DES Grandsons Hypospadias ; True Transgenerational Effect ?

image of Grandson

Hypospadias in sons of women exposed to diethylstilbestrol: a true trans-generational effect?

2005 Study Abstract

In May 2005, Pons et al. reported on an increased risk of hypospadias in male children of women exposed to diethylstilbestrol (DES) in utero. The authors have retrospectively reviewed the electronic files from 17 633 deliveries of male neonates in a 10-year period. The mothers of 240 male neonates had reported in utero DES exposure, three of whom (1.23%) presented with hypospadias vs 44/17 393 (0.5%) in the remaining male neonates (from non-DES-exposed mothers). The authors conclude that there is an increased risk of hypospadias in the male children of women exposed in utero to DES due to the transgenerational effects of DES. Although these results apparently compare favourably with the initial Dutch cohort, we would like to address the authors with our concerns regarding the interpretation of these additional data.

In utero DES exposure has been associated not only with an increased risk of preterm labour but also with an increased risk of intra-uterine growth retardation (IUGR). In turn, an increased risk of cryptorchidism and hypospadias has been associated with decreased birth weight. One might expect a higher rate of IUGR in the subgroup of neonates of women exposed in utero to DES compared with the control group in the Parisian cohort, as previously observed in the Dutch one. This information, essential to the interpretation of the data, may avoid causing the patients undue concern about hypothetical transgenerational adverse effects of DES (i.e. genetic or epigenetic changes in either germ or somatic cells).

Sources

  • Hypospadias in sons of women exposed to ditheylstilbestrol: a true trans-generational effect?, Prenatal diagnosis, NCBI PubMed PMID: 16302166, 2005 Nov.
  • Featured image credit Johan Mouchet.
DES DIETHYLSTILBESTROL RESOURCES

Increased breast cancer risk in DES daughters, granddaughters and great granddaughters

The effects of maternal diethylstilbestrol exposure are not limited to the F1 generation

2014 Study Abstract

The idea that susceptibility to breast cancer is determined not only through inherited germline mutations but also by epigenetic changes induced by alterations in hormonal environment during fetal development is gaining increasing support. Using findings obtained in human and animal studies, this review addresses the mechanisms that may explain why daughters of mothers who took synthetic estrogen diethylstilbestrol (DES) during pregnancy have two times higher breast cancer risk than women who were not exposed to it. The mechanisms likely involve epigenetic alterations, such as increased DNA methylation and modifications in histones and microRNA expression. Further, these alterations may target genes that regulate stem cells and prevent differentiation of their daughter cells. Recent findings in a preclinical model suggest that not only are women exposed to DES in utero at an increased risk of developing breast cancer, but this risk may extend to their daughters and granddaughters as well. It is critical, therefore, to determine if the increased risk is driven by epigenetic alterations in genes that increase susceptibility to breast cancer and if these alterations are reversible.

Gene expression can be altered as a consequence of mutations or epigenetic changes. In contrast to gene mutations within the DNA, epigenetic changes involve post-transcriptional modifications; that is, methylation of gene promoter regions, histone modifications, deposition of certain histone variants along specific gene sequences and microRNA (miRNA) expression. Although both changes are heritable, an important distinction between the two is that mutations are not reversible, but epigenetic modifications generally are.

Developing germ cells undergo epigenetic erasure when they, as primordial germ cells, enter into the fetal gonads around embryonic day 10 to 11 (in mice and rats), and then undergo gender-specific reprogramming as germ cells. It is now clear that reprogramming of these cells is susceptible to modifications caused by changes in fetal hormonal environment, such as resulting from an exposure to DES or other endocrine disruptors. Consequently, these exposures can leave a permanent biochemical footprint on the genome of the F1 generation germ cells, and this change may be inherited by the F2 generation germ line and several subsequent generations.

Some researchers have begun to investigate whether the effects of maternal DES exposure during pregnancy extend to the third generation in humans. Although there is no evidence that DES granddaughters have cervical and ovarian abnormalities similar to DES daughters, there is evidence that they may have more menstrual irregularities and a higher rate of infertility than non-exposed granddaughters. In addition, DES granddaughters may have a slightly higher risk of ovarian cancer. The granddaughters are still too young to assess whether they might also be at an increased risk of developing breast cancer.

Millions of women in the US, Europe and Australia have been exposed to DES in the womb, and consequently exhibit about a two times higher breast cancer risk than unexposed women. The increase in risk may not be limited to the DES-exposed daughters, but could also increase breast cancer risk in granddaughters and great granddaughters. Such outcome would be consistent with the findings we obtained in studies using a synthetic estrogen ethinyl estradiol (EE2). If DES has similar effects to ethinyl estradiol on the transgenerational increase in breast cancer risk, it is urgent to find ways to stop the cycle of inheritance, and also prevent breast cancer in DES-exposed granddaughters and great granddaughters.

To achieve this goal, we need to understand how maternal DES exposure during pregnancy increases a daughter’s breast cancer risk. A plausible model is proposed in feature image. It is evident from studies done in animal models that in utero DES exposure induces epigenetic changes in reproductive tract tissues and the breast. DES exposure might also have induced epigenetic changes in primordial germ cells and consequently germ cells, and further be detectable in the somatic cells in granddaughters and great granddaughters. We are not aware of any study that has compared epigenetic changes in germ cells and the next generation somatic cells in individuals exposed to DES or other endocrine disruptors in utero. Second, we should investigate whether the transgenerational increase in breast cancer risk can be prevented with drugs that reverse epigenetic modifications. Our preliminary studies in mice suggest that this is achievable in daughters by using the well-tolerated and non-toxic histone deacetylase inhibitor valproic acid and DNMT inhibitor hydralazine. However, whether these compounds also prevent an increase in granddaughters and great granddaughters in experimental models remains to be investigated.

In summary, women exposed to DES in utero are destined to be at an increased risk of developing breast cancer, and this risk may extend to their daughters and granddaughters as well. It is of critical importance to determine if the increased risk is driven by epigenetic alterations in genes that increase susceptibility to breast cancer and if these alterations are reversible.

Sources

  • Maternal exposure to diethylstilbestrol during pregnancy and increased breast cancer risk in daughters, Breast Cancer Research, NCBI PubMed PMC4053091, 2014 Apr 30.
  • Proposed model to explain an increase in breast cancer risk in daughters, and possibly granddaughters and great granddaughters, of mothers who took diethylstilbestrol during pregnancy : featured image credit PMC4053091/figure/F1.
DES DIETHYLSTILBESTROL RESOURCES

Birth Defects in DES Grandsons

Birth defects in children of men exposed in utero to diethylstilbestrol (DES)

2018 Study Abstract

OBJECTIVE
Prenatal exposure to diethylstilbestrol (DES) is associated with adverse effects, including genital anomalies and cancers in men and women. Animal studies showed birth defects and tumors in the offspring of mice prenatally exposed to DES. In humans, birth defects, such as hypospadias were observed in children of prenatally exposed women. The aim of this research was to assess the birth defects in children of prenatally exposed men.

METHODS
In a retrospective study conceived by a patients’ association (Réseau DES France), the reports of men prenatally exposed to DES on adverse health effects in their children were compared with those of unexposed controls and general population.

RESULTS
An increased incidence of two genital anomalies, cryptorchidism (OR=5.72; 95% CI 1.51-21.71), and hypoplasia of the penis (OR=22.92; 95% CI 3.81-137.90), was observed in the 209 sons of prenatally exposed men compared with controls, but hypospadias incidence was not increased in comparison with either the controls or the general population. No increase of genital anomalies was observed in daughters.

CONCLUSION
With caution due to the methods and to the small numbers of defects observed, this work suggests an increased incidence of two male genital tract defects in sons of men prenatally exposed to DES. This transgenerational effect, already observed in animals and in the offspring of women prenatally exposed to DES, could be the result of epigenetic changes transmitted to the subsequent generation through men.

Sources

  • Birth defects in children of men exposed in utero to diethylstilbestrol (DES), Therapie, NCBI PubMed PMID: 29609831, 2018 Mar 3.
  • Featured image credit Danielle MacInnes.
DES DIETHYLSTILBESTROL RESOURCES

Effects of DES in a Third Generation

Exposure to Diethylstilbestrol during Sensitive Life Stages: A legacy of heritable health effects

2013 Selected Abstracts

Walker and Haven predicted that “if the high intensity of DES multigenerational carcinogenicity seen in mice is applicable to the human population, this is a health problem of major proportions.” They go on to say that it “could take over 50 years” to detect the effects in future generations, due to the length of time required for diseases such as cancer to manifest. It is predicted that cross-generational responses to DES exposure are possible due to epigenetic changes in the DNA and that the “germ cell pool could have become massively contaminated”. For example, early exposure to EDCs, like DES, is thought to reprogram mouse female reproductive tract development and affect how the reproductive tract responds to endogenous estrogens later in life. They suggest that “environmental estrogens may be more potent than previously suspected, due to synergistic action from concurrent exposures.”

The studies on the cohort of men (grandsons) and women (granddaughters) whose mothers were exposed prenatally to DES (grandchildren had no direct exposure) are limited as they are just beginning to reach the age when relevant health problems can be studied. Studies that have been performed contain preliminary data, as the power is low. Therefore, the main sources of information for third generation effects are rodent studies. In general, multi-generational mouse studies have shown an increased susceptibility to tumor formation in the third generation which suggests that DES grandchildren are also at an increased risk for cancer.

Granddaughters

Currently there are no human studies that definitively show any adverse effects of DES for the third generation of females. A small cohort study of 28 DES granddaughters found no abnormalities in the lower genital tract and no cases of CCA. These results led authors to conclude that third generation effects were unlikely even after they acknowledged that the number of participants was too small and the women were too young for the findings to have any real significance.

Multigenerational rodent studies, as a primary source for information on the effects of DES exposure, disagree with those preliminary findings in humans. Although severe effects of DES were apparent in the first round of CD-1 mouse offspring (second generation), the third generation did not exhibit the same subfertility, regardless of exposure timing or dose. However, these studies have found an increased susceptibility to tumor formation in the third generation. Aged third generation female mice had increased risks for uterine cancers, benign ovarian tumors, and lymphomas. One study found cervical adenocarcinomas, which are not generally seen in untreated mice, in third generation females similar to those induced by direct prenatal DES exposure. In the same study, third generation female mice had increases in ovarian, uterine, and mammary tumors with the total number of reproductive tumors being statistically significant from the control mice.

Grandsons

The early reports of DES grandsons show an increase in hypospadias in this population. Hypospadias occurred twenty times more frequently in the DES grandsons’ cohort, which suggests that their mothers (DES daughters) may have had a disturbed hormonal balance during their reproductive life that interfered with the genital development of the male fetus. The prevalence of hypospadias was found to be >3% in DES grandsons but the risk of the defect is still low. Mouse studies in the third generation DES-exposed male population have found an increased susceptibility for reproductive tumor formation, specifically in the testes, prostate, and seminal vesicles. No effect on reproductive capacity or other deformities was seen in DES grandsons.

Sources

  • Full study (free access) : Exposure to Diethylstilbestrol during Sensitive Life Stages: A legacy of heritable health effects, Birth defects research. Part C, Embryo today : reviews, NCBI PubMed PMC3817964, 2013 Nov 5.
  • Featured image credit Oskars Sylwan.
DES DIETHYLSTILBESTROL RESOURCES

Epigenetics and transgenerational effects of DES

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

Selected Abstracts

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.

DES caused histone deacetylation in the promoter region of the cytochrome P450 side chain cleavage (P450scc) gene.

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.

Studies in mice showed that DES induced vaginal adenosis by down-regulating RUNX1, which inhibits the BMP4/activin A-regulated vaginal cell fate decision; induced epithelial cell proliferation and inhibited stromal cell proliferation; and caused persistent down-regulation of basic-helix-loop-helix transcription factor expression (Hes1, Hey1, Heyl) in the vagina, leading to estrogen-independent epithelial cell proliferation. Neonatal exposure to DES caused persistent changes in expression of IGF-1 and its downstream signaling factors in mouse vaginas. It also up-regulated Wnt4, a factor correlated with the stratification of epithelial cells, in mouse vaginas. Interestingly, the simultaneous administration of vitamin D attenuated the ability of DES to cause hyperplasia of the vagina in neonatal mice.

In mice treated prenatally with DES there was a significant increase in enhancer of Zeste homolog 2 (EZH2) protein and EZH2 activity (measured by increased mammary histone H3 trimethylation)—a histone methyltransferase that may be linked to breast cancer risk and epigenetic regulation of tumorigenesis, as well as an increase in adult mammary gland EZH2.

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). For environmental chemicals, several ongoing projects need continued funding.

Sources

  • Full study (free access) : EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals, NCBI PubMed PMC4702494, 2015 Nov 6.
  • Featured image credit Craig Whitehead.
DES DIETHYLSTILBESTROL RESOURCES

Endocrine Disruptor Induction of Epigenetic Transgenerational Inheritance of Disease

DES phenotypes found to be due to abnormal epigenetic programming of some organs and critical genes

2014 Study Abstract

Environmental exposures such as toxicants, nutrition and stress have been shown to promote the epigenetic transgenerational inheritance of disease susceptibility. Endocrine disruptors are one of the largest groups of specific toxicants shown to promote this form of epigenetic inheritance. These environmental compounds that interfere with normal endocrine signaling are one of the largest classes of toxicants we are exposed to on a daily level. The ability of ancestral exposures to promote disease susceptibility significantly increases the potential biohazards of these toxicants. Therefore, what your great-grandmother was exposed to during pregnancy may influence your disease development, even in the absence of any exposure, and you are going to pass this on to your grandchildren. This non-genetic form of inheritance significantly impacts our understanding of biology from the origins of disease to evolutionary biology. The current review will describe the previous studies and endocrine disruptors shown to promote the epigenetic transgenerational inheritance of disease.

DES Epigenetics

One of the best examples of a human model is in the late 1950’s and early 1960’s when women in the late stages of pregnancy were exposed to the pharmaceutical diethylstilbesterol (DES) which was shown to promote abnormal uterine and cervical development in the female offspring and grand-offspring. Subsequently the phenotypes were found to be due to abnormal epigenetic programming of these organs and critical genes.

Sources

  • Full study (free access) : Endocrine disruptor induction of epigenetic transgenerational inheritance of disease, Molecular and cellular endocrinology, NCBI PubMed PMC4262585, 2014 Jul 31.
  • Epigenetic and genetic cascade of events involved in development featured image credit PMC4262585/figure/F1.
DES DIETHYLSTILBESTROL RESOURCES

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