Hsp90 and environmental impacts on epigenetic states

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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

DES-induced chromosomal trisomy

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Detection of chromosomal aberrations by fluorescence in situ hybridization in cervicovaginal biopsies from women exposed to diethylstilbestrol in utero

2006 Study Abstract

Epidemiologic studies have associated estrogens with human neoplasms such as those in the endometrium, cervix, vagina, breast, and liver.

Perinatal exposure to natural (17beta-estradiol [17beta-E(2)]) and synthetic (diethylstilbestrol [DES]) estrogens induces neoplastic changes in humans and rodents.

Previous studies demonstrated that neonatal 17beta-E(2) treatment of mice results in increased nuclear DNA content of cervicovaginal epithelium that precedes histologically evident neoplasia.

In order to determine whether this effect was associated with chromosomal changes in humans, the frequencies of trisomy of chromosomes 1, 7, 11, and 17 were evaluated by the fluorescence in situ hybridization (FISH) technique in cervicovaginal tissue from 19 DES-exposed and 19 control women.

The trisomic frequencies were significantly elevated in 4 of the 19 (21%) DES-exposed patients. One patient presented with trisomy of chromosomes 1, 7, and 11, while trisomy of chromosome 7 was observed in one patient. There were two patients with trisomy of chromosome 1. Trisomy of chromosomes 1, 7, 11, and 17 was not observed in the cervicovaginal tissue taken from control patients.

These data suggest that DES-induced chromosomal trisomy may be an early event in the development of cervicovaginal neoplasia in humans.

Sources and more information
  • Detection of chromosomal aberrations by fluorescence in situ hybridization in cervicovaginal biopsies from women exposed to diethylstilbestrol in utero, International journal of gynecological cancer : official journal of the International Gynecological Cancer Society, NCBI PubMed PMID: 16445652, 2006 Jan-Feb;16.
  • Trisomy 7 featured image credit researchgate.
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Early Life Adverse Environmental Exposures Increase the Risk of Uterine Fibroid Development

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Role of Epigenetic Regulation

Abstract

Currently, there is a remarkable lack of knowledge regarding the involvement of chromatin assembly in the process by which adverse environmental exposures increase the overall risk of UF development. The precise mechanism underlying EDC-dependent effects on myometrial cell physiology are not adequately understood.

Uterine Fibroids [UF(s), AKA: leiomyoma] are the most important benign neoplastic threat to women’s health. They are the most common cause of hysterectomy imposing untold personal consequences and 100s of billions of healthcare dollars, worldwide. Currently, there is no long term effective FDA-approved medical treatment available, and surgery is the mainstay.

The etiology of UFs is not fully understood. In this regard, we and others have recently reported that somatic mutations in the gene encoding the transcriptional mediator subunit Med12 are found to occur at a high frequency (∼85%) in UFs. UFs likely originate when a Med12 mutation occurs in a myometrial stem cell converting it into a tumor-forming stem cell leading to a clonal fibroid lesion.

Although the molecular attributes underlying the mechanistic formation of UFs is largely unknown, a growing body of literature implicates unfavorable early life environmental exposures as potentially important contributors. Early life exposure to EDCs during sensitive windows of development can reprogram normal physiological responses and alter disease susceptibility later in life. Neonatal exposure to the EDCs such as diethylstilbestrol (DES) and genistein during reproductive tract development has been shown to increase the incidence, multiplicity and overall size of UFs in the Eker rat model, concomitantly reprogramming estrogen-responsive gene expression.

Importantly, EDC exposure represses enhancer of zeste 2 (EZH2) and reduces levels of histone 3 lysine 27 trimethylation (H3K27me3) repressive mark through Estrogen receptor/Phosphatidylinositide 3-kinases/Protein kinase B non-genomic signaling in the developing uterus. Considering the fact that distinct Mediator Complex Subunit 12 (Med12) mutations are detected in different fibroid lesions in the same uterus, the emergence of each Med12 mutation is likely an independent event in an altered myometrial stem cell. It is therefore possible that a chronic reduction in DNA repair capacity eventually causes the emergence of mutations such as Med12 in myometrial stem cells converting them into fibroid tumor-forming stem cells, and thereby leads to the development of UFs.

Advancing our understanding of the mechanistic role epigenetic regulation of stem cells plays in mediating risk and tumorigenesis will help in pointing the way toward the development of novel therapeutic options.

Sources and more information
    • Full study (free access) Early Life Adverse Environmental Exposures Increase the Risk of Uterine Fibroid Development: Role of Epigenetic Regulation, Frontiers in Pharmacology, doi.org/10.3389/fphar.2016.00040, 01 March 2016.
DES DIETHYLSTILBESTROL RESOURCES

DES effects on T-cell differentiation in the thymus

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Diethylstilbestrol alters positive and negative selection of T cells in the thymus and modulates T-cell repertoire in the periphery

2006 Study Abstract

Prenatal exposure to diethylstilbestrol (DES) is known to cause altered immune functions and increased susceptibility to autoimmune disease in humans.

In the current study, we investigated the effects of DES on T-cell differentiation in the thymus using the HY-TCR transgenic (Tg) mouse model in which the female mice exhibit positive selection of T cells bearing the Tg TCR, while the male mice show negative selection of such T cells.

In female HY-TCR-Tg mice, exposure to DES showed more pronounced decrease in thymic cellularity when compared to male mice. Additionally, female mice also showed a significant decrease in the proportion of double-positive (DP) T cells in the thymus and HY-TCR-specific CD8+ T cells in the periphery. Male mice exhibiting negative selection also showed decreased thymic cellularity following DES exposure. Moreover, the male mice showed increased proportion of double-negative (DN) T cells in the thymus and decreased proportion of CD8+ T cells. The density of expression of HY-TCR on CD8+ cells was increased following DES exposure in both females and males. Finally, the proliferative response of thymocytes to mitogens and peripheral lymph node T cells to male H-Y antigen was significantly altered in female and male mice following DES treatment.

Taken together, these data suggest that DES alters T-cell differentiation in the thymus by interfering with positive and negative selection processes, which in turn modulates the T-cell repertoire in the periphery.

Sources and more information
  • Diethylstilbestrol alters positive and negative selection of T cells in the thymus and modulates T-cell repertoire in the periphery, Toxicology and applied pharmacology, NCBI PubMed PMID: 16122773, 2006 Apr.
  • T-cell differentiation featured image credit researchgate.
DES DIETHYLSTILBESTROL RESOURCES

Induction of apoptosis in murine fetal thymocytes following perinatal exposure to DES

image of thymocytes

DES induces thymic atrophy and apoptosis resulting from upregulation of Fas and Fas ligand expression

2006 Study Abstract

Perinatal exposure to diethylstilbestrol (DES) is known to cause thymic atrophy in mice, although the precise mechanism remains unclear.

In the current study the authors investigated whether perinatal exposure to DES would trigger apoptosis in thymocytes.

To this end, C57BL/6 pregnant mice were injected intraperitoneally (i.p.) on gestational day (gd)-15 and -16 with 5 microg/kg DES. Analysis of thymi harvested from mice on gd-17, gd-19 and postnatal day (PD)-1, showed a significant reduction in thymic cellularity on gd-17 only. Additionally, DES treatment significantly altered the proportion and absolute number of T-cell subsets, particularly on gd-17.

Apoptosis was increased in DES-treated thymocytes when compared to the controls and was seen only on gd-17. Moreover, DES-treated gd-17 thymocytes had increased Asp-Glu-Val-Asp (DEVDase) activity. Microarray analysis of 96 apoptotic genes in gd-17 thymocytes revealed that exposure to DES increased the expression of several apoptotic genes primarily belonging to tumor necrosis factor (TNF) and TNF receptor (TNFR) family.

Taken together, these results suggest that DES-induced thymic atrophy following perinatal exposure may result, at least in part, from increased apoptosis mediated by death receptor pathway involving TNF family members.

Sources and more information
  • Induction of apoptosis in murine fetal thymocytes following perinatal exposure to diethylstilbestrol, International journal of toxicology, NCBI PubMed PMID: 16510352, 2006 Jan-Feb.
  • Thymocytes featured image credit dartmouth.
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 In Utero Exposure and Blood DNA Methylation

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First study to evaluate possible effects of in utero DES exposure on genome-wide DNA methylation in humans

2015 Study Abstract

In utero exposure to diethylstilbestrol (DES) has been associated with increased risk of adverse health outcomes such as fertility problems and vaginal as well as breast cancer. Animal studies have linked prenatal DES exposure to lasting DNA methylation changes. We investigated genome-wide DNA methylation and in utero DES exposure in a sample of non-Hispanic white women aged 40–59 years from the Sister Study, a large United States cohort study of women with a family history of breast cancer. Using questionnaire information from women and their mothers, we selected 100 women whose mothers reported taking DES while pregnant and 100 control women whose mothers had not taken DES. DNA methylation in blood was measured at 485,577 CpG sites using the Illumina HumanMethylation450 BeadChip. Associations between CpG methylation and DES exposure status were analyzed using robust linear regression with adjustment for blood cell composition and multiple comparisons. Although four CpGs had p<105, after accounting for multiple comparisons using the false discovery rate (FDR), none reached genome-wide significance. In conclusion, adult women exposed to DES in utero had no evidence of large persistent changes in blood DNA methylation.

Previously reported candidate genes affected by DES

We also specifically report on nine genes (EMB, WNT11 TGFB1, ERBB2, EGFR, LTF, EGF, FOS, and JUN) that have previously been shown to have differential gene expression in mice uterine or vaginal tissue following pre or perinatal exposure to DES :

We compared the mean β-value for 75 CpGs in the 5’ region (as annotated by Illumina) of these genes for women with and without DES exposure histories.

Epigenome wide study

In our primary model, adjusted for multiple testing and blood cell composition, four CpGs in two different genes (KIFC3, and DCAKD) had nominal p values < 10-5, and an additional 18 CpGs in 18 different genes had nominal p values < 10-4 (featured image). None of these 22 CpGs achieved genome-wide significance after considering multiple comparisons (q<0.05). Additional adjustments for age at menarche, BMI and parity did not affect the results.

Discussion

In this study we used DNA from blood rather than from a known target tissue such as vagina, cervix or endometrium. Histologic changes in target tissues, including vaginal epithelium, are known to correlate with dose of DES exposure and with increased risk of adverse outcomes such as vaginal carcinoma. Although it may be possible to compare methylation in target tissues using archival formalin-fixed paraffin-embedded (FFPE) samples, it is difficult to find adequate FFPE samples from DES-exposed women. In addition, methylation analysis of degraded and cross-linked FFPE samples present substantial technical challenges.

In conclusion, although we cannot rule out the possibility of effects at younger ages or in other tissues, our study finds no evidence of large persistent effects of in utero DES exposure on blood DNA methylation.

Sources and more information
  • Full text (free access) : In Utero Exposure to Diethylstilbestrol and Blood DNA Methylation in Women Ages 40–59 Years from the Sister Study, PLOS one, doi.org/10.1371/journal.pone.0118757, March 9, 2015.
  • Log10 transformed DES association p-values for individual CpGs are plotted in relation to their chromosome location featured image credit journal.pone.0118757.
DES DIETHYLSTILBESTROL RESOURCES

Differential gene expression in mice uterine or vaginal tissue following pre or perinatal exposure to DES

image of differential-gene-expression

Expression of estrogen receptor and proto-oncogene messenger ribonucleic acids in reproductive tracts of neonatally diethylstilbestrol-exposed female mice with or without post-puberal estrogen administration

1996 Study Abstract

Perinatal treatment of female mice with natural and synthetic estrogens including diethylstilbestrol (DES) results in estrogen-independent persistent proliferation and cornification of the vaginal epithelium.

The dynamics of the induction of estrogen receptor (ER), c-jun, c-fos and c-myc mRNAs by 17 beta-estradiol (E2) was examined in the uterus and vagina of neonatally DES-exposed and -unexposed ovariectomized adult mice.

In the uterus of neonatally DES-unexposed ovariectomized mice, the expression of ER mRNA increased within 1 h after E2 administration and declined by 12 h thereafter. ER mRNA in the vagina decreased within 1 h after the stimulation and recovered by 12 h thereafter.

In the uterus, c-jun and c-fos mRNAs increased in concentration within 1 h after E2 administration, showing a peak 3 h after the stimulation; they decreased with time thereafter. In the vagina, the concentration of c-jun and c-fos mRNAs increased rapidly, reaching a peak within 1 h after the stimulation.

However, the expression of c-myc in uterus and vagina was not changed by postpuberal E2.

These results suggest that estrogen regulation of ER and proto-oncogene mRNAs in the vagina differs from those in the uterus. In the neonatally DES-exposed ovariectomized adult mice, uterine ER mRNA expression levels were significantly higher than in the unexposed ovariectomized controls; however, vaginal levels were drastically lower than in the controls. Expression of c-jun and c-fos mRNAs was greater in both the uterus (3- and 6-fold, respectively) and the vagina (18- and 4-fold) of neonatally DES-exposed mice than in controls. The ER mRNA and the increased levels of c-fos and c-jun mRNAs in both uterus and vagina of neonatally DES-exposed ovariectomized mice were not further altered by post-puberal E2 and may be related to ovary-independent persistent changes in the genital tract.

Sources and more information
  • Expression of estrogen receptor and proto-oncogene messenger ribonucleic acids in reproductive tracts of neonatally diethylstilbestrol-exposed female mice with or without post-puberal estrogen administration, Experimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology and German Diabetes Association, DOI: 10.1055/s-0029-1211432, 1996.
DES DIETHYLSTILBESTROL RESOURCES

Epigenetic alterations induced by in utero diethylstilbestrol exposure

image of Epigenetic alterations induced by in utero diethylstilbestrol exposure

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

Altered expression of genes reported to persist in DES-lineage females

image of DES-lineage females

Endocrine disruption of oestrogen action and female reproductive tract cancers

2014 Study Abstract

Interestingly, gene expression analysis indicates that developmental DES exposure results in persistent altered gene expression of oestrogen-responsive genes in the uterus that may explain the increased susceptibility to tumour development.

Gene ontology analysis of microarray data revealed altered expression of genes involved in cell growth, differentiation and adhesion collected uterine cancer tissue RNA from DES-exposed mice by laser capture microdissection to minimise contamination with other cell types and performed targeted transcriptional profiling. Interestingly, the tumour suppressor PTEN was down-regulated in the majority of tumours, analogous to loss of PTEN expression in human tumours. In addition, genes associated with cell adhesion, such as Decorin, were down-regulated in DES-induced tumours while suppressor of cytokine signalling 3 (Socs3) was over-expressed. Other studies have also identified molecular similarities between DES-induced tumours in mice and endometrial cancer in humans, such as microsatellite instability brought about by defects in expression of DNA mismatch repair genes such as MSH2 and MSH6.

It could be argued that the apparent trans-generational effect of endocrine disruption is of greater significance. Following neonatal DES exposure in mice, the F1 generation of DES daughters have an increased incidence of uterine adenocarcinoma found that 31% of F1 females from the maternal germ cell lineage developed tumours after 18 months despite there being no exogenous endocrine exposure in these animals, highlighting the potential for future risk to the daughters of DES-exposed women. DES is reported to induce epigenetic changes. Altered methylation patterns have been reported for several uterine genes that are permanently dysregulated after developmental DES exposure; lactoferrin and c-Fos are permanently up-regulated following neonatal DES exposure to due to hypomethylation of the promoter region.

DES has been reported to promote hypermethylation of the homeobox gene Hoxa10 in mice exposed in utero to DES. DES exposure was also associated with increased expression of DNA methyltransferases 1 and 3b leading to long-term altered expression of Hoxa10. Contrary to the reported action of DES on Hoxa10, exposure to BPA in mice in utero results in hypomethylation of the Hoxa10 promoter, which leads to enhanced binding of ERα to EREs in the promoter region and an increase in an ERE-driven reporter gene in vitro.

Thus, epigenetic changes in uterine genes may indicate a possible mechanism for trans-generational effects of DES because altered expression of genes is reported to persist in DES-lineage females.

Sources and more information
  • Full text (free access) : Endocrine disruption of oestrogen action and female reproductive tract cancers, Society for Endocrinology, Endocrine Related Cancer, doi: 10.1530/ERC-13-0342, April 1, 2014.
  • Russian doll beauty and the beast featured image credit rawdonfox.
DES DIETHYLSTILBESTROL RESOURCES