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.
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Environmental factors, epigenetics, and developmental origin of reproductive disorders

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2016 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.

Sources and more information
  • Environmental factors, epigenetics, and developmental origin of reproductive disorders, Reproductive toxicology (Elmsford, N.Y.), NCBI PubMed PMID: 27421580, 2016 Jul 12.
  • 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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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

Estrogen Imprinting : When Your Epigenetic Memories Come Back to Haunt You

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Persistent hypomethylation in the promoter of nucleosomal binding protein 1 (Nsbp1) correlates with overexpression of Nsbp1 in mouse uteri neonatally exposed to diethylstilbestrol or genistein

2008 Study Abstract

The concept of developmental programming by steroid hormones has been around for decades, and may be best understood in the brain, where early-life estrogens and androgens feminize and masculinize behavior. Other end organs, such as the liver, are also programmed by steroids during early development, leading to important and necessary sexual dimorphisms in structure and function. In addition to physiological programming or imprinting by steroids, it is well established that the reproductive tract, as well as other structures, can be reprogrammed by abnormal or inadvertent steroid exposures during early life. This phenomenon is best understood by extensive studies on the synthetic estrogen, diethylstilbestrol (DES). Beginning in the early 1950s, DES was prescribed to pregnant women to prevent spontaneous abortions, and its use has been estimated at 8 million women worldwide. However, DES utilization during pregnancy was discontinued in the United States in 1972 and worldwide by 1979 when it was discovered that maternal DES usage was linked to an increased incidence of vaginal clear-cell adenocarcinoma in DES-exposed daughters. Since that time, intrauterine DES exposure has resulted in a multitude of complications in female and male offspring, including T-shaped uteri, uterine fibroids, hypospadias, subfertility and infertility, early menopause, immune system disorders, psychosexual disturbances, and increased breast and testicular cancer risk. Although some adverse effects appear relatively early, such as vaginal cancers in young women, others are emerging as the exposed population ages, suggesting lifelong memory of early estrogen levels by cells and organs. In some instances these delayed responses may be triggered by secondary hormonal events such as onset of puberty or changing hormone levels with aging, which greatly complicates the picture. Furthermore, there is new evidence for transgenerational DES effects transmitted to DES granddaughters, the third generation, which indicates that adverse sequelae may be with us for some time.

Concern regarding fetal and neonatal exposure to estrogenic agents also continues today due to a variety of real-life circumstances that include unintended continuation of estradiol-containing birth control pills during the first months of an undetected pregnancy (estimated at > 1 million annually), exposure (sometimes daily) to environmental estrogens (e.g. bisphenol A, methoxychlor, dichlorodiphenyltrichloroethane or DDT), and consumption of high levels of phytoestrogens during pregnancy or in infant formula (e.g. soy with the isoflavone genistein), all of which have shown a variety of adverse effects in animal models . Although there is compelling evidence that soy or genistein ingestion can be chemoprotective and beneficial, as with so many things in life, this appears to depend on timing. Prepubertal genistein intake reduces breast cancer risk in humans and rodent models, whereas fetal exposure, at least in rat models, can increase risk if not accompanied by lifetime genistein intake. This serves to emphasize the subtle influences of adult life experiences on disease manifestation from early-life exposures. Together, these and a multitude of other studies indicate that early-life estrogen exposure fits the expanding paradigm of the developmental origins of human adult disease.

Over the years, researchers have conducted extensive studies on developmental reprogramming, also referred to as developmental estrogenization or estrogen “imprinting,” using rodent models that largely recapitulate, and in many instances predict, the adverse effects seen in DES-exposed humans. A major research effort during this time has been to identify the molecular underpinning whereby cells and tissues “remember” early-life estrogen exposures long after hormone withdrawal. In the late 1990s, McLachlan and colleagues provided the first clue that the process may have an epigenetic basis when they described a permanent change in uterine lactoferrin gene methylation as a function of fetal DES exposure. They went on to propose that developmental reprogramming by early-life estrogenic exposures may be mediated by altered epigenetic memory. As in lower species such as frogs in which early estrogen treatment changes gene methylation and alters the response of the vitellogenin gene to hormones later in life, they also speculated that the first hormonal experience may epigenetically alter the set point for the later hormone response in mammals. A few studies have since substantiated that epigenetic mechanisms underlie estrogen imprinting by demonstrating that early-life estrogens permanently alter DNA methylation and gene expression of specific genes in the uterus and prostate gland, which are associated with altered susceptibility to adult pathology in those tissues.

In the present issue of Endocrinology, Tang et al.  identify additional layers of complexity to epigenetic reprogramming that have not been previously appreciated. Earlier work in the laboratory of Newbold et al. determined that developmental DES resulted in a high incidence of uterine adenocarcinoma in the murine model, which could be predictive of cancers not yet observed in DES daughters. In partnership with the laboratory of Newbold et al., Tang et al.  used an unbiased approach of methylation sensitive restriction fingerprinting to identify altered DNA methylation patterns in prepubertal, adult, and aged mouse uteri that had been exposed during early life to high-dose or low-dose DES, or to genistein at doses consistent with human dietary intake. They identified 14 genes whose methylation patterns shift as a result of neonatal DES or genistein treatments, several of which were previously unrecognized participants in developmental estrogenization of this tissue. Thus, the present data further substantiate the original hypothesis and findings of McLachlan and colleagues. Tang et al.  went on to thoroughly characterize the altered DNA methylation at specific CG sites in the promoter of one gene in particular, nucleosomal binding protein 1 (Nsbp1), and directly correlated the changes with altered gene expression. This gene is particularly significant because Nsbp1 is a nucleosome-core-particle binding protein that plays a role in chromatin remodeling, which itself underpins a higher order epigenetic process. Nsbp1 is structurally similar to the conserved functional domains of the high-mobility group proteins, HMG 14/17, that interact with nucleosomes, transiently destabilize chromatin, increase access to DNA, and enhance gene transcription at targeted sites. Thus, modifications in Nsbp1 methylation and expression by early estrogens have the potential to affect higher order chromatin structure that, in turn, may alter expression of many genes and possibly drive oncogenesis. Of course, this awaits confirmation by future experiments that directly address this possibility. Nonetheless, these novel findings provide important evidence that epigenetic reprogramming by early-life estrogens may involve multiple epigenetic pathways that combine to initiate disease later in life.

However, the real novelty and significant implications of the study by Tang et al. are not in the aforementioned results, as important as they may be, but in the demonstration that epigenetic reprogramming itself involves a two-step process. Original studies by Newbold et al. showed that the onset of uterine adenocarcinoma in neonatal DES-exposed mice required a secondary hormonal “push” by pubertal steroids because prepubertally ovariectomized mice did not develop cancers. The study by Tang et al.  now shows that this is a direct function of specific DNA methylation changes induced by the secondary pubertal hormones that occur only in mice that were first exposed neonatally to DES or genistein. The authors found that the Nsbp1 promoter is moderately methylated, and the gene is expressed in uteri during prepubertal development in control animals, and becomes hypermethylated and largely silenced by early adulthood. This suggests that the role for Nsbp1 in the uterus is normally restricted to development. When mice were given DES or genistein immediately after birth, the Nsbp1 promoter became hyper or hypomethylated in a treatment-specific manner that led to decreased or increased Nsbp1 expression before puberty. This can be considered the first epigenetic modification. If the mice were prepubertally ovariectomized, these divergent methylation patterns persisted through aging. However, if the mice were allowed to undergo puberty with its secondary estradiol exposure, the Nsbp1 promoter was radically shifted to a hypomethylated state in all neonatal estrogen-exposed uteri. Importantly, this secondary epigenetic modification resulted in overexpression of Nsbp1 throughout life, which correlates with the onset of uterine adenocarcinoma in this model . Thus, in addition to overt epigenetic memories determining our fate, it appears that repressed epigenetic memories are also looming in the background, only to be epigenetically triggered by future events. These new findings stress the importance of secondary events in life that are necessary to uncover the initial cryptic modifications.

A “second hit” model has long been applied to genetic changes and the ontogeny of cancer . It now appears that a parallel two-step concept may apply to epigenetic alterations as well. A somewhat similar scenario was previously shown in Drosophila. Flies with a Krüppel gene mutation (Krif−1) exhibited a specific phenotypical outcome (eye outgrowth) only if they were “epigenetically sensitized” by chromatin modification. The authors concluded that an otherwise cryptic genetic variation can be modified epigenetically to unmask a predisposed phenotype. Ruden et al.  went on to propose further that a similar mechanism may be responsible for the transmission of early-life DES effects on uterine development and cancer. The study by Tang et al.  for the first time identifies molecular evidence to support this proposal. Moreover, the aforementioned hypothesis can now be expanded to state that cryptic epigenetic marks may be further epigenetically modified to unmask a predisposed phenotype. This begs the question whether the complexity of lifetime epigenetic interactions might be the basis for individual variability to early-life exposures or even to differential responsiveness to therapeutic interventions.

There are many things that remain to be done, not the least of which is connecting the dots between the epigenetically altered genes and the disease at hand. This is true not only for the uterus, but for other cancers (breast, testicular, prostate) and disease states that result from estrogen imprinting. Might there be other secondary triggers for repressed epigenetic memories aside from just hormones? Can we formulate an “epigenetic fingerprint” consisting of multiple genes that may be similar or unique for the separate estrogenic compounds, end organs, or second hits? If so, perhaps these could be used in the future for early detection of altered disease susceptibility as a result of known or unknown early-life exposures. Perhaps they could even be used to devise novel interventions. Clearly, we have only just begun.

Sources and more information
  • Estrogen Imprinting: When Your Epigenetic Memories Come Back to Haunt You, Endocrinology, Endocrine Society, Oxford University Press Volume 149, Issue 12, Pages 5919–5921, 1 December 2008.
  • Estrogen Imprinting featured image credit dennis43.
DES DIETHYLSTILBESTROL RESOURCES

Prospects for studying epigenetic mediation of exposure–response relationships

2012 Study Abstracts

Changes in epigenetic marks such as DNA methylation and histone acetylation are associated with a broad range of disease traits, including cancer, asthma, metabolic disorders, and various reproductive conditions. It seems plausible that changes in epigenetic state may be induced by environmental exposures such as malnutrition, tobacco smoke, air pollutants, metals, organic chemicals, other sources of oxidative stress, and the microbiome, particularly if the exposure occurs during key periods of development. Thus, epigenetic changes could represent an important pathway by which environmental factors influence disease risks, both within individuals and across generations. We discuss some of the challenges in studying epigenetic mediation of pathogenesis and describe some unique opportunities for exploring these phenomena.

Epigenetic effects in neurological disorders

Historically, the field of epigenetics has focused on elucidating mechanisms for maintaining DNA methylation in dividing cells. However, recent work has discovered dynamic DNA methylation changes in non-dividing cells including neurons, motivating studies of effects of environmental exposures on epigenetic marks in relation to neurological disorders.

Alzheimer’s disease, schizophrenia, and autism spectrum disorders show a variety of epigenetic anomalies. An epigenetic mechanism has been proposed to explain the association between famine during the prenatal period and schizophrenia risk, as well as associations between expression of imprinted genes and both autism and schizophrenia. Recent studies, moreover, report associations of both conditions with DNA methylation in additional, non-imprinted genes.

Human studies – Diethylstilbestrol (DES)

Decades ago, human males exposed in utero to DES were found to have excess occurrence of urogenital malformations, including hypospadias, and elevated prevalence of hypospadias was subsequently reported among unexposed boys whose mothers had been exposed in utero to DES. More recently, in a study addressing potential competing effects of measured environmental and genotypic factors, a notable excess of hypospadias was reported among unexposed grandsons of women who had been exposed in utero to DES. One postulated mechanism for these observations is that epigenetic changes in the androgen receptor gene are induced in primordial germ cells of DES-exposed female fetuses at the time that the reproductive system is forming, and subsequently transmitted across generations to affected sons and grandsons. To our knowledge, this mechanism has not been examined at the molecular level.

Multigenerational studies – Diethylstilbestrol offspring

DES is regarded as a potential environmental endocrine disruptor by many researchers because this compound binds steroid hormone receptors and has only exogenous sources. Because exposure occurred only in relatively controlled clinic settings, cohorts of exposed individuals could in theory provide an opportunity to trace any epigenetic effects of exposure among themselves and their descendents to the restricted time periods of their pregnancies or gestation before administration to pregnant women ended in the 1970s. Exposure and comparison group data of varying quality are or could be made available from several sources, the highest quality in theory being participants in trials of DES efficacy conducted in the 1950s, although sample sizes were limited. Nonetheless, the numerous case–control sets, DES-exposed cohorts, and interest groups that subsequently enrolled large numbers of exposed men and women to monitor and investigate health effects of exposed individuals could prove to be valuable resources for investigation of potential epigenetic and transgenerational effects of DES among exposed individuals and their descendents. Initial proof-of-concept studies could include investigation of epigenetic marks postulated to have been disrupted by DES exposures.

  • Read and download the full study (free access) Environmental epigenetics: prospects for studying epigenetic mediation of exposure–response relationships, on the NCBI, PubMed, PMC3432200, 2012 Oct.
  • Image credit Daniel Friedman.
DES DIETHYLSTILBESTROL RESOURCES

Autism and Down’s Syndrome Risk in Diethylstilbestrol-exposed Offspring

Birth Defects in the Sons, Daughters and Grand Children of Women who were Exposed in Utero to DES

2009 Study Abstract

The DES Combined Cohort Follow-Up Study

“In 1994, the first combined cohort questionnaires were mailed to 6,551 second generation women, including 4,459 exposed to DES in utero, and 2,092 unexposed.”

“Based on the mothers reports, genitourinary anomalies affecting the sons included horseshoe-shaped kidney, renal agenesis, born with one kidney; penile/testicular defects included hypospadias and testicular atrophy; skeletal anomalies included scoliosis, club foot, polydactyly, torticollis, and hip dysplasia; heart defects included heart murmur, ventricular septal disease, tetralogy of fallot, atrial septal defect, and pulmonic stenosis; neurological anomalies included cerebral palsy, ptosis, and autism; muscle or tissue anomalies included cleft palate, hernia, and torticollis; chromosomal/hereditary syndromes included Down’s Syndrome, chrondodystrophy, and adrenoleukodystrophy; eye conditions included amblyopia, cataract, and strabismux; hearing loss was unspecified; gastrointestinal defects included trache-oesophageal fistula/atresia, and intestinal or gall bladder anomalies; miscellaneous conditions (defined as conditions affecting fewer than 5 sons) included benign tumors, cysts, fistulas, skin anomalies, and blood disorders.

Based on mothers’ reports, skeletal anomalies affecting the daughters included hip dysplasia, scoliosis, club foot, missing limbs, and extra digits; heart defects included atrial septal defect, and ventricular septal defect; chromosomal/heritable conditions included Down’s syndrome, Noonan’s syndrome, and Williams syndrome; neurological anomalies included cerebral palsy, and anencephalus; genitourinary anomalies primarily involved the kidney and included double kidney, horseshoe shaped kidney, renal agenesis and dysgenesis, and born with one kidney; skin anomalies included hemangioma; miscellaneous conditions (defined as conditions affecting fewer than 5 daughters) included benign tumors, cysts, cleft palate, anomalies of the eye/vision or ear/hearing, learning disabilities, blood disorders, muscle or musculoskeletal anomalies, and gastrointestinal abnormalities.”

The DES Third Generation Cohort Study

“Questionnaire mailings to the third generation women began in August 2000 and were completed in April 2003. Questionnaires were returned by 793 (88%) of the 898 women whose contact information was provided by their mothers, including 463 (90%) exposed and 330 (86%) unexposed. The third generation questionnaire queried women for demographic information, hormonal and reproductive factors, and health conditions, including birth defects. Self-reported birth defects were skeletal anomalies including hip dysplasia and missing forearm; congenital heart conditions including heart murmur and atrial septal defect; chromosomal conditions included Down’s syndrome and cystic fibrosis; neurological conditions included cerebral palsy and hemiparesis; miscellaneous conditions (defined as conditions affecting fewer than 5 daughters) included anomalies of the eye, ear, skin, and/or blood, and pyloric stenosis.”

  • Read and download the full study (free access) Birth Defects in the Sons and Daughters of Women who were Exposed in utero to Diethylstilbestrol (DES), on the NCBI, PubMed, PMC2874639, 2009 Nov 30.
  • Image credit The NCBI PMC2874639/table/T2.
DES DIETHYLSTILBESTROL RESOURCES

Endocrine disruptors and psychiatric disorders in children exposed in utero

Evidence from a French cohort of 1002 prenatally exposed children and the example of diethylstilbestrol (DES) as a model for PE study

2016 Study Abstract

Aim of the work In utero diethylstilbestrol (DES) exposure has been demonstrated to be associated with somatic abnormalities in adult men and women as well as shown for its trangenerationnal effect.

Endocrine disruptors and psychiatric disorders in children exposed in utero: evidence from a French cohort of 1002 prenatally exposed children and the example of diethylstilbestrol (DES) as a model for PE study, Conference Paper, Research Gate, publication/293333931, January 2016.

Researchers Marie-Odile Soyer-Gobillard and Charles Sultan, image credit lamarseillaise.

Conversely, the data are contradictory regarding the association with psychological or psychiatric disorders during adolescence and adulthood.

This work was designed to determine whether prenatal exposure to DES and/or Ethinyloestradiol affects brain development and whether it is associated with psychiatric disorders in male and female adolescents and young adults.

Methods
HHORAGES Association, a national patient support group, has assembled a cohort of 1280 women (spontaneous testimonies communicated after various informations) who took DES and/or EE during pregnancy. We obtained responses to detailed questionnaire from 529 families, corresponding to 1182 children divided into three groups:

  1. Group 1 (n=180): firstborn children without DES treatment,
  2. Group 2 (n=740): exposed children,
  3. and Group 3 (n=262): children born after a previous pregnancy treated by DES and/or EE.

Key Results
No psychiatric disorders were reported in Group 1. In Group 2, the incidence of psychiatric disorders was drastically elevated (83.8%), and in Group 3, the incidence was still elevated (6.1%) compared with the general population.

Total number of psychological/psychiatric disorders among the 982 (1002-20 stillborns) DES-exposed and post-DES children
Among the 982 DES-exposed adolescents (1002-20 stillborns) (Group 2) and post-DES adolescents (Group 3):

  • Behavioral disorders, violence, aggressiveness, obsessive-compulsive disorders (n=110) (11.2%)
  • Eating disorders (n=83) (8.4%)
  • Schizophrenia (n=171) (17.4%)
  • Depression, bipolar disorders, anxiety (n=257) (26.2%)
  • Suicides (n=33) (3.4%)
  • Suicide attempts (n=642) (65.4%)

Conclusions
This work demonstrates that prenatal exposure to DES and/or EE is associated with a high risk of psychiatric disorders in adolescence and adulthood. Molecular epigenetic mechanisms subtending these toxic effects are in progress.

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Behavioral and Somatic Disorders in Children Exposed in Utero to Synthetic Hormones

A Testimony-Case Study in a French Family Troop

Using a familial case control study, Marie-Odile Soyer-Gobillard – former director emeritus at the CNRS (French National Center for Scientific Research) – and Charles Sultan show that there are serious effects on the psychological and physical health of the descendants of women treated with synthetic hormones during their pregnancy: psychiatric illnesses are often found associated with somatic disorders which are well known to be the DES and EE signature.

Behavioral and Somatic Disorders in Children Exposed in Utero to Synthetic Hormones: A Testimony-Case Study in a French Family Troop, Endocrinology and Metabolism, intechopen, DOI: 10.5772/48637, October 3, 2012.

Synthetic hormones, acting as endocrine disturbers, are toxic for humans, especially for pregnant women and their children, probably partly in relation with their toxic degradation status.

In all cases girls suffered more than boys either of somatic and/or psychiatric disorders due to the estrogen receptor alpha or beta concentration higher in female fetus than in male. It is also clear that in all the families most of the exposed children are ill while quite the unexposed are not.

2012 Study Overview

  • Materials and methods: Gathering questionnaires and the evidence
  • Results / Data Analysis / Discussion
  • A multi-generational effect? By what mechanism?
  • Conclusion

A multi-generational effect? By what mechanism?

Multi-generational carcinogenesis studies were realized on mice after diethylstilbestrol impregnation with impressive and undisputable results. Our observations presented in this present work from the French HHORAGES troop raises the question of the mechanism through with synthetic hormones as DES cause either psychiatric disorders in exposed children and/or adverse effects in subsequent generations. Since Abdomaleky et al  concluded that modulation of gene-environment interactions may be trough DNA methylation, authors put forward hypothesis that DES-induced changes in epigenetic background and alteration of DNA methylations could be significant factors. The pregnant mother’s exposure to DES at very early neurodevelopment time and/or at time of sex determination would appear to be sufficient to alter the remethylation of neuron precursors and/or of the fetus germ line. Only a few third-generation children suffering psychiatric illness are mentioned in testimonies. This is understandable because third generation exposed children are still too young (excepted in some cases) to present psychiatric disorders as schizophrenia which is not the case for hypospads that are detectable from birth in male children and grand-children. Work is already under way concerning the gene X environment DES impact hypothesis by comparing DES and EE exposed children, various genetic and epigenetic factors to those of mother and unexposed children of the same family as studied by the INSERM team U796 in collaboration with the HHORAGES families.

Conclusions

In the present familial case control study, we have shown that there are serious effects on the psychological and physical health of the descendants of women treated with synthetic hormones during their pregnancy: psychiatric illnesses are often found associated with somatic disorders which are well known to be the DES and EE signature. Synthetic hormones, acting as endocrine disturbers, are toxic for humans, especially for pregnant women and their children, probably partly in relation with their toxic degradation status. In all cases girls suffered more than boys either of somatic and/or psychiatric disorders due to the estrogen receptor alpha or beta concentration higher in female fetus than in male. It is also clear that in all the families most of the exposed children are ill while quite the unexposed are not.

So what now? As the precautionary principle was not applied in the past, and still is not in force today, and since the lessons of recent history were never taken into account , it is our common duty to repair the damage by supporting the devastated families, and by pursuing research work on the observation of trans-generational effects. Such effects are already highlighted by the demonstration that cancers are observed even in the fourth generation in mice . According to the Skinner’s mini review “the ability of an environmental compound (as DES or EE) to promote the reprogramming of the germ-line appears to be the causal factor in the epigenetic transgenerational phenotype,” we observed an increase of the genital malformations in the third generation in male infants whose mothers were treated with xenoestrogens. In the HHORAGES troop, DES and EEexposed infants are already pointed out as bodily and/or psychologically impaired after their mothers were treated with clomifene citrate (an ovulation stimulator previously used for IVF-type medically assisted procreation). Another concern is the putative future effect of ethinylestradiol containing oral estrogenic contraception on future generations due to its lipophily after its metabolization and its future release in fetus through the placenta. As for the demonstration of the causality link within the HHORAGES troop, will we have to wait for a large-scale epidemiological study, or are we allowed to think that the impressive figures that we are publishing in this work are not merely random? The only way now is to respect absolutely the precautionary principle and to delete completely or to give the less possible toxic (synthetic) hormone medication: for example Clavel Chapelon and her Endogenous Hormones and Breast Cancer Collaborative Group in Villejuif informed that natural hormone as micronized (natural) progestin associated with estrogens (synthetic alas!) is more often ordered for SHT (Substitutive Hormonal Treatment) in order to avoid breast cancer. Unfortunately, she said also that in the contrary the same SHT is not recommended to avoid the endometrium cancer …

As Newbold et al said after they reviewed the damages caused by DES ,

“only new advances in the knowledge of genetic and epigenetic mechanisms of the disruptions of fetal development will enable us to be aware of the risks entailed by the other estrogenic disruptors which are present around us and in ourselves, even at very low doses”

, whilst Theo Colborn insists on the fact that the foetus cannot be protected against endocrine disruptors, whatever they may be, except at zero level.

Click to download the complete PDF.

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