DES Developmental Programming and Fetal Origins of Adult Disease

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

Introduction

The 2007 Summit on “Environmental Challenges to Reproductive Health and Fertility” convened scientists, health care professionals, community groups, political representatives and the media to hear presentations on the impact of environmental contaminants on reproductive health and fertility and to discuss opportunities to improve health through research, education, communication and policy. Environmental reproductive health focuses on exposures to environmental contaminants, particularly during critical periods of development, and their potential effects on future reproductive health, including conception, fertility, pregnancy, adolescent development and adult health. Approximately 87,000 chemical substances are registered for use in commerce in the US, with ubiquitous human exposures to environmental contaminants in air, water, food and consumer products. Exposures during critical windows of susceptibility may result in adverse effects with lifelong and even intergenerational health impacts. Effects can include impaired development and function of the reproductive tract and permanently altered gene expression, leading to metabolic and hormonal disorders, reduced fertility and fecundity and illnesses such as testicular, prostate, uterine and cervical cancers later in life. This executive summary reviews effects of pre- and post-natal exposures on male and female reproductive health and provides a series of recommendations for advancing the field in the areas of research, policy, health care and community action.

Abstracts

Developmental Programming and Fetal Origins of Adult Disease

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.

Reproductive Effects of Early Life Exposures in Females

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, NCBI PubMed, PMC2440710, 2009 Feb 1.
  • Featured image credit Kiệt Hí.
DES DIETHYLSTILBESTROL RESOURCES

Occurrence of tumours in the descendants of male prenatally exposed to DES

Multigenerational effects of DES have been reported through the paternal lineage

1992 Study Abstract

There is well documented evidence both in humans and in experimental animals that exposure to diethylstilbestrol (DES) during pregnancy results in an increased incidence of tumours in the progeny.

The increased cancer risk has been reported to persist in the second generation descendants of DES-exposed pregnant mice. In the present experiment, female mice of the CBA strain were treated at day 17 of pregnancy with 1 microgram/g body weight of DES.

The descendants of DES-treated mothers, described as F1DES, were mated among each other or with untreated animals.

  • The F1DES females were found to be sterile when mated with either F1DES or untreated males.
  • F1DES males were successfully mated with untreated females.
    • In the female offspring so obtained, but not in the male, a statistically significant increased incidence of tumours was observed, in particular of uterine sarcomas, and also of benign ovarian tumours and of lymphomas.

Sources

  • Occurrence of tumours in the descendants of CBA male mice prenatally treated with diethylstilbestrol, International journal of cancer, NCBI PubMed, PMID: 1728603, 1992 Jan.
  • Featured image credit Ousa Chea.
DES DIETHYLSTILBESTROL RESOURCES

Intensity of Multigenerational Carcinogenesis from DES

Multigenerational effects of DES have been reported through the maternal lineage

We were told that it “could take over 50 years” to detect the effects of DES exposure in future generations, due to the length of time required for diseases to manifest. It is predicted that cross-generational responses to the exposure of DES are possible due to epigenetic changes in the DNA.

1997 Study Abstract

Mice exposed prenatally to diethylstilbestrol (DES-exposed mice) can transmit a carcinogenic influence to the next generation (DES-lineage mice) when mated to control mice.

The persistence of this effect was studied one generation further (DES-lineage-2 mice) by mating DES-lineage female mice to control males. The interaction of maternal dietary fat levels with DES was also tested by feeding high and low levels of dietary fat during the pregnancies that produced the final two generations.

DES-lineage-2 mice, exposed to low or high fat maternal diets, had significantly more tumors than control mice with corresponding dietary fat exposure. The frequency of tumors in DES-lineage-2 mice was not significantly lower than in DES-lineage mice from a previous experiment.

Thus, the multigenerational effect of DES is relatively intense in mice. If this type of carcinogenesis can occur in the human population, it poses a major threat to future generations.

Sources

  • Intensity of multigenerational carcinogenesis from diethylstilbestrol in mice, Carcinogenesis, NCBI PubMed, PMID: 9111216, 1997 Apr.
  • Featured image credit Alisa Olaivar.
DES DIETHYLSTILBESTROL RESOURCES

Increased susceptibility for tumors transmitted to DES subsequent generations

Proliferative lesions and reproductive tract tumors in male descendants of mice exposed developmentally to diethylstilbestrol

2000 Study Abstract

Prenatal exposure to diethylstilbestrol (DES) is associated with reproductive tract abnormalities, subfertility and neoplasia in experimental animals and humans.

Studies using experimental animals suggest that the carcinogenic effects of DES may be transmitted to succeeding generations.

To further evaluate this possibility and to determine if there is a sensitive window of exposure, outbred CD-1 mice were treated with DES during three developmental stages:

  1. group 1 was treated on days 9-16 of gestation (2.5, 5 or 10 microg/kg maternal body weight) during major organogenesis;
  2. group II was treated once on day 18 of gestation (1000 microg/kg maternal body weight) just prior to birth;
  3. and group III was treated on days 1-5 of neonatal life (0.002 microg/pup/day).

DES-exposed female mice (F(1)) were raised to maturity and bred to control males to generate DES-lineage (F(2)) descendants. The F(2) males obtained from these matings are the subjects of this report; results in F(2) females have been reported previously. Reproductive performance of F(2) males when bred to control females was not different from control males. However, in DES F(2) males killed at 17-24 months, an increased incidence of proliferative lesions of the rete testis and tumors of the reproductive tract was observed. Since these increases were seen in all DES treatment groups, all exposure periods were considered susceptible to perturbation by DES.

These data suggest that, while fertility of the DES F(2) mice appeared unaltered, increased susceptibility for tumors is transmitted from the DES ‘grandmothers’ to subsequent generations.

Sources

  • Proliferative lesions and reproductive tract tumors in male descendants of mice exposed developmentally to diethylstilbestrol, Carcinogenesis, NCBI PubMed, PMID: 10874014, 2000 Jul.
  • Featured image credit Руслан Гамзалиев.
DES DIETHYLSTILBESTROL RESOURCES

The Role of Parental and Grandparental Epigenetic Alterations in Familial Cancer Risk

image of arental and Grandparental Epigenetic

Some epigenetic alterations that influence cancer risk are inherited through the germline from the DES-exposed to offspring and are observed in multiple DES generations of victims

2008 Study Abstract

Epigenetic alterations of the genome such as DNA promoter methylation and chromatin remodeling play an important role in tumorigenesis. These modifications take place throughout development with subsequent events occurring later in adulthood. Recent studies, however, suggest that some epigenetic alterations that influence cancer risk are inherited through the germline from parent to child and are observed in multiple generations. Epigenetic changes may be inherited as Mendelian, non-Mendelian, or environmentally induced traits. Here, we will discuss Mendelian, non-Mendelian, and environmentally induced patterns of multigenerational epigenetic alterations as well as some possible mechanisms for how these events may be occurring.

Diethylstilbestrol

One example of multiple generations in families showing effects of an environmental agent are daughters of mothers who were exposed to diethylstilbestrol (DES) during the first trimester.

The daughters show developmental abnormalities and an increased risk of developing a rare type of clear-cell adenocarcinoma. DES daughters also show a 2.5-fold increase in breast cancer risk after 40 years of age. To prove that this indeed is an inherited transgenerational effect, granddaughters and great granddaughters of the exposed mothers will need to show a DES phenotype. This analysis has not yet been completed.

Mouse studies have shown that the F2 generation from a DES-exposed pregnant female had strikingly similar effects as the F1 generation, including abnormal uterine development and uterine cancer. The proposed mechanism of action of DES is aberrant CpG methylation of key uterine cancer genes. The changes in CpG methylation may be stable throughout gametogenesis, providing insight into the transgenerational effects of DES.

Sources and more information
  • Full study (free access) : The Role of Parental and Grandparental Epigenetic Alterations in Familial Cancer Risk, Perspectives in Cancer Research, NCBI PubMed PMC4423451, 2008 Nov.
  • Epigenetics featured image credit NestleNutritionInstitute.
DES DIETHYLSTILBESTROL RESOURCES

Hsp90 and environmental impacts on epigenetic states

image of hsp90

A model for the trans-generational effects of diethylstibesterol on uterine development and cancer

2005 Study Abstract

Hsp90 is a chaperone for over 100 ‘client proteins’ in the cell, most of which are involved in signaling pathways. For example, Hsp90 maintains several nuclear hormone receptors, such as the estrogen receptor (ER), as agonist-receptive monomers in the cytoplasm.

In the presence of agonist, Hsp90 dissociates and the receptors dimerize, enter the nucleus and ultimately activate transcription of the target genes. Increasing evidence suggests that Hsp90 also has a role in modifying the chromatin conformation of many genes. For example, Hsp90 has recently been shown to increase the activity of the histone H3 lysine-4 methyltransferase SMYD3, which activates the chromatin of target genes. Further evidence for chromatin-remodeling functions is that Hsp90 acts as a capacitor for morphological evolution by masking epigenetic variation. Release of the capacitor function of Hsp90, such as by environmental stress or by drugs that inhibit the ATP-binding activity of Hsp90, exposes previously hidden morphological phenotypes in the next generation and for several generations thereafter.

The chromatin-modifying phenotypes of Hsp90 have striking similarities to the trans-generational effects of the ER agonist diethylstilbesterol (DES). Prenatal and perinatal exposure to DES increases the predisposition to uterine developmental abnormalities and cancer in the daughters and granddaughters of exposed pregnant mice.

In this review, we propose that trans-generational epigenetic phenomena involving Hsp90 and DES are related and that chromatin-mediated WNT signaling modifications are required. This model suggests that inhibitors of Hsp90, WNT signaling and chromatin-remodeling enzymes might function as anticancer agents by interfering with epigenetic reprogramming and canalization in cancer stem cells.

Sources and more information
  • Hsp90 and environmental impacts on epigenetic states: a model for the trans-generational effects of diethylstibesterol on uterine development and cancer, Human molecular genetics, NCBI PubMed PMID: 15809267, 2005 Apr.
  • Hsp90 pathway featured image credit robertanagourney.
DES DIETHYLSTILBESTROL RESOURCES

Environmental factors, epigenetics, and developmental origin of reproductive disorders

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2017 Study Highlights

  • Epidemiological and model system studies support an early origin of reproductive dysfunction.
  • Estrogenic/anti-androgenic chemicals as endocrine disrupting chemicals (EDCs) have vast developmental influences on adult reproductive outcomes.
  • Gestational, perinatal, neonatal, and pubertal periods are “windows of susceptibility” for epigenetic programming.
  • EDCs induce exposure-specific epigenetic modifications in regulatory genes in organs of the reproductive system.
  • Germline epigenetic disruption is a mechanism underlying transgenerational inheritance of reproductive disorders.

Abstract

Sex-specific differentiation, development, and function of the reproductive system are largely dependent on steroid hormones.

For this reason, developmental exposure to estrogenic and anti-androgenic endocrine disrupting chemicals (EDCs) is associated with reproductive dysfunction in adulthood.

Human data in support of “Developmental Origins of Health and Disease” (DOHaD) comes from multigenerational studies on offspring of diethylstilbestrol-exposed mothers/grandmothers.

Animal data indicate that ovarian reserve, female cycling, adult uterine abnormalities, sperm quality, prostate disease, and mating behavior are susceptible to DOHaD effects induced by EDCs such as bisphenol A, genistein, diethylstilbestrol, p,p’-dichlorodiphenyl-dichloroethylene, phthalates, and polyaromatic hydrocarbons.

Mechanisms underlying these EDC effects include direct mimicry of sex steroids or morphogens and interference with epigenomic sculpting during cell and tissue differentiation.

Exposure to EDCs is associated with abnormal DNA methylation and other epigenetic modifications, as well as altered expression of genes important for development and function of reproductive tissues.

Here we review the literature exploring the connections between developmental exposure to EDCs and adult reproductive dysfunction, and the mechanisms underlying these effects.

DNA Methylation

Several studies have demonstrated the impact of developmental exposure to EDCs on DNA methylation patterns in female reproductive tissues. A paper from almost two decades ago showed that neonatal exposure to DES results in hypomethylation of specific CpGs in the promoter region of the lactoferrin (Ltf) gene. Ltf is normally an estrogen responsive gene in the uterus, but DES induced hypomethylation was correlated with aberrant expression of Ltf in the absence of estrogen throughout life, suggesting that a permanent alteration in the hormone responsiveness of the gene had occurred. A subsequent study using the same model examined uterine DNA methylation pattern differences in a non-biased way. Several gene promoter regions were found to have differential methylation following neonatal DES or genistein exposure; one of these was Nsbp1 (now named Hmgn5), a protein that plays a role in chromatin compaction. The promoter region of this gene was hypomethylated later in life (6 months of age) following developmental exposure to either DES or genistein, and this was correlated with aberrant over-expression of uterine Nsbp1. Prenatal DES exposure results in hypermethylation of the Homeobox (Hox)a10 promoter that correlates with a decrease in gene expression.

Sources and more information
  • Environmental factors, epigenetics, and developmental origin of reproductive disorders, Reproductive toxicology (Elmsford, N.Y.), NCBI PubMed PMID: 27421580, 2017 Mar.
  • Freestyle (Non-parametric) featured image credit Daniel Friedman.
DES DIETHYLSTILBESTROL RESOURCES

Neonatal acute myeloid leukemia in a DES grandchild infant

Neonatal acute myeloid leukemia in an infant whose mother was exposed to diethylstilboestrol in utero

Neonatal acute myeloid leukemia in an infant whose mother was exposed to diethylstilboestrol in utero

2009 Study Abstract

We report on an acute myeloid leukemia in a neonate whose mother was exposed to diethylstilboestrol in utero.

The newborn presented with leukemia cutis, hemorrhagic skin lesions, hyperleucocytosis and disseminated intravascular coagulation. A bone marrow examination confirmed the diagnosis of acute monocytic leukemia with a t(11;19) MLL-ELL fusion transcript. Chemotherapy was initiated but the child developed a bilateral pulmonary infection that led to fatal respiratory distress.

This case shows acute myeloid leukemia and the third pediatric leukemia reported after maternal diethylstilboestrol exposure.

Sources and more information
  • Neonatal acute myeloid leukemia in an infant whose mother was exposed to diethylstilboestrol in utero, Pediatric blood & cancer, NCBI PubMed PMID: 19405140, 2009 Aug.
  • Acute myeloid leukemia featured image credit omicsonline.
DES DIETHYLSTILBESTROL RESOURCES

Effects of Low-Dose Diethylstilbestrol Exposure on DNA Methylation in Mouse Spermatocytes

Low doses of DES inhibits the proliferation of GC-2 cells, alters cell cycle progression, triggers cell apoptosis, and induces male reproductive toxicity

2015 Study Abstract

Evidence from previous studies suggests that the male reproductive system can be disrupted by fetal or neonatal exposure to diethylstilbestrol (DES). However, the molecular basis for this effect remains unclear. To evaluate the effects of DES on mouse spermatocytes and to explore its potential mechanism of action, the levels of DNA methyltransferases (DNMTs) and DNA methylation induced by DES were detected.

The results showed that low doses of DES inhibited cell proliferation and cell cycle progression and induced apoptosis in GC-2 cells, an immortalized mouse pachytene spermatocyte-derived cell line, which reproduces primary cells responses to E2. Furthermore, global DNA methylation levels were increased and the expression levels of DNMTs were altered in DES-treated GC-2 cells. A total of 141 differentially methylated DNA sites were detected by microarray analysis. Rxra, an important component of the retinoic acid signaling pathway, and mybph, a RhoA pathway-related protein, were found to be hypermethylated, and Prkcd, an apoptosis-related protein, was hypomethylated.

These results showed that low-dose DES was toxic to spermatocytes and that DNMT expression and DNA methylation were altered in DES-exposed cells. Taken together, these data demonstrate that DNA methylation likely plays an important role in mediating DES-induced spermatocyte toxicity in vitro.

Effects of DES on GC-2 Cell Viability and Proliferation

To explore the potential cytotoxic effects of DES on GC-2 cells, cell proliferation was assessed. GC-2 cells were treated with various concentrations (0~10−4 M) of DES for 24 h, 48 h, or 72 h. DES exposure clearly reduced the viability of GC-2 cells in a dose-dependent manner within a certain concentration and time range. In addition, cell proliferation was significantly decreased when cells were exposed to different DES concentrations. The proportion of newborn (newly divided) cells decreased with increasing DES concentrations, indicating that the DNA replication capacity of GC-2 cells was decreased following DES exposure. Notably, even at a DES concentration of as low as 2×10−7 M, the proportion of newborn cells was lower than that in the DMSO group, suggesting that low doses of DES had adverse effects on mouse spermatocytes.

Effect of DES on GC-2 Cell Cycle Progression

Cell cycle progression is an important factor influencing cell proliferation. We analyzed the influence of DES on cell cycle progression to evaluate its antiproliferative activity. The proportion of cells in S phase increased for the DES-exposed cells compared with that for the DMSO-exposed cells. In particular, the proportion of S phase cells for the DMSO group was 28.97±1.21%, while those for the 2×10−7, 2×10−6, and 2×10−5 M DES-treated groups were 33.72±3.35%, 35.68±3.50%, and 43.44±3.65%, respectively. These results showed that DES changed the proportion of cells in the S cell phase and affected GC-2 cell cell cycle progression.

DES Induced Apoptosis in GC-2 Cells

Apoptosis is another important factor contributing to cell proliferation. Thus, we further examined apoptosis in GC-2 cells treated with or without DES. Apoptotic cells were recognized by their fragmented, degraded nuclei and apoptotic bodies. DES-treated GC-2 cells showed nucleolus pyknosis, and at increasing doses of DES, more nuclear fragmentation was observed. Flow cytometric analysis also produced similar results. After treatment with 0, 2×10−7, 2×10−6, and 2×10−5 M DES, the apoptosis rates were (1.3±0.52)%, (2.4±0.95)%, (2.7±0.68)%, and (16.8±1.34)%, respectively. Altogether, these results demonstrated that DES induced apoptosis in GC-2 cells.

Effects of DES on Global DNA Methylation in GC-2 Cells

Given the important effects of DNA methylation on gene regulation, transcriptional silencing and development, we sought to determine whether the DNA methylation patterns varied between GC-2 cells with and without DES exposure. We performed 5-mC dot blot DNA hybridizations to analyze the methylation statuses of GC-2 cells with and without exposure to this compound. The results of this analysis, with the density of band indicating relative DNA methylation levels, the global DNA methylation level in GC-2 cells was slightly increased following exposure to 2×10−7 and 2×10−6 M DES, and it was significantly increased in GC-2 cells exposed to 2×10−5 M DES. These data indicated that the global DNA methylation level increased with increasing concentrations of DES. They further suggested that DNA methylation might be crucial for the GC-2 cell toxicity observed following low-dose DES exposure.

Effects of DES on DNMT Expression

Because DNMTs were found to play important roles in establishing and maintaining DNA methylation patterns, we determined the expression levels of Dnmt1, Dnmt3a and Dnmt3b. Compared with the control, Dnmt1 and Dnmt3a expression was slightly increased in GC-2 cells exposed to 2×10−7 M DES and significantly increased in cells exposed to 2×10−6 and 2×10−5 M DES. In contrast, Dnmt3b expression decreased sharply with increasing doses of DES.

Analysis of Differential DNA Methylation following DES Exposure

To further explore DES-induced alterations in methylation, we screened differentially methylated DNA sites using an Affymetrix Mouse Promoter 1.0R Array. Data are available at GEO datasets (GEO number: GSE71311). A total of 141 differentially methylated sites (including 130 hypermethylated and 11 hypomethylated sites) were found in cells with and without 2×10−5 M DES exposure (fold change>3) by microarray analysis. The methylation statuses at some differential methylation sites were verified by MSP, and mRNA expression levels were detected by real-time PCR. In brief, compared with control cells, retinoid X receptor α (rxra) was hypermethylated in cells exposed to 2×10−7, 2×10−6, and 2×10−5 M DES and its mRNA expression was downregulated with increasing doses of DES. Myosin-binding protein H (mybph) was hypermethylated in cells exposed to 2×10−5 M DES, and its expression level was also reduced significantly. Protein kinase C δ (prkcd) was hypomethylated in cells exposed to 2×10−5 M DES, and its mRNA expression was increased. These results indicated that the methylation statuses of these genes were inversely correlated with their mRNA expression levels in DES-exposed GC-2 cells, suggesting that DNA methylation was involved in the regulation of mRNA expression in these cells.

Discussion

The present study has provided several lines of evidence demonstrating that low doses of DES induce spermatocyte toxicity by triggering apoptosis, inhibiting proliferation, and affecting cell cycle progression. We have further found that DNA methylation might play an important role in DES-induced spermatocyte toxicity.

DES has long been known to affect the male reproductive system by causing alterations, such as reproductive organ dysplasia, and germ cell damage. With regard to germ cells, abnormal spermatogenesis is the most common type of DES-induced effect. Some researchers have found that exposure of mice to 5 μg DES results in major morphological alterations to the testes, as reflected by the absence of germ cells in several tubules. Another study has reported that this compound (1.0 mg/kg) induces spermatogenic apoptosis in adult male rats. In our study, the apoptosis rate of GC-2 cells exposed to 2×10−5 M DES was significantly increased compared with that of DMSO-treated cells, and these results are in agreement with those of previous studies. GC-2 cell cycle progression was also altered following exposure to 2×10−5 M DES. Specifically, the percentage of DES-treated cells in the S phase of the cell cycle was greater than that of DMSO-treated cells, indicating that DES induced S phase arrest in spermatocytes. Further analysis using an EDU Cell Proliferation Kit indicated that the percentage of newborn cells was decreased following DES exposure, even at a DES concentration of as low as 2×10−7 M. EDU is readily incorporated into cellular DNA during DNA replication. Mammalian spermatogenesis is a unique process involving successive differentiation steps, including spermatogonial mitosis, spermatocyte meiosis and spermiogenesis. Each primary spermatocyte duplicates its DNA and subsequently undergoes meiosis I to produce two haploid secondary spermatocytes, which later divide once more into haploid spermatids. Interestingly, EDU incorporated into DES-treated spermatocyte cells less frequently than untreated cells. Based on these data, we proposed that low doses of DES can cause spermatocyte toxicity.

DNA methylation has been implicated in the regulation of spermiogenesis. DNA methylation at promoter regions plays a role in gene silencing, and during spermiogenesis, methylation occurs to silence retrotransposons and imprinted genes. Therefore, we proposed that DNA methylation might be involved in DES-induced spermatocyte toxicity. First, we detected the genome-wide methylation statuses of GC-2 cells exposed to 2×10−7, 2×10−6, or 2×10−5 M DES and found a tendency of increased methylation in these cells, even following exposure to low doses of DES. DNMTs, including Dnmt1, Dnmt3a, and Dnmt3b, were found to be involved in DNA methylation. Dnmt1 is responsible for the maintenance of DNA methylation during DNA synthesis, and Dnmt1-deficient embryos have been shown to have 30% less genomic methylation than that found in embryos. This phenomenon was also embodied in our experimental results. Dnmt1 protein expression was increased in GC-2 cells treated with 2×10−7, 2×10−6, or 2×10−5 M DES, consistent with the increase in the global DNA methylation level. Previous studies demonstrated that ERα could upregulate Dnmt1 expression by directly binding to the DNMT1 promoter region in ER-positive human breast cancer cells MFC-7 cells. DES has strong estrogenic activity, can activate ERα, and increase the expression of Dnmt1, which is coincidence with our results. Dnmt3a and Dnmt3b are de novo enzymes that establish methylation patterns. Spermatogonia deficient in Dnmt3a and Dnmt3b display variations in methylation patterns at paternally imprinted regions, which may impair spermatogenesis to an extent. Our results showed that low doses of DES were toxic to spermatocytes in vitro and caused alterations in the Dnmt3a and Dnmt3b protein expression levels. Taken together, our results suggest that DNA methylation plays a role in low-dose DES-induced male reproductive toxicity.

To further explore the potential mechanism of action of DES, DNA microarray technology is a useful tool for mapping methylation changes at multiple CpG loci. Microarray analysis performed in this study revealed the presence of thousands of variations in DNA methylation between GC-2 cells with and without DES exposure. The genes that were found to be differentially methylated are involved in the following processes: DNA repair, including mnd1 and nono; cell cycle progression, including hbp1 and ccno; apoptosis and proliferation, including rnf5, prkcd, jtb, nlrx1, mybph and rhoa; male reproductive development, including mybph, cldn11 and fkbp6; and other processes. The MSP assay results confirmed that the methylation statuses of some of the abovementioned genes were associated with low-dose DES-induced GC-2 cell toxicity. Rxra, an important component of the retinoic acid signaling pathway, is a key regulator of embryonic development and has been linked to several birth defects. Rxra knockout animals showed an increase in apoptosis, resulting in abnormal morphogenesis during development, in addition to abnormal cell proliferation, cell differentiation, and cell death processes in adult differentiated tissues. The two zinc fingers of the rxra DNA binding domain fold to form a single structural domain that consists of two perpendicularly oriented helices, which resembles the corresponding regions of ER. What’s more, Angelika Rosenauer et al indicated that transient expression in ER-negative human breast cancer cells MDA-MB-231 of wild-type ER directly stimulated the transcriptional response to RA(retinoic acid). Importantly, this activation was greater than that obtained by transfection of RAR(RA receptor), RXR(retinoid X receptor), or RAR combined with RXR, and the DNA binding domain of ER plays a key role in the response to RA-induced transcription. These researches suggested that ER had relation with rxra, and DES, as a strong ER agonist, had effect on the express of rxra. Mybph directly inhibits rock1 and plays important roles in cell motility and proliferation. In our study, the rxra and mybph promoters were hypermethylated, and their mRNA levels were reduced in low-dose DES-treated GC-2 cells. Accordingly, the viability of DES-treated cells was decreased, suggesting that decreases in the mRNA levels of rxra and mybph due to hypermethylation played important roles in low-dose DES-induced GC-2 cell toxicity. Prkcd is involved in the regulation of a variety of cellular functions, including apoptosis and cell growth and differentiation. Its overexpression has been shown to induce phenotypic changes indicative of apoptosis in several cell types. Our results indicated that prkcd was hypomethylated and that its high expression in DES-exposed cells was correlated with the increased apoptosis rate, similar to the previously reported theoretical results. These findings suggested that DNA methylation played an important role in low-dose DES-induced male reproductive toxicity.

As is known to all, DES has multigenerational effects. Some researches found that there is a high prevalence of hypospadias in the sons of women exposed to DES in utero. A nationwide cohort study in collaboration with a French association of DES-exposed women showed that a significant proportion of boys born to DES daughters exhibited hypospadias with no other molecular defects identified. DES-induced changes in epigenetic background and alteration of DNA methylation could be significant factors in the susceptibility to disease development. Epigenetic changes in the some genes, transmitted through the DES daughter, could explain such a finding. In our study, low dose of DES could change methylation status of many genes in GC-2 cells. Based on these, we deduced that low dose of DES could affect the methylation of germ cells in the same way, and many of the epigenetic changes would transmitted from father to grandson. Therefore, it is necessary to make further study related to low DES exposure and DNA methylation in germ cells.

In conclusion, our results showed that low doses of DES inhibited the proliferation of GC-2 cells, altered cell cycle progression, triggered cell apoptosis, and induced male reproductive toxicity. Through molecular studies, we have found that global DNA methylation and DNMT expression vary in DES–exposed GC-2 cells. Additionally, differentially methylated DNA sites were found in GC-2 cells treated with DES compared with those treated with DMSO. These results suggested that epigenetic modification might be a potential mechanism of low-dose DES-induced male reproductive toxicity.

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DES DIETHYLSTILBESTROL RESOURCES

Epigenetic alterations induced by in utero diethylstilbestrol exposure

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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

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.

Probably the most common mechanism of epigenetic gene silencing is methylation, and it might also be the most important. DNA methyltransferases (DNMTs) catalyze the methylation of genomic DNA by adding a methyl group (CH3) onto the 5-carbon of the cytosine ring within CpG dinucleotides. Histone modifications are complex, as they involve not just histone methylation but also acetylation, deacetylation and other post-translational changes. These modifications occur in the amino-terminal tails of histones and affect the ‘openness’ of the chromatin, which determines whether a gene is expressed or silenced (for example, acetylation allows transcription, while deacetylation represses transcription). Trimethylation of histone H3 at lysine K27 is catalyzed by the Polycomb group (PcG) protein enhancer of Zeste-2 (EZH2) and results in gene silencing. PcG/H3K27me3 interact with DNMTs, and together they establish and maintain silencing of PcG target genes. Over 2,000 different PcG target genes have been identified and they include some tumor suppressor genes. Many of the PcG target genes regulate cell fate, including apoptosis, proliferation and stem cell differentiation. As discussed in more detail below, methylation of PcG target genes is linked to increased breast cancer risk.

DNMTs may be key players in regulating histones and the entire epigenomic machinery, since DNA methylation events often precede histone modifications. Upregulation of DNMTs increases the expression of EZH2 and other polycombs; this may happen by DNMTs inducing methylation of non-coding miRNAs that target the polycombs.

We and others have observed that the expression of DNMTs is persistently altered in estrogen-regulated tissues following estrogenic exposures during early life. In utero exposure to DES is reported to increase the expression of DNMT1 in the epididymis and uterus . We found that DNMT1 expression is increased in the mammary glands of adult rat offspring of dams exposed to ethinyl estradiol during pregnancy. These changes provide a key regulatory layer to influence gene expression in the mammary gland and perhaps breast tumors of individuals exposed to DES or other estrogenic compounds in utero.

Promoter methylation

In utero DES exposure alters methylation patterns of several genes in estrogen’s target tissues, including Hox genes, c-fox, and Nsbp1, but it has not been studied whether changes in methylation patterns occur in the mammary gland. We have explored changes in methylation in the mammary glands of adult rats exposed in utero to the synthetic estrogen ethinyl estradiol using global sequencing approaches. Among the genes that exhibited increased promoter methylation were several PcG target genes, suggesting that a maternal exposure to synthetic estrogens during pregnancy causes long-lasting changes in the methylation of genes that regulate cell fate, including stem cell differentiation.

Histone modifications

As an increase in EZH2 expression in the mammary glands of mice exposed to DES in utero has been reported, histone modifications also seem to be influenced by maternal exposure to synthetic estrogens during pregnancy. Jefferson and colleagues recently investigated whether upregulation of lactoferrin and sine oculis homeobox 1 (Six1) in the uterus of adult mice exposed to DES neonatally is caused by histone modifications. Their data indicate that neonatal DES exposure induces changes during the early postnatal period in the expression of multiple chromatin-modifying proteins but these changes do not last to adulthood. However, alterations in epigenetic marks at the Six1 locus in the uterus were persistent. Similarly, changes in the methylation of Nsbp1 and expression of DNMTs in the uterus of DES-exposed offspring are different in the early postnatal period compared to adulthood. This suggests that some epigenetic alterations are further influenced by factors operating during postnatal development, such as a surge of estrogens and progesterone from the ovaries at puberty onset.

microRNAs

Maternal exposures during pregnancy have been found to induce persistent changes in miRNA expression in the offspring. miRNAs are short non-coding single-stranded RNAs composed of approximately 21 to 22 nucleotides that regulate gene expression by sequence-specific base-pairing with the 3’ untranslated region of target mRNAs. miRNA binding induces post-transcriptional repression of target genes, either by inducing inhibition of protein translation or by inducing mRNA degradation. Expression of many miRNAs is suppressed by estrogens. Although the effects of maternal DES exposure during pregnancy on miRNA expression in the offspring have not been investigated, it is known that many other manipulations, such as maternal low protein diet, alter miRNA patterns among the offspring. We recently found that in utero exposure to ethinyl estradiol lowers the expression of many of the same miRNAs in the adult mammary gland as are downregulated by E2 in MCF-7 human breast cancer cells. Since miRNAs can be silenced by methylation or as a result of increased PcG expression, and they target DNMTs, histone deacetylases and polycomb genes, the observed increase in DNMT expression, histone marks and EZH2 in the in utero DES-exposed offspring may be a result of epigenetic silencing of miRNAs that target them.

2014 Study Conclusions

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 and more information
  • Full text (free access) : 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