The trans-generational effects of DES exposure may pose a risk for future generations

Endocrine disruption of oestrogen action and female reproductive tract cancers

2014 Study Abstract

Endocrine disrupting chemicals (EDC) are ubiquitous and persistent compounds that have the capacity to interfere with normal endocrine homoeostasis. The female reproductive tract is exquisitely sensitive to the action of sex steroids, and oestrogens play a key role in normal reproductive function. Malignancies of the female reproductive tract are the fourth most common cancer in women, with endometrial cancer accounting for most cases. Established risk factors for development of endometrial cancer include high BMI and exposure to oestrogens or synthetic compounds such as tamoxifen. Studies on cell and animal models have provided evidence that many EDC can bind oestrogen receptors and highlighted early life exposure as a window of risk for adverse lifelong effects on the reproductive system. The most robust evidence for a link between early life exposure to EDC and adverse reproductive health has come from studies on women who were exposed in utero to diethylstilbestrol. Demonstration that EDC can alter expression of members of the HOX gene cluster highlights one pathway that might be vulnerable to their actions. In summary, evidence for a direct link between EDC exposure and cancers of the reproductive system is currently incomplete. It will be challenging to attribute causality to any single EDC when exposure and development of malignancy may be separated by many years and influenced by lifestyle factors such as diet (a source of phytoestrogens) and adiposity. This review considers some of the evidence collected to date.

  • ER signalling
  • ER expression and actions of oestrogens in reproductive tract tissues
  • Oestrogen biosynthesis and ER expression in benign and malignant endometrial tract disorders
  • Evidence that oestrogens increase the risk of developing a reproductive cancer
  • Endocrine disruptors implicated in reproductive tract disorders and cancer
Diethylstilbestrol

DES, a synthetic non-steroidal oestrogen, is often regarded as the archetypal endocrine disruptor. From about 1940 to 1970, DES was given to pregnant women in the mistaken belief that it would reduce the risk of pregnancy complications. Herbst et al. reported a probable link between DES and vaginal clear cell adenocarcinoma in girls and young women who had been exposed to this drug. It is estimated that five to ten million people were exposed to DES, including the pregnant mothers who received treatment and their offspring. Of the several million women exposed to DES in utero, a cohort of 4653 DES-exposed women have been followed up to investigate the long-term consequences of exposure (Hoover et al. 2011). Patient data stratified to account for the extent of exposure or dose effects of DES identified an association between treatment of mothers earlier during their pregnancy and adverse vaginal epithelial changes at a younger age in their offspring. While DES exposure is associated with increased risk of breast and cervical/vaginal clear cell adenocarcinoma, several studies have indicated that there is no associated risk of endometrial or ovarian cancer. As endometrial cancer is most likely to present after menopause, many of the DES-exposed women may not yet be old enough to determine whether they are at excess risk, as in the 2011 report only 27% were older than 50 years. Although to date epidemiological data indicate that DES-exposed women may not be at increased risk of developing endometrial cancer, studies in animal models provide evidence to the contrary.

In the early 1990s, Newbold et al. developed a mouse model for investigating hormonal carcinogenesis in mice by investigating the effects of neonatal exposure to oestrogens on cancer development. Treatment of CD1 neonatal mice with DES on postnatal days 1–5, which correspond to late prenatal human development, resulted in 90% of DES-exposed mice developing uterine adenocarcinomas after 18 months while none of the control animals had neoplastic lesions. Crucially, while administration of DES increased the risk of uterine adenocarcinoma, endogenous oestrogen was required for tumour development with prepubertal ovariectomy preventing tumour development. In DES-exposed women, vaginal and cervical carcinomas were only detected post-menarche consistent with a requirement for endogenous oestrogen in tumour development. ERα knockout mice (ERKO) did not develop tumours following neonatal DES exposure (Couse & Korach 2004); transgenic mice overexpressing ERα displayed accelerated tumour development but mice with a dominant negative isoform of ERα (ERΔ3) were not protected, highlighting the complexity of the molecular signalling mechanisms involved.

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. Kabbarah et al. 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. Newbold et al.  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.

  • Evidence that expression of HOX genes can be altered by exposure to EDC
  • Evidence that early life exposure to EDC can alter onset of puberty or timing of menopause
  • Evidence that lifestyle factors can increase the influence of EDC on lifetime risk of developing reproductive tract cancers
  • Summary and future perspectives

Sources

  • Endocrine disruption of oestrogen action and female reproductive tract cancers, Endocrine-Related Cancer, endocrinology-journals, doi: 10.1530/ERC-13-0342, April 1, 2014.
  • Featured image chuttersnap.
DES DIETHYLSTILBESTROL RESOURCES

DES effects on the female reproductive system

image of female on sofa

Epigenetic effects of endocrine-disrupting chemicals on female reproduction: An ovarian perspective

2010 Study Abstract

The link between in utero and neonatal exposure to environmental toxicants, such as endocrine-disrupting chemicals (EDCs) and adult female reproductive disorders is well established in both epidemiological and animal studies. Recent studies examining the epigenetic mechanisms involved in mediating the effects of EDCs on female reproduction are gathering momentum. In this review, we describe the developmental processes that are susceptible to EDC exposures in female reproductive system, with a special emphasis on the ovary. We discuss studies with select EDCs that have been shown to have physiological and correlated epigenetic effects in the ovary, neuroendocrine system, and uterus. Importantly, EDCs that can directly target the ovary can alter epigenetic mechanisms in the oocyte, leading to transgenerational epigenetic effects. The potential mechanisms involved in such effects are also discussed.

DES effects on the female reproductive system

DES is a nonsteroidal synthetic estrogen that was prescribed to pregnant women at doses of 5–150 mg/day to prevent miscarriages from 1940s to 1970s). Even though early on DES was shown to be an ineffective drug, it was continued in use till the 1970s. Numerous abnormalities in the reproductive, cardiovascular, and immune systems have since been reported in both male and female offspring of women treated with DES, and validated in animal models. There are limited reports that these effects are being observed in the granddaughters of DES-treated women as well. While DES caused vaginal clear cell adenocarcinoma in only 0.1% of the female offspring, over 95 % reported reproductive tract dysfunction and poor pregnancy outcomes. Since there is evidence of multi-generational effects, epigenetic mechanisms could play an important role and were therefore investigated.

Mice injected with a single dose of 10 μg/kg DES on E15 and examined at 7 months of age had no CL and numerous atretic follicles. They were also found to have vacuolated interstitial tissue with lipid droplet inclusions. Other studies with varying doses of DES (5 μg/kg to 100 μg/kg) administered either in utero (E9-E16), or neonatally (PND1-PND5) , demonstrated that adult DES ovaries developed similar hypertrophy and vacuolation of interstitial tissue, hemorrhagic cysts and lack of corpora lutea. These animals also had high levels of testosterone. There was a dose-dependent reduction in the number of the litters as well as the number of oocytes ovulated after stimulation with exogenous gonadotropins. The oocytes derived from such treated ovaries and used in IVF showed lower levels of fertilizability, suggesting reduced oocyte quality. However, 5 µg/day DES-treated ovaries transplanted into untreated ovariectomized host mice were able to give rise to normal female offspring that in turn gave birth to normal size litters and had normal uterine morphology, suggesting that the DES treatment effects were not mediated via germ cells. However, the age at which these animals were sacrificed was 8–12 weeks, and other studies have shown that DES-treated animals do develop epithelial cancers of the uterus by 18 months of age.

DES can bind to both ERs with many fold higher affinity than estradiol. Multiple studies from Iguchi and colleagues showed that in utero (E15–18) and neonatally (PND1-5) DES-treated mice had ovaries containing excessive number of MOFs by adulthood. MOFs were also observed in ovaries that were treated in vitro at PND1-5, following their transplantation to untreated mice, suggesting a direct effect of DES in the ovary. Recent studies showed that neonatal exposure to 3 μg/kg DES induced MOFs, a process mediated by ERβ and not ERα. DES exposure was shown to reduce oocyte apoptosis (potentially suppressing oocyte nest breakdown) via ERβ signaling mechanisms. Furthermore, it was hypothesized that such alterations in the germ cell and somatic cell populations may affect the invasion of pregranulosa cells and basement membrane remodeling during primordial follicle formation. Interestingly, the incidence of MOFs has been reported with other EDC exposures as well).

The effects of DES on the sexual dimorphism of the brain have been documented. In utero and postnatal exposures increased the size of SDN-POA in females thereby defeminizing the region. It was also found that PND1-10 treatment led to a significant reduction in the levels of LH secreted although a similar effect was not found when the exposure was prenatal (E16-20), highlighting the importance of DES actions on the neuroendocrine circuits.

It is well known that DES caused T-shaped uteri and clear cell adenocarcinoma of the uterus, cervix, and vagina in women whose mothers were exposed to DES during pregnancy. There are numerous animal studies validating these human reports. For example, progeny of DES-treated mice have shown malformations of the uterus, squamous metaplasia of the luminar and glandular epithelium, endometrial hyperplasia and leiomyomas, and oviductal proliferative lesions. Ovariectomized animals when supplemented with estradiol are able to respond by a transient increase in gene expression and concomitant uterine proliferation and growth. When such a stimulus is removed, the uterus returns to its unstimulated state. However, when DES or estradiol is administered during neonatal development, expression of immediate early genes such as lactoferrin, EGF, and proto-oncogenes such as c-fos, c-jun, and c-myc is upregulated even into adulthood. Inversely, expression of genes that are necessary for uterine development, such as the Abdominal B (AbdB) Hox gene, Hoxa-10, (known to be controlled by estradiol and progesterone,), Wnt7a as well as Msx2 are repressed leading to structural abnormalities of the reproductive tract. Numerous studies have been conducted to assess the methylation patterns of promoters of several of these estrogen-responsive genes associated with uterine development.

Neonatal DES exposure in mice caused nearly 90% incidence of epithelial cancers of the uterus by 18 months of age. In mice similarly treated with DES, the promoter region of the lactoferrin gene was found to be hypomethylated in the adult uterus. However, if the animals were exposed for the same length of time during adulthood, no such methylation or expression defects were observed. Subsequently, it was also found that exon 4 of the c-fos gene was extensively hypomethylated while the promoter region and intron 1 was unaffected, thereby potentially allowing for the upregulation of c-fos expression. QPCR studies performed by Sato and colleagues examining the expression of Dnmts in neonatally DES exposed C57BL/6 mice, revealed that expression of Dnmt1 and Dnmt3b was decreased at PND5 in DES-treated mice, and the pattern continued until PND14. Interestingly, it was found that human leiomyoma samples had alterations in the levels of Dnmts as well, with concomitant global hypomethylation.

As mentioned above, DES down-regulates Hoxa gene expression. These effects are akin to those associated with uterine abnormalities found in Hoxa KO mice. The predominant phenotype is the loss of boundary between the oviduct and uterus. It has been shown that the anterior to posterior specific pattern of Hoxa-9 is essential for the normal development and function of the uterus and that DES causes a posterior shift of Hoxa-9 and Hoxa-10 expression and homeotic anterior transformations. A recent report by Bromer and colleagues has shown that after in utero (E9-16) exposure to 10 μg/kg DES, there is hyper-methylation in the promoter and intron 1 regions of Hoxa-10 gene, in the caudal part of the uterus with a concomitant increase in the Hoxa-10 expression in the same region. While these data are interesting from the point of epigenetic regulation of the regionalization of the uterus, the authors suggest that the apparently conflicting data i.e., increased methylation vs increased gene expression might be due to differential binding to transcriptional repressors. Since previous studies have shown that no epigenetic changes were detected in the promoters of Hoxa-10 and Hoxa-11 genes, continuation of these studies is warranted.

Alworth and colleagues showed that in utero exposure (E12-18) of CD-1 mice to DES doses of 0.1–100 μg/kg followed by estradiol administration at 7–8 months of age caused opposite responses between low and high doses. The lower dose enhanced the response to exogenous estrogens, resulting in an increase in uterine weight, while the high dose dampened the response resulting in lighter uteri. A global methylation assay was conducted employing DMH, which was suitable to detect hypermethylation events. Over 300 CpG island loci were examined and five candidates were identified in 18S rDNA and 45S pre-rDNA methylation, suggesting a role for ribosomal assembly and protein synthesis in the mediation of DES effects.

Couse and colleagues have shown that ERα is essential for the mediation of DES effects in the uterus: αERKO female mice exhibited a complete resistance to the effects of DES while βERKO mice did not. Additionally, as mentioned earlier, ERα induction is necessary for activation of estrogen responsive gene expression including that of the lactoferrin and c-fos genes. Since these genes are all downstream of ERα signaling, it is imperative to thoroughly examine the potential role of epigenetic mechanisms in the regulation of ERα expression after EDC exposure. Interesting new studies by Bredfeldt and colleagues have now provided a link between ER signaling and regulation of histone modifications. It was found that rapid PI3K/Akt signaling downstream of membrane-associated ER, in response to estradiol as well as DES, caused reduction in trimethylation of H3K27. More interestingly, activation of this non-genomic signaling caused reprogramming of the uterine gene expression profile. Whether such altered epigenetic mechanisms are transgenerational would be of great interest to the field.

Tang and colleagues recently investigated whether neonatal DES/genistein exposure could cause epigenetic changes and alter gene expression in adult uteri and whether there are interactions between adult ovarian hormones and such epigenetic reprogramming. CD-1 mice were exposed to DES (1 μg and 1000 μg/kg) or genistein (50 mg/kg) from PND1-5. Subsequently, some animals were sacrificed at PND19 while others were aged to 6 and 18 months with or without ovariectomies. Genome-wide methylation analysis was conducted with MSRF and candidate genes were identified. Of interest was the nucleosomal binding protein 1 (Nsbp1), which was shown to be hypomethylated at PND19 and hyper-methylated by puberty, in the control. Low-dose DES- and genistein- treated vs high-dose DES-treated animals had opposing methylation patterns. Furthermore, it was shown that in the aged animals, both DES and genistein caused hyper-methylation in the ovariectomized animals but remained hypomethylated in non-ovariectomized animals. These data suggest that Nsbp1 is hyper-methylated in intact mice with age and that DES and genistein have opposing effects on the methylation patterns in intact vs ovariectomized aging animals (hypomethylation vs hyper-methylation), respectively. These studies highlighted the age-dependent aspect of epigenetic reprogramming and also its interaction with steroid hormones.

Transgenerational epigenetic effects associated with DES exposures

Documentation of the transgenerational epigenetic effects of EDCs has been accumulating such as the case of transgenerational effects of DES exposure reported in humans. Exposure to DES while in utero was shown to affect the female F2 generation but not males in mice. Later studies with DES exposure showed that it leads to reproductive tract tumors in both F2 generation males and female mice.

Sources

  • Full text (free access) : Epigenetic effects of endocrine-disrupting chemicals on female reproduction: An ovarian perspective, Frontiers in neuroendocrinology, NCBI PubMed, PMC3009556, 2010 Jul 4.
  • Featured image credit Luis Sarabia.
DES DIETHYLSTILBESTROL RESOURCES

DES Developmental Programming and Fetal Origins of Adult Disease

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

Introduction

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

Abstracts

Developmental Programming and Fetal Origins of Adult Disease

The DES Example

Prenatal exposure to diethylstilbestrol (DES), a synthetic estrogen and thus EDC, provides an unfortunate example of developmental programming. DES was given to U.S. pregnant women between 1938 and 1971 under the erroneous assumption that it would prevent pregnancy complications. In fact, in utero exposure to DES alters the normal programming of gene families, such as Hox and Wnt, that play important roles in reproductive tract differentiation. As a result, female offspring exposed to DES in utero are at increased risk of clear cell adenocarcinoma of the vagina and cervix, structural reproductive tract anomalies, infertility and poor pregnancy outcomes, while male offspring have an increased incidence of genital abnormalities and a possibly increased risk of prostate and testicular cancer. These observed human effects have been confirmed in numerous animal models which have also provided information on the toxic mechanisms of DES. Animal experiments have also predicted changes later found in DES-exposed humans, such as oviductal malformations, increased incidence of uterine fibroids and second-generational effects such as increased menstrual irregularities and possibly ovarian cancer in DES-granddaughters and increased hypospadias in DES-grandsons.

DES is but one example of how exposure to EDCs can disrupt developing organ systems and cause abnormalities, many of which only appear much later in life or in the subsequent generation, such as endometriosis, fibroids and breast, cervical and uterine cancer in women; poor sperm quality and increased incidence of cryptorchidism and hypospadias in men; and subfertility and infertility in men and women.

Reproductive Effects of Early Life Exposures in Females

Uterus Development and the Environment

Women exposed to DES in utero during critical periods of reproductive tract development developed several types of reproductive tract abnormalities, as well as an increased incidence of cervical-vaginal cancer later in life. Animal studies that simulate the human DES experience have since shown that exposure of the developing reproductive tract of CD-1 mice to DES imparts a permanent estrogen imprint that alters reproductive tract morphology, induces persistent expression of the lactoferrin and c-fos genes and induces a high incidence of uterine adenocarcinoma. Experiments in rats have shown exposure to DES during the critical window of uterine development leaves a hormonal imprint on the developing uterine myometrium in rats that were genetically predisposed to uterine leiomyoma, increasing the risk for adult uterine leiomyoma from 65% to greater than 90% and increasing tumor multiplicity and size. DES-induced developmental programming appears to require the estrogen receptor α, suggesting that signaling through this receptor is crucial for establishing developmental programming.

Sources

  • Full study (free access) : Proceedings of the Summit on Environmental Challenges to Reproductive Health and Fertility: Executive Summary, Fertility and sterility, NCBI PubMed, PMC2440710, 2009 Feb 1.
  • Featured image credit Kiệt Hí.
DES DIETHYLSTILBESTROL RESOURCES

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

Multigenerational effects of DES have been reported through the paternal lineage

1992 Study Abstract

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

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

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

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

Sources

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

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

image of arental and Grandparental Epigenetic

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

2008 Study Abstract

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

Diethylstilbestrol

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

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

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

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

Hsp90 and environmental impacts on epigenetic states

image of hsp90

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

2005 Study Abstract

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

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

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

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

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

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

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

Do breast tissue of women exposed in utero to DES exhibit genetic abnormalities ?

image of breast-neoplasms

In utero exposure to diethylstilbestrol (DES) does not increase genomic instability in normal or neoplastic breast epithelium

2006 Study Abstract

BACKGROUND
In 1992, the National Cancer Institute (NCI) established the Continuation of Follow-Up of DES-Exposed Cohorts to study the long-term health effects of exposure to diethylstilbestrol (DES). Genetic effects on human breast tissue have not been examined. The authors investigated whether breast tissue of women exposed in utero to DES might exhibit the genetic abnormalities that characterize other DES-associated tumors.

METHODS
Subjects enrolled in the NCI Cohort were queried about breast biopsies or breast cancer diagnoses. Available tissue blocks were obtained for invasive cancers (IC), in situ cancers (CIS), or atypical hyperplasia (AH). Exposure status was blinded, lesions were microdissected, and their DNA was analyzed for microsatellite instability (MI) and loss of heterozygosity (LOH), or allele imbalance (AI), at 20 markers on 9 chromosome arms.

RESULTS
From 31 subjects (22 exposed, 9 unexposed), 273 samples were analyzed (167 normal epithelium, 16 AH, 30 CIS, 60 IC). Exposed and unexposed subjects exhibited no differences in breast cancer risk factors or demographic characteristics, except for age at diagnosis (exposed vs. unexposed: 43.2 vs. 48.8 years of age, P = .02). The authors found that MI was rare and that AI was common, with frequencies consistent with previous reports. The global age-adjusted relative rate (RR) of AI was 1.3, 95% CI = 0.8-2.4. No statistically significant associations were observed after adjustment for risk factors or after stratification by histology or by chromosome arm.

CONCLUSIONS
In utero DES exposure does not appear to significantly increase genomic instability in breast epithelium, as measured by MI and AI. Breast tissue may respond differently from that of the reproductive tract to in utero DES exposure. Consequences of in utero DES exposure on the breast may be mediated by proliferative effects of estrogen.

Sources and more information
  • In utero exposure to diethylstilbestrol (DES) does not increase genomic instability in normal or neoplastic breast epithelium, Cancer, NCBI PubMed PMID: 16998936, 2006 Nov.
  • Full text: wiley: DOI: 10.1002/cncr.22223,
    Cancer, Volume 107, Issue 9, pages 2122–2126, 1 November 2006.
  • Breast neoplasms featured image credit vmshashi.
DES DIETHYLSTILBESTROL RESOURCES