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

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

Diethylstilbestrol-Induced Mouse Hypospadias: “Window of Susceptibility”

image pf hypospadias in human fetal penises

Defining a DES “programming window”

2016 Study Abstract

Hypospadias, an abnormality affecting the penile urethra, is one of the most prevalent congenital malformations afflicting human males. The morphology of hypospadias is markedly different in humans versus mice reflecting substantial differences in penile development in humans and mice. Estrogens such as diethylstilbestrol (DES) elicit mouse penile malformations, but the types of penile abnormalities differ depending on whether DES treatment is prenatal or neonatal.

A thorough investigation of the effects of DES over a wide age range of treatment may

  • elucidate the morphogenetic mechanisms involved in generating abnormal penile morphology and hypospadias
  • and reveal those penile elements more (or less) sensitive on a temporal basis to developmental exposure to DES.

Such an approach may also explain why certain effects of DES elicited and expressed during development resolve to normality in adulthood.

To define the actual “window of susceptibility” to the adverse effects of DES, pregnant mice and their neonatal pups were injected subcutaneously with 200ng/gbw DES every other day

  • from embryonic day 12 to 18 (DES E12-E18),
  • postnatal day 0 to 10 (DES P0-P10),
  • embryonic day 12 to postnatal day 10 (DES E12 to P10),
  • postnatal day 5 to 15 (DES P5 to P15),
  • and postnatal day 10 to 20 (DES P10 to P20).

Aged-matched controls received sesame oil vehicle. After euthanasia at 10, 15, 20 and 60 days, penises were analyzed by gross morphology, histology and morphometry.

Penises of all 5 groups of DES-treated mice were reduced in size, which was confirmed by morphometric analysis of internal penile structures, and are presumably mediated via signaling through estrogen receptors alpha and/or beta (ERα and ERβ), which have been previously detected in all of the structures affected by DES.

The most profound effects were seen in the DES E12-P10, DES P0-P10, and DES P5-P15 groups, thus defining a DES “programming window”.

For all parameters, DES treatment from P10-P20 showed the most mild of effects.

Adverse effects of DES on the MUMP cartilage and erectile bodies observed shortly after the last DES injection reverted to normality in the DES P5-P15, but not in the E12-P10 and P0-P10 groups, in which MUMP cartilage and erectile body malformations persisted into adulthood, again emphasizing a “window of susceptibility” in the early neonatal period.

Sources

  • Full study (free access) : Diethylstilbestrol-Induced Mouse Hypospadias: “Window of Susceptibility”, Differentiation, NCBI PubMed PMC4803596, 2016 Jan 20.
  • Scanning electron micrographs of human fetal penises at 7 and 10 weeks of gestation. In (A) note the prominent urethral groove. In (B) the edges of the urethral groove are fusing in the midline to form the urethra, but the distal urethral groove is still widely open. Featured image credit PMC4803596/figure/F3.
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

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

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

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