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 and third-generation hypospadias

image of third-generation hypospadias

Prevalence of hypospadias in grandsons of women exposed to diethylstilbestrol during pregnancy: a multigenerational national cohort study

2011 Study Abstract

Prenatal diethylstilbestrol (DES)-exposed mice have raised the suspicion of a transgenerational effect in the occurrence of genital malformation in males. This nationwide cohort study in collaboration with a French association of DES-exposed women studied 529 families and showed that a significant proportion of boys born to DES daughters exhibited hypospadias with no other molecular defects identified.

Although the role of fetal androgens is crucial to male genital development during the first trimester of pregnancy, defects in the synthesis or molecular action of testosterone are rare in isolated hypospadias (Hypospadias may be a multifactorial defect arising from genetic, hormonal, and environmental factors). It has been hypothesized that changes in androgen/estrogen balance due to endogenous or exogenous hormonal factors during the critical period of penile and urethral development contribute to this malformation.

Men who were exposed in utero to diethylstilbestrol (DES), a synthetic estrogen, may exemplify the effects of environmental chemicals with endocrine-disrupting activity on genital development (DES was prescribed for pregnant women from the late 1930s to the 1970s in the mistaken belief that it would prevent miscarriage or premature birth). Unfortunately, DES was found to be not only ineffective but also harmful. Daughters born from DES-related pregnancies often show abnormalities in the Müllerian structures and have elevated risks of peripubertal vaginal and cervical clear-cell adenocarcinoma, fertility problems, ectopic pregnancies, miscarriages, and premature births (The risk of reproductive tract abnormalities also appears to be increased for DES sons, who may present hypoplastic testis, epididymal cysts, cryptorchidism, or hypospadias).

After several studies in animals, a question emerged as to whether the harmful effects of DES can be “transmitted” to subsequent generations. Newbold and colleagues reported an increased incidence of reproductive tract tumors in male and female descendants of mice developmentally exposed to DES. In the human, Klip et al. reported an increased risk of hypospadias in sons of DES daughters in a cohort of women diagnosed with fertility problems. Other studies either confirmed or questioned these results. However, the direct implication of DES in the occurrence of hypospadias remains debatable, since many other uncontrolled factors, especially environmental and genetic, are implicated in this malformation. We studied the prevalence of hypospadias in the grandsons of DES-treated and -untreated women and ruled out other environmental and genetic factors that could have been associated with the malformation in these patients.

A nationwide cohort study was conducted in collaboration with a French association of DES-treated women (HHORAGES Association). The reason the women joined this association was not the presence of hypospadias in the second or third generation but rather psychological disturbances, vaginal and cervical clear-cell adenocarcinomas, miscarriages, and other abnormalities. Five hundred twenty-nine families were included. All of the second- and third-generation offspring were accounted for and included in the study. No one declined to participate. DES exposure was reported in 1,000 out of 1,180 pregnancies. The featured image details the patient groups. The clinical diagnosis of hypospadias was standardized and based on a detailed operative report or direct clinical examination by a pediatrician and/or urologist. The malformation was characterized as severe (proximal, penoscrotal) or nonsevere (glandular, subcoronal, distal, midshaft). Each mother with a hypospadiac son was contacted and responded to a short questionnaire validated in Europe for data collection (no. QLK4-1999-01422) to determine whether other occupational exposure had occurred during the pregnancy. To exclude a defect of the androgen pathway, we performed molecular analysis of the genes known to be associated with hypospadias such as androgen receptor (AR), 5α reductase (srd5A2), and MAMLD1 genes in DNA from peripheral blood. The local university hospital ethics committee approved this study (ID RCB No. 2008-A00781-54), and each patient gave informed consent through the Hhorage Association.

The prevalence of hypospadias was low in boys unexposed to DES in utero (0/180), whereas it was high in the in utero–exposed boys (3.57%, 16/448, P=.02). In the third generation, the prevalence of hypospadias in boys born to DES daughters was high when compared with boys born to unexposed parents (8.2%, n = 8/97 vs. n = 0/360; P<.001). The hypospadiac patients of the second and third generations were not related. The results are summarized in the featured image.

Neither mutations nor polymorphisms of the AR and MAMLD1 genes were found among hypospadiac boys of the third generation. Only one polymorphism of the srd5A2 gene was detected (A49T) in a boy. The mothers of the third-generation affected boys indicated little environmental or occupational exposure to endocrine-disrupting chemicals during pregnancy (no professional activity, n = 2; sales clerk in a food or clothing shop, n = 3; office worker, n = 3), and such exposure was therefore unlikely to have contributed to the occurrence of hypospadias. Two mothers of hypospadiac sons exhibited hypoplastic or bifid uterus.

The main effect of DES is profound disturbance in the androgenic/estrogenic balance of animal and human fetuses since it has both estrogenic and antiandrogenic actions by competing with natural androgens for the ligand-binding domain of the androgen receptor. In utero exposure to DES during the critical period of reproductive tract development is known to induce genital malformation in mice. In utero–exposed sons show greater risks of structural urogenital abnormalities like hypospadias, epididymal cysts, micropenis, and cryptorchidism. The present study reinforces these data with a prevalence of hypospadias greater than 3%, although it should be noted that the second-generation population included only 180 controls from the same families since this series was specifically designed to study the third-generation boys.

More interesting is the hypothesis of a transgenerational effect of DES. Animal studies first suggested that DES might increase transgenerational susceptibility to malignant tumors of the female reproductive tract, presumably by damage to germ cells and abnormal imprinting. In human beings, DES exposure may also lead to permanently altered germ cells. The suggestion of a transgenerational effect of DES in human beings was based on the observation of a high prevalence of hypospadias, particularly with severe phenotypes, in the sons of women exposed to DES in utero.

But variations in the definition of the control population may explain the wide range of odds ratios reported in the literature to date. Palmer et al. reported a prevalence 6 times higher than that of Klip et al. The present study, which shows a high prevalence of hypospadias of various severities in the third generation, tried to limit this bias and included DES-free pregnancies and DES-exposed pregnancies from the same families. Nevertheless, two limitations should be noted:

  1. DES-exposed women without problems were not included;
  2. and the fertility status of the exposed and non-exposed couples, the age at pregnancy, and the parity for each women were variable, and this may have hidden fertility problems or greater use of contraception.

The low fertility rates of the DES sons may also be explained by other findings, such as severe psychotic disorders or oligospermia in cases of hypospadias with additional defects.

We did not identify any genetic or environmental factors that would have explained the hypospadias in DES grandsons. Our results thus raise the question of the mechanism through which DES causes adverse effects in subsequent generations. The frequency of transmission both observed in our series of hypospadiac grandsons and previously reported in generations examined for various disease states secondary to DES exposure is particularly high. This frequency of a transgenerational phenotype is such that a mutational event involving the DNA sequence could not be implicated. DES-induced changes in epigenetic background and alteration of DNA methylation could be significant factors in the susceptibility to disease development. The primordial germ cells undergo demethylation during migration and early colonization of the embryonic gonad, followed by remethylation starting at the time of sex determination in a sex-specific manner. The pregnant mother’s exposure to DES at the time of fetal sex determination appears to be sufficient to alter the remethylation of the germ line in the male fetus and permanently reprogram the imprinted pattern of DNA methylation. The transmission of multigenerational DES effects would thus occur through the paternal lineage. But our findings indicated that most of the third-generation hypospadiac boys were born to DES daughters. This agreed with previous studies (although paternal transmission of DES effects is not excluded). Epigenetic changes in the AR gene, transmitted through the DES daughter, could explain such a finding since the antiandrogen effect of DES is known to modify the phosphorylation level of AR.

The association between hypospadias in grandsons and uterine abnormalities in their mothers suggests other hypotheses for the transgenerational mechanisms of DES. First, DES daughters may have displayed disturbed hormonal balance during their reproductive life or placental malfunction that might have interfered with the genital development of a male fetus. Second, the estrogen receptor gene ERα and estrogen-responsive genes that contribute to the development of both female internal genitalia and hypospadias may also be involved since ERα is implicated in the induction of abnormalities after DES exposure. Last, the genes involved in the structural differentiation of both the female and male reproductive tracts may be altered by DES exposure. DES has been reported to delay expression of HOXa family genes during Müllerian duct development. DES could also interfere with HOX gene expression during penile formation.

For many authors, DES is an experimental environmental xenoestrogen. Despite the bias that could not be fully eliminated and the difficulty of extrapolating the risks of exposure (no monotonic dose-response relationship, varying effects depending on the timing of exposure in the developing organism, manifestations delayed until later in life), this clinical study strengthens the suspicion of the transgenerational effects of environmental endocrine disruptors.

Sources

  • Full text (free access) : Prevalence of hypospadias in grandsons of women exposed to diethylstilbestrol during pregnancy: a multigenerational national cohort study, CHU Montpellier and Universite Montpellier, France, hhorages, February 23, 2011.
  • Featured image : Detailed patient groups included in the study. In the second generation, the phenotype of affected boys was isolated hypospadias in all cases, severe in 12 cases (posterior or penoscrotal), and not severe in four cases (mild or anterior). In the third generation, the hypospadias was severe in five cases and not severe in three cases. Bilateral cryptorchidism was present in one case. *The size of the population was under 1,000, and the prevalence of hypospadias of about 1/1,000, as seen in the general population, could not be represented.
DES DIETHYLSTILBESTROL RESOURCES

Hypospadias: a transgenerational effect of diethylstilbestrol?

image of des grandson

Is there an increased risk of hypospadias for DES grandsons?

2005 Study Abstract

BACKGROUND
In 2002, an increased risk of hypospadias was reported for sons of women exposed to diethylstilbestrol (DES) in utero, suggesting transgenerational effects of DES. The aim of this study was to further assess the association between parental DES exposure and hypospadias in a case-referent study.

METHODS
Cases with hypospadias were retrieved from the hospital information system. Referents were recruited via the parents of cases. Both parents completed postal questionnaires. Associations were estimated by odds ratios (OR) with 95% confidence intervals (CI). Additionally, conditional logistic regression analyses were performed for a matched subset of parents.

RESULTS
The final database included 583 cases and 251 referents. In the initial analyses, an indication was found for an increased risk of hypospadias when mothers were exposed to DES in utero: OR=2.3 (95% CI 0.7-7.9). Conditional logistic regression resulted in a stronger risk estimate: OR=4.9 (95% CI 1.1-22.3). Paternal exposure to DES did not increase the risk.

CONCLUSIONS
The results confirm an increased risk of hypospadias when mothers were exposed to DES in utero. However, the excess risk appears to be of much smaller magnitude than in the 2002 study. Further research on the potential health risks for the third generation is of great importance.

Sources

  • Full text (free access) : Hypospadias: a transgenerational effect of diethylstilbestrol?, Human reproduction (Oxford, England), NCBI PubMed, PMID: 16293648, 2005 Nov.
  • Featured image credit Todd Cravens.
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

Increased susceptibility for tumors transmitted to DES subsequent generations

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

2000 Study Abstract

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

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

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

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

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

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

Sources

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

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.

Sources and more information
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.

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