the main epidemiological studies investigating the relationship between BPA and isolated premature thelarche.

Table 2

7. Bisphenol A (BPA) and Male Puberty

Only 2 studies were identified belonging to this specific category. The first work was carried out by Ferguson et al. in 2014, who studied the correlation between prenatal exposure to phthalates and bisphenol A by evaluating maternal urine collected in the third month of pregnancy [ 128]. They also considered postnatal exposure by sampling the urine of children born to those mothers, with levels of sex hormones and development of secondary sexual characteristics evaluated in 113 boys aged between 8 and 14 years. Prenatal exposure to BPA did not show a clear association with any of the analysed hormones, in contrast to prenatal exposure to phthalates, which seemed to have an inverse relationship with levels of DHEAS and inhibin B and a direct relationship with SHBG (while there was no correlation with the levels of oestradiol, free testosterone, or total testosterone). With regard to exposure during childhood, BPA seemed to be positively associated with levels of SHBG and inversely correlated with total values of testosterone and free testosterone. The most interesting result, probably correlated with the anti-androgenic nature of BPA, was that prenatal exposure to BPA seemed to be associated with a reduced age of onset of adrenarche [ 128].

The second study, conducted by Wang et al. in 2016, evaluated urinary BPA in 671 Chinese boys aged between 9 and 18 years and their pubertal development [ 129]. The group consisted of young people from three stages of puberty: early puberty, late mid-puberty, and puberty (100% of 13-year-old patients enrolled in the study had begun pubertal development at the time of recruitment and had testicular volume >3 mL, Tanner stage G2). The results showed that moderate levels of urinary BPA seemed to correlate with the early appearance of pubic hair and testicular development, although this latter association was not statistically significant. On the other hand, there was a direct correlation between values of urinary BPA and the delayed appearance of genital development's final stages. According to this study, BPA exposure in boys anticipated both pubarche and adrenarche [ 129]. However, it slowed the progression of puberty, especially with regard to the maturation of genitals. Additional information offered by this study was the apparent importance of BMI as not interfering in the relationship between BPA and pubertal development, emphasizing the uncertain role of fat in male puberty in contrast to in females [ 130, 131].

shows the main epidemiological studies investigating the relationship between BPA and male precocious puberty.

Table 3

8. Critical Analysis of the Relationship between Bisphenol A (BPA) and Puberty

The first data on BPA and puberty was obtained from studies conducted on animals. A growing number of animal experiments, especially on rats, has highlighted that early puberty onset may be caused by exposure to BPA in the female population [ 132, 133, 134, 135, 136]. Other studies have published results that are inconsistent with these data, in particular those referring to BPA exposure in utero and during breastfeeding [ 137]. However, other studies on rats have shown no clear correlation with puberty early onset, even among works evaluating postnatal exposure to BPA [ 138, 139].

Among the limited number of human studies, similar equivocal results have been found. Only 7 of the 19 scientific articles considered in this review showed a correlation between BPA and pubertal development. Surveys that evaluated female patients with central precocious puberty and isolated premature breast development aged 2 months to 4 years have confirmed that early exposure to EDCs may lead to risk of developing signs of early puberty. However, a direct relationship between BPA levels and age of menarche or pubertal stage has not been found. The reason for the elevated serum or urine values of BPA in children affected by central precocious puberty has not yet been explained.

The underlying molecular mechanism is not clear: only 2 studies evaluated serum levels of kisspeptin, and both found no association with BPA levels. This might suggest that BPA action on precocious puberty plays a different role towards the activation of kisspeptin's network. In fact, even if an indirect action of BPA on puberty through interference with the GnRH axis is possible, some studies have ruled out any correlation between blood levels of BPA, FSH, LH, and oestradiol. However, the possibility of identifying elevated levels of BPA in obese or overweight girls is accepted as is the possible obesogenic effect of exposure to BPA and the close relationship between urinary BPA, body weight, and early puberty [ 101]. Another consideration can be made about Wang's study in Chinese boys, as a delay in sexual development was found. This result seems to confirm the idea that children with premature pubertal onset undergo a compensatory slowdown in the late-pubertal stage [ 140, 141].

The data obtained from surveys are conflicting, perhaps because of the lack of standardization of studies. A further explanation might be the different geographical areas where the epidemiological investigations have been carried out. The most studied populations were in America and Asia, in 5 and 13 papers, respectively. None of the American studies found an effect of BPA on puberty, whereas 7 of the 13 Asian studies found a correlation. The different exposures based on geographic location can probably explain this difference. Furthermore, different pathological expressions resulting from exposure to EDC can be suspected in various human ethnic groups [ 142]. The association between BPA and early puberty could reflect changes in personal habits, such as familiar and socio-economic conditions or variable pharmacokinetics and metabolism that are difficult to quantify. Zawatski and Lee have advanced another hypothesis for the conflicting results: EDCs show non-monotonic dose-response relationships instead of the fluctuation of hormone levels during pubertal transition; thus, measurements cannot be considered representative of the hormonal condition [ 43].

In the end, the different results of epidemiological studies may be attributed to the various methods used for the determination of BPA exposure. Regarding BPA metabolism, it has a short half-life (5-6 h) in our bodies and is almost completely eliminated after 24 h [ 143]. Regarding urinary BPA determinations, several studies have demonstrated that single spot samples of urine give an average value of prolonged exposure to such EDCs [ 144, 145]. Teitelbaum stated that single measurements of urinary phenols are fairly representative of 6-12 months of exposure in children [ 146]. Another advantage of BPA urinary determination is the easy detection (more than 90% of the general population, according to some studies) [ 147].

9. Conclusions

BPA plays a role only in female patients with precocious puberty or premature thelarche and shows no correlation with pubertal development in healthy subjects. Instead, studies on its role in male puberty are still limited and did not provide definitive data. The molecular mechanism underlying its activity as a puberty disruptor is not clear, but a potential interference with metabolism has been hypothesized. However, this is only speculation because the currently available data do not allow establishment of a clear role for EDCs, BPA in particular, on pubertal development. Moreover, the considered populations are often limited, and many final considerations are derived from cross-sectional studies. All findings related to BPA and puberty should therefore be interpreted with caution with respect to these limitations, highlighting the need for more research on the potential role of any EDCs in adverse endocrine outcomes.

Acknowledgments

This study was supported by a grant from the World Association of Infectious Diseases and Immunological Disorders (WAidid).

Author Contributions

Alberto Leonardi wrote the first draft of the manuscript; Marta Cofini, Laura Lucchetti and Clelia Cipolla performed the literature search; Donato Rigante, Laura Penta and Susanna Esposito revised the draft and made a substantial scientific contribution. All the authors read and approved the final version of the manuscript.

Conflicts of Interest

The authors declare that they have no competing interests.

References

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The Effect of Bisphenol A on Puberty: A Critical Review of the Medical Literature

# data[].attributes.attachment_link.description
Many scientific studies have revealed a trend towards an earlier onset of puberty and have disclosed an increasing number of children that display precocious puberty. As an explanation, some authors have considered the global socio-economic improvement across different populations, and other authors have considered the action of endocrine disrupting chemicals (EDCs).

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