Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits

DES adverse impact on fetal germ cells, impairing neurodevelopment of offspring

Abstracts

We conducted a large-scale cohort analysis to assess the association between use of diethylstilbestrol during pregnancy and third-generation ADHD. The observed associations were robust to covariate adjustment and sensitivity analyses. Despite animal evidence of adverse multigenerational consequences—including neurodevelopmental disorders—of EDC exposure, to date only a few studies have explored the potential multigenerational implications of EDC exposure in humans. These studies have only considered diethylstilbestrol exposure, and none has studied neurodevelopmental outcomes. Some studies have reported increased risk of hypospadias in grandsons of women exposed to diethylstilbestro during pregnancy. Titus-Ernstoff et al found delayed menstrual regularization, higher odds of irregular menstrual periods, and fewer live births among women whose grandmothers used diethylstilbestrol during pregnancy. Birth defects have also been found in grandchildren of women who used diethylstilbestrol when pregnant.

2018 Study Key Points

Question
Is exposure to diethylstilbestrol during pregnancy associated with adverse multigenerational neurodevelopmental outcomes?

Findings
A cohort study of 47 450 women in the Nurses’ Health Study II found significantly elevated odds for attention-deficit/hyperactivity disorder in the grandchildren (third generation) of users of diethylstilbestrol, a potent endocrine disruptor.

Meaning
Exposure to endocrine disruptors during pregnancy may be associated with multigenerational neurodevelopmental deficits.

IMPORTANCE

Animal evidence suggests that endocrine disruptors affect germline cells and neurodevelopment. However, to date, the third-generation neurodevelopmental outcomes in humans have not been examined.

OBJECTIVE

To explore the potential consequences of exposure to diethylstilbestrol or DES across generations—specifically, third-generation neurodevelopment.

DESIGN, SETTING, AND PARTICIPANTS

This cohort study uses self-reported health information, such as exposure to diethylstilbestrol during pregnancy and attention-deficit/hyperactivity disorder (ADHD) diagnosis, from 47 540 participants enrolled in the ongoing Nurses’ Health Study II. The 3 generations analyzed in this study were the participants (F1 generation), their mothers (F0 generation), and their live-born children (F2 generation). MAIN OUTCOMES AND MEASURES Participant- and mother-reported exposure to diethylstilbestrol during pregnancy and physician-diagnosed child ADHD.

RESULTS

The total number of women included in this study was 47 540. Of the 47 540 F0 mothers, 861 (1.8%) used diethylstilbestrol and 46 679 (98.2%) did not while pregnant with the F1 participants. Use of diethylstylbestrol by F0 mothers was associated with an increased risk of ADHD among the F2 generation: 7.7% vs 5.2%, adjusted odds ratio (OR), 1.36 (95% CI, 1.10-1.67) and an OR of 1.63 (95% CI, 1.18-2.25) if diethylstilbestrol was taken during the first trimester of pregnancy. No effect modification was observed by the F2 children’s sex.

CONCLUSIONS AND RELEVANCE

This study provides evidence that diethylstilbestrol exposure is associated with multigenerational neurodevelopmental deficits. The doses and potency level of environmental endocrine disruptors to which humans are exposed are lower than those of diethylstilbestrol, but the prevalence of such exposure and the possibility of cumulative action are potentially high and thus warrant consideration.

Sources

  • Full study (free access) : Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits, JAMA Pediatrics doi:10.1001/jamapediatrics.2018.0727, May 21, 2018.
  • Featured image by Andre Hunter.
DES DIETHYLSTILBESTROL RESOURCES

DES exposure can disrupt developing organ systems and cause abnormalities that only appear in the subsequent generation

Proceedings of the Summit on Environmental Challenges to Reproductive Health and Fertility: Executive Summary

2008 Manuscript Abstracts

The DES Example – Prenatal exposure to diethylstilbestrol (DES), a synthetic estrogen and thus EDC, provides an unfortunate example of developmental programming. DES was given to U.S. pregnant women between 1938 and 1971 under the erroneous assumption that it would prevent pregnancy complications.

In fact, in utero exposure to DES alters the normal programming of gene families, such as Hox and Wnt, that play important roles in reproductive tract differentiation.

As a result, female offspring exposed to DES in utero are at increased risk of clear cell adenocarcinoma of the vagina and cervix, structural reproductive tract anomalies, infertility and poor pregnancy outcomes, while male offspring have an increased incidence of genital abnormalities and a possibly increased risk of prostate and testicular cancer. These observed human effects have been confirmed in numerous animal models which have also provided information on the toxic mechanisms of DES. Animal experiments have also predicted changes later found in DES-exposed humans, such as oviductal malformations, increased incidence of uterine fibroids and second-generational effects such as increased menstrual irregularities and possibly ovarian cancer in DES-granddaughters and increased hypospadias in DES-grandsons.

DES is but one example of how exposure to EDCs can disrupt developing organ systems and cause abnormalities, many of which only appear much later in life or in the subsequent generation, such as endometriosis, fibroids and breast, cervical and uterine cancer in women; poor sperm quality and increased incidence of cryptorchidism and hypospadias in men; and subfertility and infertility in men and women.

Epigenetic studies have also shown that DES causes alterations in uterine tissue architecture and morphology and heightens susceptibility to uterine adenocarcinoma by inducing permanent changes in several estrogen-responsive uterine genes. These are but a few examples of how the field of epigenetics has and will continue to contribute to our mechanistic understanding of the impact of environmental contaminants on reproductive health.

Uterus Development and the Environment – Women exposed to DES in utero during critical periods of reproductive tract development developed several types of reproductive tract abnormalities, as well as an increased incidence of cervical-vaginal cancer later in life. Animal studies that simulate the human DES experience have since shown that exposure of the developing reproductive tract of CD-1 mice to DES imparts a permanent estrogen imprint that alters reproductive tract morphology, induces persistent expression of the lactoferrin and c-fos genes and induces a high incidence of uterine adenocarcinoma. Experiments in rats have shown exposure to DES during the critical window of uterine development leaves a hormonal imprint on the developing uterine myometrium in rats that were genetically predisposed to uterine leiomyoma, increasing the risk for adult uterine leiomyoma from 65% to greater than 90% and increasing tumor multiplicity and size. DES-induced developmental programming appears to require the estrogen receptor α, suggesting that signaling through this receptor is crucial for establishing developmental programming.

Sources

  • Full study (free access) : Proceedings of the Summit on Environmental Challenges to Reproductive Health and Fertility: Executive Summary, Fertility and Sterility, PMC2440710, 2008 Feb.
  • Featured image Dmitry Ratushny.
DES DIETHYLSTILBESTROL RESOURCES

Epigenetics, Evolution, Endocrine Disruption, Health, and Disease

image of Epigenetics, Evolution, Endocrine Disruption, Health, and Disease

A possible mechanism for how DES exerts its action epigenetically has been proposed recently

2006 Study Abstracts

Endocrine-disrupting chemicals (EDCs) in the environment have been linked to human health and disease. This is particularly evident in compounds that mimic the effects of estrogens. Exposure to EDCs early in life can increase risk levels of compromised physical and mental health. Epigenetic mechanisms have been implicated in this process. Transgenerational consequences of EDC exposure is also discussed in both a proximate (mechanism) and ultimate (evolution) context as well as recent work suggesting how such transmission might become incorporated into the genome and subject to selection. We suggest a perspective for exploring and ultimately coming to understand diseases that may have environmental or endocrine origins.

That epigenetic mechanisms may play a role in endocrine disruption helps explain the transgenerational effects of some hormonally active chemicals. Treatment with diethylstilbestrol (DES) during pregnancy results in vaginal adenocarcinoma in female offspring in humans and mice. Female offspring of mice exposed to DES during pregnancy, when mated to control males, produce a second generation of females who, although not exposed to DES themselves, express this same rare genital tract cancer. This transgenerational transmission of a specific reproductive tract lesion would be hard to explain without invoking an epigenetic mechanism for heritable change and, given the finding of altered DNA methylation patterns in a specific uterine gene in mice treated developmentally with DES, we think a strong case can be made for such a conclusion. Newbold and colleagues next showed that specific, rare genital tract cancers (rete testes cancers) are also expressed and therefore transmitted to the male offspring of females treated in utero with DES. In colloquial terms, this demonstrated the occurrence of reproductive tract tumors in the grandsons and granddaughters of mothers treated with DES. A possible mechanism for how DES exerts its action epigenetically has been proposed recently. The transmission of uniquely specific changes in the program of development in mice has implications for similarly exposed humans as well as the biology of hormonally induced disease.

We have already mentioned that DES treatment of mice during development results in reproductive tract cancers, persistent up-regulation of key estrogen-responsive genes, and altered patterns of gene methylation in the affected genes and that the cancer can be transmitted through two generations. In addition to DES, methoxychlor has been reported to increase global DNA methylation in uterine ribosomal DNA after in utero exposure; the alteration in methylation remains months after treatment.

To understand the role of estrogens in development, there is hardly a more powerful model than that of the outcomes observed in humans and mice developmentally exposed to the synthetic estrogen DES. Female offspring of humans or mice exposed prenatally to DES have a risk for vaginal clear cell adenocarcinoma. The mechanisms underlying these developmentally induced lesions have been sought for three decades. There was the suggestion by clinical investigators that DES had altered the normal differentiation of the epithelial cells of the fetal cervix and vagina such that they responded abnormally to estrogen at puberty, because no cancers had been seen in prepubescent girls. Similarly, ovariectomy of developmentally DES-treated mice prevented the subsequent expression of uterine adenocarcinomas.

Epigenetic change in the molecular program of cell differentiation in the affected tissues may be a common mechanism. The clear cell cancers of the vagina in DES-exposed women displayed genetic instability consistent with epigenetic imprints in the absence of any expected mutation in classical oncogenes or tumor suppressor genes. Using a well validated mouse model for DES genital tract tumors, Li and colleagues discovered that one of the estrogen-inducible genes in the mouse uterus, lactotransferrin, that had been shown earlier to be persistently up-regulated by developmental DES exposure, had an altered pattern of CpG methylation in the promoter region of the gene upstream from the estrogen response element. Subsequent work demonstrated that other developmentally up-regulated genes such as fos and jun also had persistent changes in the pattern of methylation of the gene after DES exposure during development. These experiments raise the possibility that DES (and other environmental estrogens) alter the program of differentiation of estrogen target cells in the reproductive tract through an epigenetic mechanism.

Other studies support this hypothesis. In addition to cervicovaginal adenocarcinomas in female mice and humans exposed prenatally to DES and uterine adenocarcinoma in mice, it has been shown that developmental exposure to DES results in excess risk of uterine leiomyomas (fibroids) in mice, rats, and women. It was also recently reported that sea lions in areas contaminated with EDCs have a higher prevalence of uterine fibroids. The Eker rat carries a germ-line mutation in a tumor suppressor gene and is predisposed to uterine leiomyoma. Cook and colleagues used this model system to demonstrate a DES-induced alteration in developmental imprinting as analyzed by tumor suppressor gene penetrance, concluding that developmental programming by estrogen works in concert with preexisting genetic change. In a population of 819 black and 504 white women, fibroid status was determined by ultrasound screening or surgical record review, whereas prenatal DES exposure was determined by interview. DES-exposed women had a significantly greater risk for uterine fibroids and tended to have larger tumors. The authors conclude that their study, as well as animal studies, indicate a role for prenatal estrogen in the etiology of uterine leiomyoma in women.

Sources

  • Full study (free access) : Epigenetics, Evolution, Endocrine Disruption, Health, and Disease, Endocrinology, Volume 147, Issue 6, Pages s4–s10, doi.org/10.1210/en.2005-1122, June 2006.
  • Featured image academic.oup.
DES DIETHYLSTILBESTROL RESOURCES

Epigenetics and transgenerational effects of DES

Endocrine Society banners

EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals

2015 Paper Abstract

The mechanisms of action of EDCs are varied and not entirely understood, but recent evidence suggests that some EDCs may cause epigenetic changes, which in turn may lead to transgenerational effects of EDCs on numerous organ systems. Epigenetic changes are described as heritable changes in gene expression that are not due to changes in DNA sequence (ie, not due to mutation). Several possible mechanisms of epigenetic change exist, including methylation of cytosine residues on DNA, post-translational modification of histones, and altered microRNA expression. To date, most studies on the effects of EDCs on epigenetic changes have focused on DNA methylation, but recent studies have also addressed the effects of EDCs on histone modifications and microRNA expression.

DNA methylation is a process in which methyl groups are attached to cytosine residues by DNA methyltransferase enzymes (DNMTs), usually in cytosine-guanosine dinucleotide pairs (CpG sites), although DNA methylation can occur on non-CpG residues. DNA methylation is important for several normal developmental and reproductive processes such as gametogenesis and embryogenesis. Hypermethylation in a promoter region is thought to repress gene transcription because the methylated promoter region has a decreased affinity for transcription factors and an increased affinity for methylated DNA-binding proteins, methyltransferases, histone deacetylases (HDACs), and/or corepressors.

Histone modification is a process in which specific amino acids in the N-terminal ends of histones undergo post-translational modification, including acetylation, methylation, phosphorylation, sumoylation, and ubiquitination by enzymes such as histone acetyltransferases, deacetylases, methyltransferases, and demethylases. These modifications determine whether the DNA wrapped around histones is available for transcription and play roles in determining the rate of transcription. Histone modifications also help regulate replication, recombination, and higher-order organization of the chromosomes. Changes to these modifications are often found in diseases such as cancer and are best studied for those diseases.

The molecular mechanisms by which microRNAs and other noncoding RNAs affect gene expression are not entirely understood, but it is likely that microRNAs play a role in gene regulation and chromatin organization. MicroRNAs often bind to the 3′ end of gene transcripts and initiate mRNA degradation or suppression of protein translation. Studies also suggest that microRNAs can affect the expression of other epigenetic regulators such as DNMTs and histone-modification enzymes.

Both hormones and EDCs cause DNA methylation, histone modifications, and altered microRNA expression. These epigenetic changes often cause phenotypic changes in organisms, which may appear immediately or long after EDC exposure. These properties are dictated by the timing of exposure. When EDCs introduce epigenetic changes during early development, they permanently alter the epigenome in the germline, and the changes can be transmitted to subsequent generations. When an EDC introduces epigenetic changes during adulthood, the changes within an individual occur in somatic cells and are not permanent or transmitted to subsequent generations. For an EDC to have truly transgenerational effects, exposure must occur during development, and the effects need to be observed in the F3 generation. This is because when a pregnant F0 female is exposed to an EDC, germ line cells in her F1 fetus are directly exposed to the EDC. These exposed F1 germ line cells are then used to produce the F2 generation, and thus, the F2 generation was directly exposed to the EDC via the germ cells. This exposure scenario makes the F3 generation the first generation that was not directly exposed to the EDC.

EDC-induced epigenetic changes are also influenced by dose of exposure, and they are tissue specific. Thus, it is important to consider both dose of EDC and the tissue before making firm conclusions about the epigenetic effects of EDCs. DNA methylation changes are the best-studied mechanism in this regard. For example, prenatal exposure to DES caused hypermethylation of the Hoxa10 gene in the uterus of mice and was linked to uterine hyperplasia and neoplasia later in life. Beyond the effects of prenatal exposure to DES on the daughters exposed in utero are suggestions that this leads to transgenerational effects of the chemical on the reproductive system, although whether this is linked to DNA methylation changes in humans is unknown.

Little is known about the ability of EDCs to cause histone modifications and whether this leads to transgenerational effects in animals or humans. DES caused histone deacetylation in the promoter region of the cytochrome P450 side chain cleavage (P450scc) gene. Further studies, however, need to be conducted to identify other EDCs causing histone modifications in animals and humans and to determine whether such modifications lead to transgenerational effects.

Synthetic estrogens are well known disruptors of uterine structure and function in humans and animals. Consistent with previous studies, recent data indicate that neonatal DES exposure caused endometrial hyperplasia/dysplasia in hamsters and increased uterine adenocarcinoma and uterine abnormalities in Donryu rats. Neonatal DES exposure also caused the differential expression of 900 genes in one or both layers of the uterus. Specifically, DES altered multiple factors in the PPARγ pathway that regulate adipogenesis and lipid metabolism, and it perturbed glucose homeostasis, suggesting that DES affects energy metabolism in the uterus. In the mouse uterus, DES altered the expression of chromatin-modifying proteins and Wnt signaling pathway members, caused epigenetic changes in the sine oculis homeobox 1 gene, and decreased the expression of angiogenic factors. DES also altered the expression of genes commonly involved in metabolism or endometrial cancer in mice, and it activated nongenomic signaling in uterine myometrial cells and increased the incidence of cystic glands in rats.

EDC exposures to pregnant animals have been shown to cause multigenerational or transgenerational effects on a number of disease endpoints, particularly reproduction, neurobehavior, and adiposity. This work needs much more follow-up to better determine the underlying mechanisms, which are likely to include epigenetic molecular programming changes. Moreover, research is needed in human populations. Some work has been conducted in grandchildren of DES-exposed women who took this estrogenic pharmaceutical during pregnancy. The consequences on the offspring (F1 generation) are well-studied, and research is beginning to be published on the grandchildren (F2 generation).

In summary, a prominent mechanism for increased disease risk in adulthood as a function of early-life EDC exposure is attributed to epigenomic reprogramming, a result of high plasticity as the epigenetic code is installed during development. Furthermore, the environment-gene interface must be considered as a basis for individual disease susceptibility whereby EDC-induced modifications of the epigenetic code early in life permit cryptic genetic variants or low-penetrant mutations to emerge and to manifest a phenotype at later life stages, long after the initial EDC exposure.

Sources

  • Full study (free access) : EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals, Endocrine Reviews, NCBI PubMed PMC4702494, 2015 Dec.
  • Featured image by archive.transgenderuniverse.
DES DIETHYLSTILBESTROL RESOURCES

Transgenerational neuroendocrine disruption of reproduction

2011 summary of epigenetic and transgenerational effects of DES

Key points

  • The hypothalamic neuroendocrine systems develop in a sexually dimorphic manner, largely because of differences in levels of gonadal steroids
  • Environmental endocrine disrupting chemicals (EDCs) impair the function of the neuroendocrine systems that control reproduction
  • Developmental exposure to EDCs, particularly during embryonic and early postnatal periods, permanently impairs functions and predisposes individuals to disease later in life owing to altered epigenetic programming
  • The mechanisms of EDC action include effects on the epigenetic molecular machinery that controls gene expression in hypothalamic and reproductive tissues
  • Effects of EDCs may be transmitted transgenerationally through molecular changes to the germline or through context-dependent modifications to somatic cells by continued exposures to EDCs or the individual’s social or environmental context

DES and epigenetic transmission

Transgenerational epigenetic effects of the estrogenic EDC diethylstilbesterol (DES) began with observations of rare vaginal clear-cell carcinomas and reproductive tract abnormalities in young women whose mothers had been prescribed DES in a misguided effort to avert miscarriage. These observations provided the first evidence for developmental programming or the fetal basis of adult disease caused by exogenous estrogens in humans. A potent estrogenic pharmaceutical, DES not only failed to reduce miscarriage risk, but it also exposed the developing daughters and sons to high levels of prenatal estrogens and predisposed them to adult diseases. Animal studies have replicated many of these effects of prenatal DES treatment and have begun to reveal the molecular mechanisms by which DES programs developing tissues. The case of DES is important because it is one of the first to link fetal exposure to a hormonally active compound with the latent development of disease years or even decades after the insult. This example in humans has laid the groundwork for much of the work on the effect of other EDC exposures to the fetus.

Epidemiological studies have also found that DES is associated with a small but significant increase in birth defects in grandchildren of women given DES during pregnancy. The granddaughters reported an increase in heart conditions,  slight but significant differences in their menstrual cycles and a reduction in live births when compared with women whose grandmothers were not exposed to DES.  Another study found an increase in the risk of hypospadias in the sons of women who were exposed to DES in utero,  and there was the single clinical observation that the 15-year-old granddaughter of a woman who took DES while she was pregnant developed a small-cell carcinoma of the ovary  Although the sample sizes in the last two studies were exceedingly small (28 and 1, respectively), they provide preliminary evidence for the potential of transgenerational effects of EDCs in the human population.

Rodent studies have revealed transgenerational epigenetic effects of DES. DES exposure of the F1 generation during embryonic and early postnatal development at a range of dosages had adverse consequences in the F2 generations. In F2 males, the incidence of proliferative lesions of the testes was increased, and serum estradiol concentration was reduced. In F2 females, the incidence of uterine adenocarcinomas and other tumors of the reproductive tract was increased in a subset of the dosage groups. Exposure of newborn (postnatal day 1–5) F1 female mice to 2 μg of DES led to demethylation of the estrogen-sensitive gene lactotransferrin promoter at two CpG dinucleotides (−464 and −454) in the uterus, and to upregulation of uterine expression of lactotransferrin in both F1 female mice and their F2 female offspring.  Female F1 animals exposed to the same dosing paradigm had decreased methylation of exon 4 of the Fos gene on postnatal day 5 and increased Fos mRNA expression in the uterus in adulthood.  Additionally, mice exposed gestationally to 10 μg/kg of DES from embryonic days 9–16 resulted in increased methylation of the homeobox A10 promoter and intron and increased Hox-A10 protein expression in the postnatal uterus (day 14).

Treatment of newborn, female and male mice on post-natal days 1–5 with 3 μg per pup per day of DES changed total DNA methylation in reproductive tissues. DES also altered expression of the enzymes that regulate DNA methylation. For example, expression of DNA methyltransferases 1, 3a and 3b was increased in the epididymis, and DNA methyltransferases 1 and 3b were increased in the uterus of mice exposed to DES compared with animals exposed to the vehicle control. Finally, in rats treated with 1 mg/kg DES on postnatal days 10–12, the exposure decreased global histone trimethylation at lysine 27 in the uterus on postnatal day 12 and altered the expression of several estrogen-sensitive genes in the uterus of the neonatal and adult exposed animals. Although most of these studies have only been conducted in tissues from F1 individuals, they provide insight into the potential targets for molecular epigenetic modifications that merit further study in subsequent generations in rodents.

Sources

  • Transgenerational neuroendocrine disruption of reproduction, Endocrinology, NCBI PubMed PMC3976559, 2011 Jan 25.
  • Summary of epigenetic and transgenerational effects of DES featured image PMC3976559/table/T1.
DES DIETHYLSTILBESTROL RESOURCES

Epigenetic mechanisms in the actions of endocrine-disrupting chemicals

Gonadal effects and role in female reproduction

2014 Study Abstract

There is a heightened interest and concern among scientists, clinicians, and regulatory agencies as well as the general public, regarding the effects of environmental endocrine-disrupting chemicals (EDCs). In this review, we identify the main epigenetic mechanisms and describe key ovarian processes that are vulnerable to the epigenetic actions of EDCs. We also provide an overview of the human epidemiological evidence documenting the detrimental effects of several common environmental EDCs on female reproduction. We then focus on experimental evidence demonstrating the epigenetic effects of these EDCs in the ovary and female reproductive system, with an emphasis on methoxychlor, an organochlorine pesticide. We conclude the review by describing several critical issues in studying epigenetic effects of EDCs in the ovary, including transgenerational epigenetic effects.

Diethylstilbestrol DES

The most convincing human evidence that estrogenic EDC exposure during development can permanently affect female reproduction comes from reports of the effects of DES, a nonsteroidal synthetic estrogen. From the 1940s to the 1970s, DES was prescribed at doses of 5–150 mg⁄ day to prevent miscarriages. Numerous abnormalities in the reproductive, cardiovascular, and immune systems have since been reported in both male and female offspring of women treated with DES, and similar effects have been demonstrated in animal models. Some of these effects, such as irregular cylicity or ovarian cancer, are being observed in the granddaughters of DES-treated women as well. In light of this multigenerational aspect, whether epigenetic mechanisms are involved is a significant question.

Experimental evidence from in vivo studies regarding the effects of EDCs in the ovary

Characteristic sets of defects in the ovary are the hallmark of EDC exposure in rodent models. For example, mice injected as little as a single dose of 10 μg/kg DES on E15 and examined at 7 months of age had numerous atretic follicles, and no corpora lutea (an indicator of ovulation). Other studies in rodents of varying doses of DES administered either in utero (E9-16) or neonatally (PND1-5) demonstrated similar defects by adulthood. Estrous cyclicity was completely disrupted and high levels of testosterone were found. A vacuolated interstitial tissue with lipid droplet inclusions and hemorrhagic cysts were also observed. A recent study proposes that while increased lipid droplet are caused by impaired steroidogenesis due to suppressed LH levels, the hemorrhagic follicles are results of direct effects of DES in the ovary. Furthermore, there was a dose-dependent reduction in the number of litters as well as the number of oocytes ovulated after stimulation with exogenous gonadotropins with such oocytes when used in IVF showing lower levels of fertilizability, suggesting reduced oocyte quality. Most striking of all, an excessive number of MOFs is found in adult ovaries; such a finding is considered to be an indicator of reduced reproductive lifespan.

Such estrogenic actions of these EDCs are mediated via the ER signaling pathway. Recent studies have shown that MOFs induced by neonatal exposure to 3 μg/kg DES is mediated by ERβ and not ERα. DES exposure was shown to reduce oocyte apoptosis (potentially suppressing oocyte nest breakdown) via ERβ signaling mechanisms. Furthermore, it was hypothesized that alterations in the germ cell–to–somatic cell ratio may affect the invasion of pregranulosa cells and basement membrane remodeling during primordial follicle formation. In contrast to ERβ signaling mechanisms involved in mediating ovarian effects, Couse and colleagues reported that ERα is essential for the mediation of DES effects in the uterus: αERKO female mice exhibited a complete resistance to the effects of DES while βERKO mice did not.

Epigenetic mechanisms associated with the female reproductive system

Although there are now well-documented studies of the physiological and morphological effects of EDCs on the ovary, the early research providing evidence of an epigenetic component of EDC actions was predominantly on the uterus. For example, it is well known that DES caused T-shaped uteri and clear cell adenocarcinoma of the uterus, cervix, and vagina in women whose mothers were exposed to DES during pregnancy. In the numerous animal studies validating these human reports, developmentally DES-treated mice manifest malformations of the uterus, squamous metaplasia of the luminar and glandular epithelium, endometrial hyperplasia and leiomyomas, and oviductal proliferative lesions. Ovariectomized animals when supplemented with E2 are able to respond by a transient increase in gene expression and concomitant uterine proliferation and growth. When such a stimulus was removed, the uterus returned to its unstimulated state. However, when DES or E2 is administered during neonatal development, expression of immediate early genes such as c-fos, c-jun, and c-myc as well as lactoferrin and EGF are upregulated even into adulthood. This was associated with hypomethylation of the promoter region of the lactoferrin gene in the adult uterus. However, if animals were exposed for the same interval during adulthood, no such methylation or expression defects were observed, indicating the importance of developmental exposure. Subsequently, it was also found that exon 4 of the c-fos gene was extensively hypomethylated while the promoter region and intron 1 were unaffected, thereby potentially allowing for the upregulation of c-fos expression. Furthermore, recent work by Block and coworkers has shown that DES disrupts the regionalization of expression of Hoxa-9 and Hoxa-10 and the homeotic anterior transformations associated with hypermethylation in the promoter and intron 1 regions of Hoxa-10 gene. Additionally, ERα induction is necessary for activation of estrogen-responsive gene expression in the uterus, including that of the lactoferrin and c-fos genes. Since many of the above genes are downstream of ER signaling, which is involved in direct actions of EDCs, it is imperative to thoroughly examine the potential role of epigenetic mechanisms in the regulation of ER expression after EDC exposure. In addition, PI3K/Akt signaling downstream of membrane-associated ER signaling caused reduction in trimethylation of the histone H3K27 in response to E2 and DES exposures. More interestingly, activation of this non-genomic signaling caused reprogramming of the uterine gene expression profile . Another example is that of the investigation by Tang and colleagues, wherein it was demonstrated that neonatal DES/genistein exposure reduced DNA methylation and increased gene expression of nucleosomal binding protein 1 in adult uteri. These studies highlighted the age-dependent aspect of epigenetic reprogramming and also its interaction with steroid hormones.

Sources

  • Full study (free access) : Epigenetic mechanisms in the actions of endocrine-disrupting chemicals: Gonadal effects and role in female reproduction, HHS Author Manuscripts, NCBI PubMed PMC4151320, 2014 Sep 2.
  • Featured image by Christian Perner.
DES DIETHYLSTILBESTROL RESOURCES

Environmental Epigenetics and Its Implication on Disease Risk and Health Outcomes

image of Environmental Epigenetics

2012 Study Abstracts

Introduction

This review focuses on how environmental factors through epigenetics modify disease risk and health outcomes. Major epigenetic events, such as histone modifications, DNA methylation, and microRNA expression, are described. The function of dose, duration, composition, and window of exposure in remodeling the individual’s epigenetic terrain and disease susceptibility are addressed. The ideas of lifelong editing of early-life epigenetic memories, transgenerational effects through germline transmission, and the potential role of hydroxylmethylation of cytosine in developmental reprogramming are discussed. Finally, the epigenetic effects of several major classes of environmental factors are reviewed in the context of pathogenesis of disease. These include endocrine disruptors, tobacco smoke, polycyclic aromatic hydrocarbons, infectious pathogens, particulate matter, diesel exhaust particles, dust mites, fungi, heavy metals, and other indoor and outdoor pollutants. We conclude that the summation of epigenetic modifications induced by multiple environmental exposures, accumulated over time, represented as broad or narrow, acute or chronic, developmental or lifelong, may provide a more precise assessment of risk and consequences. Future investigations may focus on their use as readouts or biomarkers of the totality of past exposure for the prediction of future disease risk and the prescription of effective countermeasures.

Implications of Lifelong Editing of Early-Life Epigenetic Memories

The concept of continued editing of early-life epigenetic markings or memories during adult life has been proposed on the basis of evidence from limited experimental studies. Exposure of mice to diethylstilbestrol (DES, a xenoestrogen) or genistein (a phytoestrogen) during the perinatal period induced specific epigenetic markings in their uteri. However, some of these epigenetic markings (hypomethylation of Nsbp1) remained “hidden” during prepuberty life and appeared in adulthood only in the exposed intact females but not in their ovariectomized counterparts, suggesting that adult exposure to ovarian steroids may cause these markings to “surface.” Coincidentally, the prevalence of uterine cancer was higher in neonatally exposed intact mice, but not in mice ovariectormized before puberty.

Epigenetic Factors Shown to Trigger Epigenetic Events and Affect Disease States

Exposure to EDCs during early developmental periods is a major health concern because it can cause persistent changes in gene expression through epigenetic reprogramming in somatic cells, as well as germ-line cells, and subsequently promote transgenerational inheritance. The xenoestrogen DES was widely used in cattle and other livestock industries and is still an EDC in many populations. Early-life exposure of mice to DES increases risk of uterine cancer that is accompanied by demethylation of an estrogen-responsive gene, lactoferrin, in the mouse uterus. In utero exposure of mice to DES triggered hypermethylation of the homeobox A10 with attended uterine hyperplasia and neoplasia in later life. A more recent report documented hypermethylation of nucleosome binding protein 1 (Nsbp1 or Hmgn5) as a hidden uterine epigenetic mark after neonatal DES exposure that only appeared upon sexual maturation of the exposed mice but failed to manifest if the animals were ovarietomized before puberty. Of significant interest is the transgenerational effect of developmental exposure of mice to DES that promoted c-fos expression, hypomethylation of specific exon CpGs, and increased susceptibility to tumorigenesis in the F2 generation. These experimental data support the hypothesis that epigenetic reprogramming is responsible for the devastating consequences observed in the offspring of women who took DES during pregnancy. The DES effects include female genital abnormalities, vaginal cancer, and male urogenital disorders. The adverse effects may be reverberating in the grandchildren of these women.

Sources

  • Full study (free access) : Environmental Epigenetics and Its Implication on Disease Risk and Health Outcomes, ILAR Journal, NCBI PubMed PMC4021822, 2012 Dec.
  • Featured image by h heyerlein.
DES DIETHYLSTILBESTROL RESOURCES

Transgenerational Epigenetic Inheritance: Focus on Endocrine Disrupting Compounds

image of transgenerational-epigenetic-inheritance

2014 Study Abstract

The classic case of an EDC is diethylstilbestrol (DES), an estrogen agonist and androgen receptor antagonist synthesized first in the 1930s and prescribed to at least 5 million women at risk for miscarriage or experiencing other reproductive problems, from 1938 up to 1975. Instead of the desired effects, use of this compound lead to increased incidence of breast, vaginal, and cervical cancers.

In addition, maternal exposure has documented adverse affects on daughters. These include the same types of cancers as well as a variety of difficulties conceiving and maintaining pregnancies, reproductive tract malformations, abnormal menstrual cycles, early puberty, and behavioral issues. The vast variety of effects is probably related to complexities introduced by the timing of DES treatment and doses. For example, a recent large study of women exposed prenatally to DES revealed a strong correlation between DES, particularly in the first trimester, and noncancerous uterine fibroids. Offspring of rodents exposed to DES during pregnancy recapitulate many of these effects.

Although the initial clinical studies were limited to female offspring, correlations between DES exposure and hypospadias, cryptorchidism, and testicular cancer have been reported in F1 and F2 sons and grandsons of women given DES. Analyses of the clinical studies suggest that the male reproductive illnesses are related to but not necessarily caused by estrogen actions. An alternative hypothesis is that DES produces low-birth-weight babies, and these infants are more prone to testicular dysgenesis syndrome. Multigenerational work in mice has demonstrated that high, but clinically relevant, doses of DES increase the incidence of uterine and other reproductive tract tumors in females and lesions in the male rete testes in F2 offspring. Few data on the critical F3 generation in humans are available nor are there experimental data from rodent models.

EDCs, such as DES, share many properties with steroid hormones: they act at low doses (picograms) and can act in a nonmonotonic manne. Like hormones, they are particularly effective during development, at which time they can modify the course of reproductive tract and brain development. Importantly, the EDCs are more promiscuous than steroids and bind to a larger variety of receptors than normal ligands, albeit with reduced affinities.

Sources

  • Transgenerational Epigenetic Inheritance: Focus on Endocrine Disrupting Compounds, Endocrinology, NCBI PubMed PMC4098001, 2014 Aug.
  • Featured image Matt Artz.
DES DIETHYLSTILBESTROL RESOURCES

Epigenomics in Environmental Health

image of Epigenomics

DES establishes altered epigenetic marks capable of transgenerational inheritance

2011 Study Abstract

Exposures to endocrine disrupting chemicals such as the model toxicant diethylstilbestrol (DES) and the omnipresent bisphenol A (BPA) are of particular concern in the context of development.

In utero exposure of mice to DES has been shown to result in the hypermethylation of the developmentally critical (specifically to uterine organogenesis) Hoxa10 gene.

Although not a genotoxic agent, epidemiologic evidence from individuals exposed to DES during the first 3 months in utero indicates an increase in vaginal clear cell carcinoma incidence and reproductive disorders as adults.

In addition, grandchildren of DES exposed women reported higher incidences of rare reproductive disorders; whether this reflects detection bias or possibly implicates a role for epigenetic transgenerational inheritance remains to be clarified.

Ruden et al. drawing similarities between DES and Hsp90 has proposed a mechanism through which DES establishes altered epigenetic marks capable of transgenerational inheritance, wherein DES plays a role in modifying H3K4 methylation by increasing the activity of the H3K4 methyltransferase SMYD3, thereby altering epigenetic control of various genes.

Sources

  • Full study (free access) : Epigenomics in Environmental Health, Journal of andrology, Frontiers in Genetics, NCBI PubMed PMC3268636, 2011.
  • Featured image by sciencemag.
DES DIETHYLSTILBESTROL RESOURCES

DES multigenerational assay data interpretation

Comparison of the developmental and reproductive toxicity of diethylstilbestrol administered to rats in utero, lactationally, preweaning, or postweaning

2002 Study Abstract

The objective of the study was to determine which period of exposure produces the most marked effects on the reproductive capacity and sexual development of the rat, with particular emphasis on the relative sensitivity of in utero and postnatal exposures.

The endocrine active chemical, diethylstilbestrol (DES) was used as an agent known to affect many of the endpoints examined. Hitherto, such comparisons have been made between studies, rather than within a study. Our data will be helpful in the interpretation of future multigenerational assay data.

In preliminary studies, DES was shown to be active in the immature rat uterotrophic assay with a lowest detected dose of 0.05 mg DES/kg body weight by sc injection and 10 mg DES/l (1.6 mg DES/kg body weight) by administration in drinking water. A dose of 60 microg DES/l drinking water ( approximately 6.5mg DES/kg body weight/day) was selected for the main study since this represented the midpoint of the drinking water uterotrophic dose response and produced no overt maternal toxicity. The study used 10 groups of concomitantly pregnant animals, including 2 control groups. The first comparison was between the effects of exposure to DES in utero, and exposure from conception to weaning. Another group of animals was exposed to DES in utero and cross-fostered to untreated pregnant females to prevent lactational transfer of DES to pups. Two groups were exposed to DES neonatally, either from birth to postnatal day (PND) 10 (pups thus having only lactational exposure), or from birth until weaning (PND 21; pups thus having both lactational exposure and self-exposure via drinking water). In addition, a dose response study to DES was conducted on animals exposed from weaning to PND 100, when the first phase of the study was terminated. Pups exposed to DES in utero and pups exposed from weaning to PND 100 were bred to assess fertility of the F1 animals and the sexual development of F2 offspring. This last comparison was to determine the extent to which weanling rats could be used in endocrine toxicity studies to assess their potential to show activity in utero. The most sensitive period of exposure for inducing developmental effects in F1 animals was from weaning onwards. The neonatal to weaning period (PND 1-21) was the next most sensitive. Essentially no effects were induced in F1 animals exposed in utero. No effects of any kind were observed in animals only exposed over the early neonatal period of PND 1-10. The mean day of vaginal opening, testes descent, and prepuce separation was only altered in groups where postnatal exposure to DES continued beyond PND 10, or was started at weaning. No changes were observed in anogenital distance or caudal sperm counts. Some changes in organ weights were observed, but the interpretation of these was often confused by concomitant changes in body weight. In general, histopathological examination of tissues yielded no additional information. In breeding studies with animals exposed to DES in utero, or from weaning, reduced litter sizes and marginal advances in the day of vaginal opening were observed in the offspring, together with changes in organ weights. However, no unique sensitivity was noted for exposure in utero. Evaluation of the several exposure periods and the many markers monitored in this study may have individual strengths in individual cases, but when rigorously compared using the reference estrogen DES, many preconceptions regarding their absolute or relative value were not upheld. Further, each of these markers is subject to natural variability, as demonstrated by comparisons made among the 5 separate control groups available in parts of the present study. This variability increases the chance that small changes observed in endocrine toxicity studies employing small group sizes and a single control group, or no concomitant control group, may be artifactual. The most marked effects observed in this study were on the developmental landmarks in the F1 animals induced by exposures after PND 10. Some effects on developmental landmarks and organ weights were observed in F2 animals following exposure either in utero or postweaning.

This study therefore does not establish a unique role for exposures in utero or during the early neonatal period.

Sources

  • Comparison of the developmental and reproductive toxicity of diethylstilbestrol administered to rats in utero, lactationally, preweaning, or postweaning, Toxicological sciences : an official journal of the Society of Toxicology, NCBI PubMed PMID: 12075118, 2002 Jul.
  • Featured image NASA.
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