Fertility disorders attributable to the use of diethylstilbestrol during intrauterine life

Women treated with distilbene during pregnancy : it seems that the increased number of miscarriages, extra-uterine pregnancies and perinatal deaths are probably due to the maternal treatment which is also responsible for semen abnormalities

1984 Study Abstract

In the early 1970s, the elevated rate of abnormalities in children of the 2-3 million US women and 260,000 French women treated with diethylstilbestrol (DES) during pregnancy began to be recognized.

Four kinds of cervicovaginal anomalies have been observed in women exposed to DES in utero:

  1. 22-58% have been estimated to have morphologic anomalies with the timing of exposure to DES more important than the total dose
  2. a high proportion has an insufficient cervical mucus not corrected by exogenous administration of estrogen
  3. a high proportion develop cervical stenosis after cryosurgery, electrocoagulation, or conization
  4. and the increased incidence of prematurity in infants of DES-exposed mothers has been attributed to cervical incompetence.

69% of 267 women studied had hysterographically demonstrated uterotubal malformations. A characteristic aspect was a T-shaped uterus but other anomalies were noted. Hysterographic anomalies were correlated with cervico-isthmic anatomic anomalies, anomalies of the vaginal epithelium, and with the date of 1st exposure to DES in utero. The total dose of DES did not affect the frequency of genital anomalies. Possible tubal anomalies have not been well studied, although 1 author has observed short and narrow tubes and other abnormalities.

The number of extrauterine pregnancies is known to be elevated in women exposed to DES in utero. The possibility of an increased incidence of menstrual irregularity, dysmenorrhea, or oligomenorrhea in DES-exposed women has been suggested but remains controversial.

The responsibility of DES exposure in utero for later reduced fertility is also in dispute. Higher rates of spontaneous abortion, extrauterine pregnancy, prematurity, and perinatal death have been reported in DES-exposed women.

Increased incidence of stenosis of the meatus, hypospadias, epididymal cysts, testicular hypoplasia and other anomalies, cryptorchidism, microphallus, and varicocele have been reported in men exposed to DES in utero. Reduced sperm counts and anomalies in the volume of the ejaculate, percentage of sperm mobile, and sperm morphology have been reported in exposed men. Sperm anomalies may be responsible for reduced fertility in exposed men, but the exact extent is unknown.

Sources

  • Fertility disorders attributable to the use of diethylstilbestrol during intrauterine life, NCBI PubMed PMID: 6397835, 1984 Apr.
  • Featured image credit Edward Cisneros.
DES DIETHYLSTILBESTROL RESOURCES

Increased breast cancer risk in DES daughters, granddaughters and great granddaughters

The effects of maternal diethylstilbestrol exposure are not limited to the F1 generation

2014 Study Abstract

The idea that susceptibility to breast cancer is determined not only through inherited germline mutations but also by epigenetic changes induced by alterations in hormonal environment during fetal development is gaining increasing support. Using findings obtained in human and animal studies, this review addresses the mechanisms that may explain why daughters of mothers who took synthetic estrogen diethylstilbestrol (DES) during pregnancy have two times higher breast cancer risk than women who were not exposed to it. The mechanisms likely involve epigenetic alterations, such as increased DNA methylation and modifications in histones and microRNA expression. Further, these alterations may target genes that regulate stem cells and prevent differentiation of their daughter cells. Recent findings in a preclinical model suggest that not only are women exposed to DES in utero at an increased risk of developing breast cancer, but this risk may extend to their daughters and granddaughters as well. It is critical, therefore, 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.

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.

Developing germ cells undergo epigenetic erasure when they, as primordial germ cells, enter into the fetal gonads around embryonic day 10 to 11 (in mice and rats), and then undergo gender-specific reprogramming as germ cells. It is now clear that reprogramming of these cells is susceptible to modifications caused by changes in fetal hormonal environment, such as resulting from an exposure to DES or other endocrine disruptors. Consequently, these exposures can leave a permanent biochemical footprint on the genome of the F1 generation germ cells, and this change may be inherited by the F2 generation germ line and several subsequent generations.

Some researchers have begun to investigate whether the effects of maternal DES exposure during pregnancy extend to the third generation in humans. Although there is no evidence that DES granddaughters have cervical and ovarian abnormalities similar to DES daughters, there is evidence that they may have more menstrual irregularities and a higher rate of infertility than non-exposed granddaughters. In addition, DES granddaughters may have a slightly higher risk of ovarian cancer. The granddaughters are still too young to assess whether they might also be at an increased risk of developing breast cancer.

Millions of women in the US, Europe and Australia have been exposed to DES in the womb, and consequently exhibit about a two times higher breast cancer risk than unexposed women. The increase in risk may not be limited to the DES-exposed daughters, but could also increase breast cancer risk in granddaughters and great granddaughters. Such outcome would be consistent with the findings we obtained in studies using a synthetic estrogen ethinyl estradiol (EE2). If DES has similar effects to ethinyl estradiol on the transgenerational increase in breast cancer risk, it is urgent to find ways to stop the cycle of inheritance, and also prevent breast cancer in DES-exposed granddaughters and great granddaughters.

To achieve this goal, we need to understand how maternal DES exposure during pregnancy increases a daughter’s breast cancer risk. A plausible model is proposed in feature image. It is evident from studies done in animal models that in utero DES exposure induces epigenetic changes in reproductive tract tissues and the breast. DES exposure might also have induced epigenetic changes in primordial germ cells and consequently germ cells, and further be detectable in the somatic cells in granddaughters and great granddaughters. We are not aware of any study that has compared epigenetic changes in germ cells and the next generation somatic cells in individuals exposed to DES or other endocrine disruptors in utero. Second, we should investigate whether the transgenerational increase in breast cancer risk can be prevented with drugs that reverse epigenetic modifications. Our preliminary studies in mice suggest that this is achievable in daughters by using the well-tolerated and non-toxic histone deacetylase inhibitor valproic acid and DNMT inhibitor hydralazine. However, whether these compounds also prevent an increase in granddaughters and great granddaughters in experimental models remains to be investigated.

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

  • 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

Genetic Markers in DES Exposed Daughters

A Pilot Study, 2015

In May 2014, researchers at the National Cancer Institute and Boston University began a small pilot study that is attempting to understand how prenatal DES exposure affects a person’s biology and influences health conditions.

The researchers are asking women (both DES exposed and unexposed) in the greater Boston area to provide a small blood sample at Boston University’s General Clinical Research Center.

The samples will be used to compare levels of hormones between exposed and unexposed women and to find out if there are any DES-related epigenetic changes in cells. Epigenetic changes occur in the cells during fetal development. These changes can turn genes on or off. So far, 47 women have provided samples and we are truly grateful for their help. If this pilot study is successful we hope to study this question in a larger group of women.

The findings may have important implications for the ways in which hormonal exposures in the fetus influence human health in later life.

Pilot Study Completed, 2018

Researchers at the National Cancer Institute and Boston University are currently studying whether genetic markers and reproductive hormone concentrations differ between women who were and were not prenatally exposed to DES. In a small pilot study of 60 women (40 who were exposed to DES and 20 who were not) blood samples were drawn from women participating in our long-term DES Follow-up Study of the health effects of DES exposure.

The samples were sent to a laboratory for measurements.

The findings of this pilot study may have profound implications for the ways in which endocrine disruption (chemicals that interfere with normal hormone function) in the fetus influences human health in later life.

Sources

DES DIETHYLSTILBESTROL RESOURCES

Epigenetics and transgenerational effects of DES

EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals

Selected Abstracts

Prenatal exposure to DES caused hypermethylation of the Hoxa10 gene in the uterus of mice and was linked to uterine hyperplasia and neoplasia later in life. Beyond the effects of prenatal exposure to DES on the daughters exposed in utero are suggestions that this leads to transgenerational effects of the chemical on the reproductive system, although whether this is linked to DNA methylation changes in humans is unknown.

DES caused histone deacetylation in the promoter region of the cytochrome P450 side chain cleavage (P450scc) gene.

Neonatal DES exposure also caused the differential expression of 900 genes in one or both layers of the uterus. Specifically, DES altered multiple factors in the PPARγ pathway that regulate adipogenesis and lipid metabolism, and it perturbed glucose homeostasis, suggesting that DES affects energy metabolism in the uterus. In the mouse uterus, DES altered the expression of chromatin-modifying proteins and Wnt signaling pathway members, caused epigenetic changes in the sine oculis homeobox 1 gene, and decreased the expression of angiogenic factors. DES also altered the expression of genes commonly involved in metabolism or endometrial cancer in mice, and it activated nongenomic signaling in uterine myometrial cells and increased the incidence of cystic glands in rats.

Studies in mice showed that DES induced vaginal adenosis by down-regulating RUNX1, which inhibits the BMP4/activin A-regulated vaginal cell fate decision; induced epithelial cell proliferation and inhibited stromal cell proliferation; and caused persistent down-regulation of basic-helix-loop-helix transcription factor expression (Hes1, Hey1, Heyl) in the vagina, leading to estrogen-independent epithelial cell proliferation. Neonatal exposure to DES caused persistent changes in expression of IGF-1 and its downstream signaling factors in mouse vaginas. It also up-regulated Wnt4, a factor correlated with the stratification of epithelial cells, in mouse vaginas. Interestingly, the simultaneous administration of vitamin D attenuated the ability of DES to cause hyperplasia of the vagina in neonatal mice.

In mice treated prenatally with DES there was a significant increase in enhancer of Zeste homolog 2 (EZH2) protein and EZH2 activity (measured by increased mammary histone H3 trimethylation)—a histone methyltransferase that may be linked to breast cancer risk and epigenetic regulation of tumorigenesis, as well as an increase in adult mammary gland EZH2.

EDC exposures to pregnant animals have been shown to cause multigenerational or transgenerational effects on a number of disease endpoints, particularly reproduction, neurobehavior, and adiposity. This work needs much more follow-up to better determine the underlying mechanisms, which are likely to include epigenetic molecular programming changes. Moreover, research is needed in human populations. Some work has been conducted in grandchildren of DES-exposed women who took this estrogenic pharmaceutical during pregnancy. The consequences on the offspring (F1 generation) are well-studied, and research is beginning to be published on the grandchildren (F2 generation). For environmental chemicals, several ongoing projects need continued funding.

Sources

  • Full study (free access) : EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals, NCBI PubMed PMC4702494, 2015 Nov 6.
  • Featured image credit Craig Whitehead.
DES DIETHYLSTILBESTROL RESOURCES

Alterations of Immune Function in the DES Exposed

Diethylstilbestrol Revisited: A Review of the Long-Term Health Effects

1995 Research Abstract

Alterations of Immune Function

Speculation that prenatal exposure to DES may lead to an altered immune status is based on observations that mice exposed perinatally to DES show reversible thymic and splenic atrophy, decreased T-helper cell subpopulations, and impaired natural killer-cell activity. Interestingly, two small case series have shown altered T-cell and natural killer-cell function in women exposed to DES in utero.

In reviewing data from the DESAD cohort, Noller and associates observed that reporting of a lifetime history of autoimmune diseases was increased among persons exposed to DES in utero compared with unexposed controls. Overall, the rate of any autoimmune disease was 28.6 cases per 1000 persons among the exposed daughters and 16.9 cases per 1000 persons among the unexposed women, resulting in a relative prevalence rate of 1.8 cases per 1000 persons (CI, 0.99 to 3.1). Although no individual autoimmune disease was significantly associated with exposure, Hashimoto thyroiditis was reported by 10 of the 1711 women exposed to DES compared with 1 of 922 controls. No other clinical manifestations of immune dysfunction have been established. On the basis of the results of animal studies, however, Blair has hypothesized that immune dysfunction in exposed offspring may only become clinically manifest with aging.

Sources

DES DIETHYLSTILBESTROL RESOURCES

Reductions in fertility in DES sons and daughters

Diethylstilbestrol Revisited: A Review of the Long-Term Health Effects

1995 Research Abstract

DES Sons

Some investigators have reported abnormalities of the urogenital system in DES sons, whereas others have found no increase in such abnormalities compared with men who were not exposed to DES. Gill and coworkers examined 308 men exposed to DES and 307 men receiving placebo who were traced from Dieckmann and colleagues’ cohort and found that the prevalence of epididymal cysts and hypotrophic testes was four times greater among exposed men. In men with testicular hypoplasia, cryptorchidism was observed in 65% (17 of 26) of men exposed to DES compared with 17% (1 of 6) of controls. No significant differences were found in mean circulating follicle-stimulating hormone, luteinizing hormone, or testosterone levels in the two groups. Spermatozoa were analyzed in 134 men (44%) exposed to DES and in 84 men (27%) who received placebo. The average sperm density of the group exposed to DES was lower than that of the placebo group (91 sperm cells X 106 /mL compared with 115 sperm cells X 106 /mL; P = 0.05). Semen quality was compared using the average Eliasson score, a scoring system that assesses sperm concentration, percentage of motile sperm, motility, and morphology. A score of 1 is classified as normal; a score of 5 to 10 is classified as pathologic; and a score of greater than 10 is classified as severely pathologic. The average Eliasson score was higher in the group exposed to DES than in the group exposed to placebo (4.9 compared with 2.5; P = 0.01); more men exposed to DES than controls had severe semen pathologic disorders (an Eliasson score > 10) (24 of 134 men exposed to DES [18%] compared with 7 of 87 controls [8%]; P = 0.05). In contrast, a study done by the Mayo Clinic found no significant differences between men who were and were not exposed to DES in the proportion of testicular or penile anomalies, sperm density or Eliasson score, or the number of pregnancies attained by their wives. These conflicting results may be related to differences in the maternal DES dose levels, heterogeneous hormone (non-DES) exposures, or different methods of recruiting study participants in the reported studies.

DES Daughters

Developmental abnormalities in the female reproductive tract frequently occur after DES exposure. Among DESAD participants at the Baylor College of Medicine who were exposed to DES, 50 of 282 (18%) were found to have gross anatomical changes of the cervix (absent pars vaginalis, coxcomb, hypoplastic cervix collar, or pseudopolyp). Among a subgroup of DESAD participants recruited for a fertility study, 154 of 293 (53%) were found to have abnormal hysterosalpingograms. These abnormalities included T-shaped and hypoplastic uteri; constriction of the upper, middle, or cornual regions; and irregular uterine margins. Data from the Dieckmann and colleagues’ cohort have consistently shown reductions in fertility in DES daughters. On the basis of data analyzed until 1986, 33% of the exposed women compared with 14% of the unexposed women reported primary infertility. Secondary infertility was also reported significantly more often among the exposed women. Vaginal epithelial changes and cervicovaginal ridges were found more often among the exposed women with primary infertility. In contrast, an early analysis of data from the DESAD cohort found that exposed and unexposed daughters were similar in the number achieving pregnancy, the total number of pregnancies, and age at first pregnancy. However, these women may have been studied too early in their reproductive life span to detect major differences in fertility. Kaufman and associates found no difference in the proportion of women with normal and abnormal hysterosalpingograms who had difficulty with conception, suggesting that structural abnormalities of the uterus alone did not account for failure to conceive. Some clinical studies and case reports have suggested that hormonal changes in DES daughters occur, including elevated testosterone and prolactin levels. However, a prospective cohort study suggested that although in utero DES exposure was related to a reduction in the duration and amount of menstrual bleeding, exposure did not affect cycle length and variability of cycle length. This suggests that gross endocrine function was not disturbed. Failure of implantation and alterations in ovarian steroidogenesis have also been postulated as possible causes of infertility in these women. Once pregnancy is achieved, DES daughters are at high risk for an unfavorable pregnancy outcome. In a review of English-language articles, Swan estimated that, overall, DES daughters are 8.6 times more likely to have an ectopic pregnancy, 1.8 times more likely to have a miscarriage, and 4.7 times more likely to have a premature birth than unexposed women. Among women with an abnormality of the cervix, vagina, or uterus, the relative risks for ectopic pregnancy, miscarriage, and premature birth are even higher (13.5, 2.6, and 9.6, respectively).

Sources

DES DIETHYLSTILBESTROL RESOURCES

Fetal and early postnatal environmental exposures and reproductive health effects in the female

image of fetal exposure

DES exposure induces changes in the expression of many genes such as Wnt7a, Hoxa9, Hoxa10, Hoxa11, lactoferrin and c-fos genes

2008 Study Abstracts

Current studies are investigating the possibility that neonatal estrogen exposure may alter activin signaling in the ovary, thereby leading to ovarian pathologies. We have examined the effect of neonatal DES and E2 exposure on the mRNA and protein levels of the key factors involved in activin signaling in the mouse ovary. Preliminary results demonstrate that neonatal estrogen exposure decreases activin subunit gene expression and impacts activin signaling, indicating that activin genes are targets of estrogen action in the mouse ovary. Future studies will further characterize the mechanisms underlying the effects of premature estrogen and activin exposure on adult ovarian and follicular function.

From the 1940s to 1970s, the xenoestogen DES was extensively prescribed to pregnant women at risk for miscarriage. 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. Since DES is readily metabolized and cleared within days after exposure, the persistent alterations resulting from developmental DES exposure cannot simply be explained by residual body burden of the compound. DES exposure also induces changes in the expression of several uterine genes involved in tissue patterning, such as Wnt7a, Hoxa9, Hoxa10 and Hoxa11, contributing to changes in tissue architecture and morphology. DES-induced developmental programming appears to require the estrogen receptor α (ERα), suggesting that signaling through this receptor is crucial for establishing developmental programming. These initial observations with DES firmly established the developmental period as a window of susceptibility during which an inappropriate xenoestrogen exposure can induce developmental programming and increase risk for diseases, including cancer, later in life.

We have recently demonstrated that developmental programming can increase the risk for developing uterine leiomyoma in adulthood the adult. Utilizing rats carrying a germline defect in the tuberous sclerosis complex 2 (Tsc-2) tumor suppressor gene that are predisposed to uterine leiomyomas, we found that an early life exposure to DES during development of the uterus increased risk for uterine leiomyoma from 65% to greater than 90% and increased tumor multiplicity and size in genetically predisposed animals, but failed to induce tumors in wild-type rats. Importantly, we found that DES exposure imparted a hormonal imprint on the developing uterine myometrium in both wild-type and carrier rats, causing an increase in expression of estrogen-responsive genes prior to the onset of tumors. Thus, when developmental programming of estrogen-responsive genes was combined with the presence of the Tsc-2 tumor suppressor gene defect, the result was an increased risk of developing hormone-dependent leiomyoma in adult animals. These data suggest that exposure to environmental factors during development can permanently reprogram normal physiological tissue responses and thus lead to increased tumor suppressor gene penetrance in genetically susceptible individuals. Developmental programming occurred as a result of the hormonal imprint imparted on the developing uterus by the brief early life exposure to DES.

Sources

  • Full study (free access) : Fetal and Early Postnatal Environmental Exposures and Reproductive Health Effects in the Female, Fertility and Sterility, NCBI PubMed PMC2527475, 2008 Feb.
  • Featured image Mel Elías.
DES DIETHYLSTILBESTROL RESOURCES

Developmental reprogramming of cancer susceptibility

image of cancer susceptibility

2012 Study Abstracts

Gene–environment interactions have been traditionally understood to promote the acquisition of mutations that drive multistage carcinogenesis, and, in the case of inherited defects in tumour suppressor genes, additional mutations are required for cancer development. However, the developmental origins of health and disease (DOHAD) hypothesis provides an alternative model whereby environmental exposures during development increase susceptibility to cancer in adulthood, not by inducing genetic mutations, but by reprogramming the epigenome. We hypothesize that this epigenetic reprogramming functions as a new type of gene–environment interaction by which environmental exposures target the epigenome to increase cancer susceptibility.

The developmental origins of health and disease (DOHAD) hypothesis

The developmental origins of health and disease (DOHAD) hypothesis posits that increased risk of disease in adulthood can result from an adverse developmental environment that reprogrammes cellular and tissue responses to normal physiological signals in a manner that increases disease susceptibility.

An example is altered DNA methylation that is observed in the uterus in response to in utero exposure to the xenoestrogen diethylstilbestrol (DES). The adult uterus of an animal exposed to this xenoestrogen in utero exhibits alterations in the methylation of the lactotransferrin (Ltf) gene after puberty. If ovariectomized, the aberrant DNA methylation that is observed in intact animals is not observed in the castrated female uterus. Similarly, early life exposure to DES or to the phytoestrogen genistein can reprogramme high-mobility group nucleosome-binding domain 5 (Hmgn5; also known as Nsbp1) gene methylation, causing the promoter of this gene to become hypomethylated in the adult uterus, which increases gene expression.

Mechanisms of epigenetic reprogramming

If developmental exposures are reprogramming the epigenome to increase susceptibility to diseases such as cancer, the effects of this reprogramming should be apparent in the ‘normal’ tissue prior to the development of disease. Ample evidence exists that developmental reprogramming induces epigenetic changes that can be detected in at-risk tissues prior to the development of tumours. In the reproductive tract, examples of genes in which developmental reprogramming induces persistent alterations in DNA methylation include FOS, LTF , homeobox A10 (HOXA10), HMGN5 and phosphodiesterase 4D variant 4 (PDE4D4). Early studies demonstrated that neonatal DES exposure induced aberrant methylation of specific CpG sites in Ltf and Fos in the mouse uterus. More recently, developmental exposure to several endocrine-disrupting compounds (EDCs), including xenoestrogens, was found to modulate the expression and promoter methylation of HOX genes, which have key roles in uterine development. Exposure to DES in utero results in increased promoter methylation and decreased HOXA10 expression, whereas bisphenol A (BPA) exposure results in decreased methylation of HOXA10, increased oestrogen receptor (ER) binding by HOXA10 and increased HOXA10 expression in the adult uterus.

Similarly, neonatal exposure of mice to DES or genistein also induces persistent hypomethylation of the Hmgn5 promoter and aberrant overexpression of this gene in the uterus throughout life, and exposure is associated with an increased risk of developing uterine tumours in adult female mice.

Early life environmental exposures and reproductive tract cancer

The xenoestrogen DES, a synthetic stilbene oestrogen, was administered to pregnant women from the 1940s to the 1970s to prevent complications of pregnancy. In the early 1970s, the daughters of women who took DES during the first trimester (so-called ‘DES daughters’) were frequently diagnosed with congenital reproductive tract abnormalities (specifically, a ‘T-shaped’ uterus), dysplasia and cervical intraepithelial neoplasia, and with a significantly increased rate of an otherwise rare type of vaginal clear cell adenocarcinoma. Continued follow-up of DES daughters has now revealed that they also have an increased relative risk for developing breast cancer (~twofold to threefold that of unexposed women) and uterine leiomyoma, although not all data on this point are concordant. DES sons may also be at a higher risk of developing prostate cancer; however, the final verdict on this point requires additional investigation.

Animal studies that simulate the human DES experience show that exposure of the developing reproductive tract to DES and other xenoestrogens imparts a permanent oestrogen imprint that alters reproductive tract morphology, induces persistent expression of oestrogen-responsive genes and induces a high incidence of uterine adenocarcinoma. Uterine leiomyoma, sometimes referred to as fibroids, arise from the smooth muscle layer of the uterus and are the most common benign tumour in women. In rats carrying a genetic defect in the tuberous sclerosis 2 (Tsc2) tumour suppressor gene, exposure to DES during uterine development causes the tumour suppressor defect to become fully penetrant, increasing tumour incidence from 65% to 100% (discussed below). This increased penetrance is associated with the reprogramming of oestrogen-responsive genes, which become hyper-responsive to hormone and promote the development of hormone-dependent uterine leiomyomas. DES exposure during this crucial perinatal period of development also modulates IGF signalling in the adult endometrium, decreasing negative feedback to insulin receptor substrate 1 (IRS1) and increasing the incidence of endometrial hyperplasia in rats that are genetically predisposed to develop these preneoplastic lesions.

Inappropriate exposure to environmental oestrogens, such as DES, the plasticizor BPA or the soy phytoestrogen genistein, during mammary gland development alters the susceptibility of the mammary gland to chemical carcinogenesis in adulthood. The mammary gland begins developing in utero and is not fully mature until after pregnancy and lactation. Importantly, the effect of xenoestrogen exposure during mammary gland development differs throughout life. Xenoestrogen exposure in utero can increase the risk for mammary tumorigenesis, in part by altering mammary gland architecture and increasing the number of target cells for transformation in terminal end buds (TEBs) of the mammary gland. Conversely, xenoestrogen exposure during the postnatal period can decrease susceptibility to mammary gland tumorigenesis by inducing a programme of differentiation in mammary epithelial cells that mimics the protective effects of pregnancy. In addition to xenoestrogen exposure, there is a suggestion that prenatal famine may increase breast cancer incidence in exposed individuals.

Testicular cancer, one of the features of testicular dysgenesis syndrome (TDS), which includes poor semen quality, undescended testis and hypospadias, has also been linked to early life environmental exposures. Experimental studies from animal models and human epidemiology support a causal association between TDS and exposure to compounds that disrupt the endocrine system, such as DES, and anti-androgens, such as vinclozolin, during male reproductive tract development. In humans, the majority of testicular cancers are derived from germ cells, and epidemiology data suggest in utero DES exposure may be a risk factor. Testicular cancer is preceded by carcinoma in situ, and recent studies suggest that these lesions have a gene expression pattern that is similar to gonocytes (spermatogonia) and exhibit DNA hypomethylation and altered methylation of histones H3K9, H3K27 and H3K4, which is analogous to the epigenetic pattern that is seen in fetal germ cells. These data have led to the suggestion that germ cells in the developing testis are the target for the developmental reprogramming that is associated with TDS.

The maternal environment might also play a part in susceptibility to testicular cancer. Low birth weight is a risk factor for testicular germ-cell cancer (which comprises more than 95% of testicular cancer), and a recent meta-analysis of 18 epidemiological studies indicated that low birth weight is also a risk factor for testicular cancer. Although germ-cell testicular cancer is very rare in experimental animal models, by contrast, interstitial cancers, benign tumours and adenocarcinoma of the rete testis are frequently observed in rodent testes that have been prenatally exposed to DES. In the prostate, early life exposure of rodents to xenoestrogens such as methoxychlor, BPA or genistein leads to prostate hypertrophy and increased inflammation with age in the prostate of adult animals and can also predispose to prostate carcinogenesis.

A new gene–environment interaction?

Studies in Eker rats with a defect in Tsc2 first pointed to the fact that exposure to environmental oestrogens during development could cooperate with a tumour suppressor gene defect to increase the penetrance of the defective tumour suppressor gene. In this model, brief neonatal exposure to environmental oestrogens, such as DES, during uterine development significantly increased tumour incidence (that is, penetrance), as well as tumour multiplicity and size. Neonatal xenoestrogen exposure also resulted in the reprogramming of oestrogen-responsive gene expression, which manifested as an increased expression of oestrogen-responsive genes in the adult myometrium at 5 months of age, many months prior to tumour development. This suggests that the combined increase in oestrogen responsiveness (reprogramming) and the defect in Tsc2 promoted the development of hormone-dependent leiomyoma, effectively increasing tumour suppressor gene penetrance. Importantly, in the absence of the tumour suppressor gene defect, environmental oestrogen exposure alone failed to induce tumours, even though gene expression was reprogrammed (K. L. Greathouse and C.L.W., unpublished observations). Conversely, ovariectomy almost completely ablated tumour development in genetically predisposed animals, indicating that, in the absence of ovarian hormones, the tumour suppressor gene defect was not sufficient to induce tumorigenesis. Thus, developmental reprogramming can cooperate with a tumour suppressor gene defect to increase its penetrance, thereby functioning as a new type of gene–environment interaction.

As a result of this developmental reprogramming, oestrogen-responsive genes in the uterus become hyper-sensitive to hormone, which promotes tumour development in adult animals that are neonatally exposed to environmental oestrogens. In Eker rats that are neonatally exposed to DES, more than 50% of the oestrogen-responsive genes that have been examined in the adult uterus displayed an inappropriate, exaggerated response to steroid hormones: reprogrammed genes were overexpressed during the oestrus cycle when oestrogen levels were high, and remained elevated even during periods of the oestrous cycle when hormones were at their lowest. As a result, the uterus displayed an ‘oestrogenized phenotype’ that cooperated with the tumour suppressor gene defect to increase tumour suppressor gene penetrance. Together, these data lead us to propose that developmental reprogramming is a type of gene–environment interaction that can cooperate with a genetic predisposition, not by inducing mutations, but by reprogramming the epigenome to modulate gene expression in order to promote tumour development.

Sources

  • Full study (free access) : Developmental reprogramming of cancer susceptibility, Nat Rev Cancer, NCBI PubMed, PMC3820510, 2012 Jun 14.
  • Featured image Ken Treloar.
DES DIETHYLSTILBESTROL RESOURCES

Epigenetics, Evolution, Endocrine Disruption, Health, and Disease

image of Epigenetics, Evolution, Endocrine Disruption, Health, and Disease

A possible mechanism for how DES exerts its action epigenetically has been proposed recently

2006 Study Abstracts

Endocrine-disrupting chemicals (EDCs) in the environment have been linked to human health and disease. This is particularly evident in compounds that mimic the effects of estrogens. Exposure to EDCs early in life can increase risk levels of compromised physical and mental health. Epigenetic mechanisms have been implicated in this process. Transgenerational consequences of EDC exposure is also discussed in both a proximate (mechanism) and ultimate (evolution) context as well as recent work suggesting how such transmission might become incorporated into the genome and subject to selection. We suggest a perspective for exploring and ultimately coming to understand diseases that may have environmental or endocrine origins.

That epigenetic mechanisms may play a role in endocrine disruption helps explain the transgenerational effects of some hormonally active chemicals. Treatment with diethylstilbestrol (DES) during pregnancy results in vaginal adenocarcinoma in female offspring in humans and mice. Female offspring of mice exposed to DES during pregnancy, when mated to control males, produce a second generation of females who, although not exposed to DES themselves, express this same rare genital tract cancer. This transgenerational transmission of a specific reproductive tract lesion would be hard to explain without invoking an epigenetic mechanism for heritable change and, given the finding of altered DNA methylation patterns in a specific uterine gene in mice treated developmentally with DES, we think a strong case can be made for such a conclusion. Newbold and colleagues next showed that specific, rare genital tract cancers (rete testes cancers) are also expressed and therefore transmitted to the male offspring of females treated in utero with DES. In colloquial terms, this demonstrated the occurrence of reproductive tract tumors in the grandsons and granddaughters of mothers treated with DES. A possible mechanism for how DES exerts its action epigenetically has been proposed recently. The transmission of uniquely specific changes in the program of development in mice has implications for similarly exposed humans as well as the biology of hormonally induced disease.

We have already mentioned that DES treatment of mice during development results in reproductive tract cancers, persistent up-regulation of key estrogen-responsive genes, and altered patterns of gene methylation in the affected genes and that the cancer can be transmitted through two generations. In addition to DES, methoxychlor has been reported to increase global DNA methylation in uterine ribosomal DNA after in utero exposure; the alteration in methylation remains months after treatment.

To understand the role of estrogens in development, there is hardly a more powerful model than that of the outcomes observed in humans and mice developmentally exposed to the synthetic estrogen DES. Female offspring of humans or mice exposed prenatally to DES have a risk for vaginal clear cell adenocarcinoma. The mechanisms underlying these developmentally induced lesions have been sought for three decades. There was the suggestion by clinical investigators that DES had altered the normal differentiation of the epithelial cells of the fetal cervix and vagina such that they responded abnormally to estrogen at puberty, because no cancers had been seen in prepubescent girls. Similarly, ovariectomy of developmentally DES-treated mice prevented the subsequent expression of uterine adenocarcinomas.

Epigenetic change in the molecular program of cell differentiation in the affected tissues may be a common mechanism. The clear cell cancers of the vagina in DES-exposed women displayed genetic instability consistent with epigenetic imprints in the absence of any expected mutation in classical oncogenes or tumor suppressor genes. Using a well validated mouse model for DES genital tract tumors, Li and colleagues discovered that one of the estrogen-inducible genes in the mouse uterus, lactotransferrin, that had been shown earlier to be persistently up-regulated by developmental DES exposure, had an altered pattern of CpG methylation in the promoter region of the gene upstream from the estrogen response element. Subsequent work demonstrated that other developmentally up-regulated genes such as fos and jun also had persistent changes in the pattern of methylation of the gene after DES exposure during development. These experiments raise the possibility that DES (and other environmental estrogens) alter the program of differentiation of estrogen target cells in the reproductive tract through an epigenetic mechanism.

Other studies support this hypothesis. In addition to cervicovaginal adenocarcinomas in female mice and humans exposed prenatally to DES and uterine adenocarcinoma in mice, it has been shown that developmental exposure to DES results in excess risk of uterine leiomyomas (fibroids) in mice, rats, and women. It was also recently reported that sea lions in areas contaminated with EDCs have a higher prevalence of uterine fibroids. The Eker rat carries a germ-line mutation in a tumor suppressor gene and is predisposed to uterine leiomyoma. Cook and colleagues used this model system to demonstrate a DES-induced alteration in developmental imprinting as analyzed by tumor suppressor gene penetrance, concluding that developmental programming by estrogen works in concert with preexisting genetic change. In a population of 819 black and 504 white women, fibroid status was determined by ultrasound screening or surgical record review, whereas prenatal DES exposure was determined by interview. DES-exposed women had a significantly greater risk for uterine fibroids and tended to have larger tumors. The authors conclude that their study, as well as animal studies, indicate a role for prenatal estrogen in the etiology of uterine leiomyoma in women.

Sources

  • Full study (free access) : Epigenetics, Evolution, Endocrine Disruption, Health, and Disease, Endocrinology, Volume 147, Issue 6, Pages s4–s10, doi.org/10.1210/en.2005-1122, June 2006.
  • Featured image academic.oup.
DES DIETHYLSTILBESTROL RESOURCES

Environmental Epigenetics and Its Implication on Disease Risk and Health Outcomes

image of Environmental Epigenetics

2012 Study Abstracts

Introduction

This review focuses on how environmental factors through epigenetics modify disease risk and health outcomes. Major epigenetic events, such as histone modifications, DNA methylation, and microRNA expression, are described. The function of dose, duration, composition, and window of exposure in remodeling the individual’s epigenetic terrain and disease susceptibility are addressed. The ideas of lifelong editing of early-life epigenetic memories, transgenerational effects through germline transmission, and the potential role of hydroxylmethylation of cytosine in developmental reprogramming are discussed. Finally, the epigenetic effects of several major classes of environmental factors are reviewed in the context of pathogenesis of disease. These include endocrine disruptors, tobacco smoke, polycyclic aromatic hydrocarbons, infectious pathogens, particulate matter, diesel exhaust particles, dust mites, fungi, heavy metals, and other indoor and outdoor pollutants. We conclude that the summation of epigenetic modifications induced by multiple environmental exposures, accumulated over time, represented as broad or narrow, acute or chronic, developmental or lifelong, may provide a more precise assessment of risk and consequences. Future investigations may focus on their use as readouts or biomarkers of the totality of past exposure for the prediction of future disease risk and the prescription of effective countermeasures.

Implications of Lifelong Editing of Early-Life Epigenetic Memories

The concept of continued editing of early-life epigenetic markings or memories during adult life has been proposed on the basis of evidence from limited experimental studies. Exposure of mice to diethylstilbestrol (DES, a xenoestrogen) or genistein (a phytoestrogen) during the perinatal period induced specific epigenetic markings in their uteri. However, some of these epigenetic markings (hypomethylation of Nsbp1) remained “hidden” during prepuberty life and appeared in adulthood only in the exposed intact females but not in their ovariectomized counterparts, suggesting that adult exposure to ovarian steroids may cause these markings to “surface.” Coincidentally, the prevalence of uterine cancer was higher in neonatally exposed intact mice, but not in mice ovariectormized before puberty.

Epigenetic Factors Shown to Trigger Epigenetic Events and Affect Disease States

Exposure to EDCs during early developmental periods is a major health concern because it can cause persistent changes in gene expression through epigenetic reprogramming in somatic cells, as well as germ-line cells, and subsequently promote transgenerational inheritance. The xenoestrogen DES was widely used in cattle and other livestock industries and is still an EDC in many populations. Early-life exposure of mice to DES increases risk of uterine cancer that is accompanied by demethylation of an estrogen-responsive gene, lactoferrin, in the mouse uterus. In utero exposure of mice to DES triggered hypermethylation of the homeobox A10 with attended uterine hyperplasia and neoplasia in later life. A more recent report documented hypermethylation of nucleosome binding protein 1 (Nsbp1 or Hmgn5) as a hidden uterine epigenetic mark after neonatal DES exposure that only appeared upon sexual maturation of the exposed mice but failed to manifest if the animals were ovarietomized before puberty. Of significant interest is the transgenerational effect of developmental exposure of mice to DES that promoted c-fos expression, hypomethylation of specific exon CpGs, and increased susceptibility to tumorigenesis in the F2 generation. These experimental data support the hypothesis that epigenetic reprogramming is responsible for the devastating consequences observed in the offspring of women who took DES during pregnancy. The DES effects include female genital abnormalities, vaginal cancer, and male urogenital disorders. The adverse effects may be reverberating in the grandchildren of these women.

Sources

  • Full study (free access) : Environmental Epigenetics and Its Implication on Disease Risk and Health Outcomes, ILAR Journal, NCBI PubMed PMC4021822, 2012 Dec.
  • Featured image by h heyerlein.
DES DIETHYLSTILBESTROL RESOURCES