A model for the trans-generational effects of diethylstibesterol on uterine development and cancer
2005 Study Abstract
Hsp90 is a chaperone for over 100 ‘client proteins’ in the cell, most of which are involved in signaling pathways. For example, Hsp90 maintains several nuclear hormone receptors, such as the estrogen receptor (ER), as agonist-receptive monomers in the cytoplasm.
In the presence of agonist, Hsp90 dissociates and the receptors dimerize, enter the nucleus and ultimately activate transcription of the target genes. Increasing evidence suggests that Hsp90 also has a role in modifying the chromatin conformation of many genes. For example, Hsp90 has recently been shown to increase the activity of the histone H3 lysine-4 methyltransferase SMYD3, which activates the chromatin of target genes. Further evidence for chromatin-remodeling functions is that Hsp90 acts as a capacitor for morphological evolution by masking epigenetic variation. Release of the capacitor function of Hsp90, such as by environmental stress or by drugs that inhibit the ATP-binding activity of Hsp90, exposes previously hidden morphological phenotypes in the next generation and for several generations thereafter.
The chromatin-modifying phenotypes of Hsp90 have striking similarities to the trans-generational effects of the ER agonist diethylstilbesterol (DES). Prenatal and perinatal exposure to DES increases the predisposition to uterine developmental abnormalities and cancer in the daughters and granddaughters of exposed pregnant mice.
In this review, we propose that trans-generational epigenetic phenomena involving Hsp90 and DES are related and that chromatin-mediated WNT signaling modifications are required. This model suggests that inhibitors of Hsp90, WNT signaling and chromatin-remodeling enzymes might function as anticancer agents by interfering with epigenetic reprogramming and canalization in cancer stem cells.
Sources and more information
Hsp90 and environmental impacts on epigenetic states: a model for the trans-generational effects of diethylstibesterol on uterine development and cancer, Human molecular genetics, NCBI PubMed PMID: 15809267, 2005 Apr.
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.
Proposed model to explain an increase in breast cancer risk in daughters, and possibly granddaughters and great granddaughters, of mothers who took diethylstilbestrol during pregnancy
Gene expression can be altered as a consequence of mutations or epigenetic changes. In contrast to gene mutations within the DNA, epigenetic changes involve post-transcriptional modifications; that is, methylation of gene promoter regions, histone modifications, deposition of certain histone variants along specific gene sequences and microRNA (miRNA) expression. Although both changes are heritable, an important distinction between the two is that mutations are not reversible, but epigenetic modifications generally are.
Probably the most common mechanism of epigenetic gene silencing is methylation, and it might also be the most important. DNA methyltransferases (DNMTs) catalyze the methylation of genomic DNA by adding a methyl group (CH3) onto the 5-carbon of the cytosine ring within CpG dinucleotides. Histone modifications are complex, as they involve not just histone methylation but also acetylation, deacetylation and other post-translational changes. These modifications occur in the amino-terminal tails of histones and affect the ‘openness’ of the chromatin, which determines whether a gene is expressed or silenced (for example, acetylation allows transcription, while deacetylation represses transcription). Trimethylation of histone H3 at lysine K27 is catalyzed by the Polycomb group (PcG) protein enhancer of Zeste-2 (EZH2) and results in gene silencing. PcG/H3K27me3 interact with DNMTs, and together they establish and maintain silencing of PcG target genes. Over 2,000 different PcG target genes have been identified and they include some tumor suppressor genes. Many of the PcG target genes regulate cell fate, including apoptosis, proliferation and stem cell differentiation. As discussed in more detail below, methylation of PcG target genes is linked to increased breast cancer risk.
DNMTs may be key players in regulating histones and the entire epigenomic machinery, since DNA methylation events often precede histone modifications. Upregulation of DNMTs increases the expression of EZH2 and other polycombs; this may happen by DNMTs inducing methylation of non-coding miRNAs that target the polycombs.
We and others have observed that the expression of DNMTs is persistently altered in estrogen-regulated tissues following estrogenic exposures during early life. In utero exposure to DES is reported to increase the expression of DNMT1 in the epididymis and uterus . We found that DNMT1 expression is increased in the mammary glands of adult rat offspring of dams exposed to ethinyl estradiol during pregnancy. These changes provide a key regulatory layer to influence gene expression in the mammary gland and perhaps breast tumors of individuals exposed to DES or other estrogenic compounds in utero.
In utero DES exposure alters methylation patterns of several genes in estrogen’s target tissues, including Hox genes, c-fox, and Nsbp1, but it has not been studied whether changes in methylation patterns occur in the mammary gland. We have explored changes in methylation in the mammary glands of adult rats exposed in utero to the synthetic estrogen ethinyl estradiol using global sequencing approaches. Among the genes that exhibited increased promoter methylation were several PcG target genes, suggesting that a maternal exposure to synthetic estrogens during pregnancy causes long-lasting changes in the methylation of genes that regulate cell fate, including stem cell differentiation.
As an increase in EZH2 expression in the mammary glands of mice exposed to DES in utero has been reported, histone modifications also seem to be influenced by maternal exposure to synthetic estrogens during pregnancy. Jefferson and colleagues recently investigated whether upregulation of lactoferrin and sine oculis homeobox 1 (Six1) in the uterus of adult mice exposed to DES neonatally is caused by histone modifications. Their data indicate that neonatal DES exposure induces changes during the early postnatal period in the expression of multiple chromatin-modifying proteins but these changes do not last to adulthood. However, alterations in epigenetic marks at the Six1 locus in the uterus were persistent. Similarly, changes in the methylation of Nsbp1 and expression of DNMTs in the uterus of DES-exposed offspring are different in the early postnatal period compared to adulthood. This suggests that some epigenetic alterations are further influenced by factors operating during postnatal development, such as a surge of estrogens and progesterone from the ovaries at puberty onset.
Maternal exposures during pregnancy have been found to induce persistent changes in miRNA expression in the offspring. miRNAs are short non-coding single-stranded RNAs composed of approximately 21 to 22 nucleotides that regulate gene expression by sequence-specific base-pairing with the 3’ untranslated region of target mRNAs. miRNA binding induces post-transcriptional repression of target genes, either by inducing inhibition of protein translation or by inducing mRNA degradation. Expression of many miRNAs is suppressed by estrogens. Although the effects of maternal DES exposure during pregnancy on miRNA expression in the offspring have not been investigated, it is known that many other manipulations, such as maternal low protein diet, alter miRNA patterns among the offspring. We recently found that in utero exposure to ethinyl estradiol lowers the expression of many of the same miRNAs in the adult mammary gland as are downregulated by E2 in MCF-7 human breast cancer cells. Since miRNAs can be silenced by methylation or as a result of increased PcG expression, and they target DNMTs, histone deacetylases and polycomb genes, the observed increase in DNMT expression, histone marks and EZH2 in the in utero DES-exposed offspring may be a result of epigenetic silencing of miRNAs that target them.
2014 Study Conclusions
In summary, women exposed to DES in utero are destined to be at an increased risk of developing breast cancer, and this risk may extend to their daughters and granddaughters as well. It is of critical importance to determine if the increased risk is driven by epigenetic alterations in genes that increase susceptibility to breast cancer and if these alterations are reversible.
Sources and more information
Full text (free access) : Maternal exposure to diethylstilbestrol during pregnancy and increased breast cancer risk in daughters, Breast Cancer Research, NCBI PubMed, PMC4053091, 2014 Apr 30.
Proposed model to explain an increase in breast cancer risk in daughters, and possibly granddaughters and great granddaughters, of mothers who took diethylstilbestrol during pregnancy featured image credit PMC4053091/figure/F1.
Persistent hypomethylation in the promoter of nucleosomal binding protein 1 (Nsbp1) correlates with overexpression of Nsbp1 in mouse uteri neonatally exposed to diethylstilbestrol or genistein
2008 Study Abstract
The concept of developmental programming by steroid hormones has been around for decades, and may be best understood in the brain, where early-life estrogens and androgens feminize and masculinize behavior. Other end organs, such as the liver, are also programmed by steroids during early development, leading to important and necessary sexual dimorphisms in structure and function. In addition to physiological programming or imprinting by steroids, it is well established that the reproductive tract, as well as other structures, can be reprogrammed by abnormal or inadvertent steroid exposures during early life. This phenomenon is best understood by extensive studies on the synthetic estrogen, diethylstilbestrol (DES). Beginning in the early 1950s, DES was prescribed to pregnant women to prevent spontaneous abortions, and its use has been estimated at 8 million women worldwide. However, DES utilization during pregnancy was discontinued in the United States in 1972 and worldwide by 1979 when it was discovered that maternal DES usage was linked to an increased incidence of vaginal clear-cell adenocarcinoma in DES-exposed daughters. Since that time, intrauterine DES exposure has resulted in a multitude of complications in female and male offspring, including T-shaped uteri, uterine fibroids, hypospadias, subfertility and infertility, early menopause, immune system disorders, psychosexual disturbances, and increased breast and testicular cancer risk. Although some adverse effects appear relatively early, such as vaginal cancers in young women, others are emerging as the exposed population ages, suggesting lifelong memory of early estrogen levels by cells and organs. In some instances these delayed responses may be triggered by secondary hormonal events such as onset of puberty or changing hormone levels with aging, which greatly complicates the picture. Furthermore, there is new evidence for transgenerational DES effects transmitted to DES granddaughters, the third generation, which indicates that adverse sequelae may be with us for some time.
Concern regarding fetal and neonatal exposure to estrogenic agents also continues today due to a variety of real-life circumstances that include unintended continuation of estradiol-containing birth control pills during the first months of an undetected pregnancy (estimated at > 1 million annually), exposure (sometimes daily) to environmental estrogens (e.g. bisphenol A, methoxychlor, dichlorodiphenyltrichloroethane or DDT), and consumption of high levels of phytoestrogens during pregnancy or in infant formula (e.g. soy with the isoflavone genistein), all of which have shown a variety of adverse effects in animal models . Although there is compelling evidence that soy or genistein ingestion can be chemoprotective and beneficial, as with so many things in life, this appears to depend on timing. Prepubertal genistein intake reduces breast cancer risk in humans and rodent models, whereas fetal exposure, at least in rat models, can increase risk if not accompanied by lifetime genistein intake. This serves to emphasize the subtle influences of adult life experiences on disease manifestation from early-life exposures. Together, these and a multitude of other studies indicate that early-life estrogen exposure fits the expanding paradigm of the developmental origins of human adult disease.
Over the years, researchers have conducted extensive studies on developmental reprogramming, also referred to as developmental estrogenization or estrogen “imprinting,” using rodent models that largely recapitulate, and in many instances predict, the adverse effects seen in DES-exposed humans. A major research effort during this time has been to identify the molecular underpinning whereby cells and tissues “remember” early-life estrogen exposures long after hormone withdrawal. In the late 1990s, McLachlan and colleagues provided the first clue that the process may have an epigenetic basis when they described a permanent change in uterine lactoferrin gene methylation as a function of fetal DES exposure. They went on to propose that developmental reprogramming by early-life estrogenic exposures may be mediated by altered epigenetic memory. As in lower species such as frogs in which early estrogen treatment changes gene methylation and alters the response of the vitellogenin gene to hormones later in life, they also speculated that the first hormonal experience may epigenetically alter the set point for the later hormone response in mammals. A few studies have since substantiated that epigenetic mechanisms underlie estrogen imprinting by demonstrating that early-life estrogens permanently alter DNA methylation and gene expression of specific genes in the uterus and prostate gland, which are associated with altered susceptibility to adult pathology in those tissues.
In the present issue of Endocrinology, Tang et al. identify additional layers of complexity to epigenetic reprogramming that have not been previously appreciated. Earlier work in the laboratory of Newbold et al. determined that developmental DES resulted in a high incidence of uterine adenocarcinoma in the murine model, which could be predictive of cancers not yet observed in DES daughters. In partnership with the laboratory of Newbold et al., Tang et al. used an unbiased approach of methylation sensitive restriction fingerprinting to identify altered DNA methylation patterns in prepubertal, adult, and aged mouse uteri that had been exposed during early life to high-dose or low-dose DES, or to genistein at doses consistent with human dietary intake. They identified 14 genes whose methylation patterns shift as a result of neonatal DES or genistein treatments, several of which were previously unrecognized participants in developmental estrogenization of this tissue. Thus, the present data further substantiate the original hypothesis and findings of McLachlan and colleagues. Tang et al. went on to thoroughly characterize the altered DNA methylation at specific CG sites in the promoter of one gene in particular, nucleosomal binding protein 1 (Nsbp1), and directly correlated the changes with altered gene expression. This gene is particularly significant because Nsbp1 is a nucleosome-core-particle binding protein that plays a role in chromatin remodeling, which itself underpins a higher order epigenetic process. Nsbp1 is structurally similar to the conserved functional domains of the high-mobility group proteins, HMG 14/17, that interact with nucleosomes, transiently destabilize chromatin, increase access to DNA, and enhance gene transcription at targeted sites. Thus, modifications in Nsbp1 methylation and expression by early estrogens have the potential to affect higher order chromatin structure that, in turn, may alter expression of many genes and possibly drive oncogenesis. Of course, this awaits confirmation by future experiments that directly address this possibility. Nonetheless, these novel findings provide important evidence that epigenetic reprogramming by early-life estrogens may involve multiple epigenetic pathways that combine to initiate disease later in life.
However, the real novelty and significant implications of the study by Tang et al. are not in the aforementioned results, as important as they may be, but in the demonstration that epigenetic reprogramming itself involves a two-step process. Original studies by Newbold et al. showed that the onset of uterine adenocarcinoma in neonatal DES-exposed mice required a secondary hormonal “push” by pubertal steroids because prepubertally ovariectomized mice did not develop cancers. The study by Tang et al. now shows that this is a direct function of specific DNA methylation changes induced by the secondary pubertal hormones that occur only in mice that were first exposed neonatally to DES or genistein. The authors found that the Nsbp1 promoter is moderately methylated, and the gene is expressed in uteri during prepubertal development in control animals, and becomes hypermethylated and largely silenced by early adulthood. This suggests that the role for Nsbp1 in the uterus is normally restricted to development. When mice were given DES or genistein immediately after birth, the Nsbp1 promoter became hyper or hypomethylated in a treatment-specific manner that led to decreased or increased Nsbp1 expression before puberty. This can be considered the first epigenetic modification. If the mice were prepubertally ovariectomized, these divergent methylation patterns persisted through aging. However, if the mice were allowed to undergo puberty with its secondary estradiol exposure, the Nsbp1 promoter was radically shifted to a hypomethylated state in all neonatal estrogen-exposed uteri. Importantly, this secondary epigenetic modification resulted in overexpression of Nsbp1 throughout life, which correlates with the onset of uterine adenocarcinoma in this model . Thus, in addition to overt epigenetic memories determining our fate, it appears that repressed epigenetic memories are also looming in the background, only to be epigenetically triggered by future events. These new findings stress the importance of secondary events in life that are necessary to uncover the initial cryptic modifications.
A “second hit” model has long been applied to genetic changes and the ontogeny of cancer . It now appears that a parallel two-step concept may apply to epigenetic alterations as well. A somewhat similar scenario was previously shown in Drosophila. Flies with a Krüppel gene mutation (Krif−1) exhibited a specific phenotypical outcome (eye outgrowth) only if they were “epigenetically sensitized” by chromatin modification. The authors concluded that an otherwise cryptic genetic variation can be modified epigenetically to unmask a predisposed phenotype. Ruden et al. went on to propose further that a similar mechanism may be responsible for the transmission of early-life DES effects on uterine development and cancer. The study by Tang et al. for the first time identifies molecular evidence to support this proposal. Moreover, the aforementioned hypothesis can now be expanded to state that cryptic epigenetic marks may be further epigenetically modified to unmask a predisposed phenotype. This begs the question whether the complexity of lifetime epigenetic interactions might be the basis for individual variability to early-life exposures or even to differential responsiveness to therapeutic interventions.
There are many things that remain to be done, not the least of which is connecting the dots between the epigenetically altered genes and the disease at hand. This is true not only for the uterus, but for other cancers (breast, testicular, prostate) and disease states that result from estrogen imprinting. Might there be other secondary triggers for repressed epigenetic memories aside from just hormones? Can we formulate an “epigenetic fingerprint” consisting of multiple genes that may be similar or unique for the separate estrogenic compounds, end organs, or second hits? If so, perhaps these could be used in the future for early detection of altered disease susceptibility as a result of known or unknown early-life exposures. Perhaps they could even be used to devise novel interventions. Clearly, we have only just begun.
Sources and more information
Estrogen Imprinting: When Your Epigenetic Memories Come Back to Haunt You, Endocrinology, Endocrine Society, Oxford University Press Volume 149, Issue 12, Pages 5919–5921, 1 December 2008.
Estrogen Imprinting featured image credit dennis43.
In utero exposure to diethylstilbestrol (DES) does not increase genomic instability in normal or neoplastic breast epithelium
2006 Study Abstract
In 1992, the National Cancer Institute (NCI) established the Continuation of Follow-Up of DES-Exposed Cohorts to study the long-term health effects of exposure to diethylstilbestrol (DES). Genetic effects on human breast tissue have not been examined. The authors investigated whether breast tissue of women exposed in utero to DES might exhibit the genetic abnormalities that characterize other DES-associated tumors.
Subjects enrolled in the NCI Cohort were queried about breast biopsies or breast cancer diagnoses. Available tissue blocks were obtained for invasive cancers (IC), in situ cancers (CIS), or atypical hyperplasia (AH). Exposure status was blinded, lesions were microdissected, and their DNA was analyzed for microsatellite instability (MI) and loss of heterozygosity (LOH), or allele imbalance (AI), at 20 markers on 9 chromosome arms.
From 31 subjects (22 exposed, 9 unexposed), 273 samples were analyzed (167 normal epithelium, 16 AH, 30 CIS, 60 IC). Exposed and unexposed subjects exhibited no differences in breast cancer risk factors or demographic characteristics, except for age at diagnosis (exposed vs. unexposed: 43.2 vs. 48.8 years of age, P = .02). The authors found that MI was rare and that AI was common, with frequencies consistent with previous reports. The global age-adjusted relative rate (RR) of AI was 1.3, 95% CI = 0.8-2.4. No statistically significant associations were observed after adjustment for risk factors or after stratification by histology or by chromosome arm.
In utero DES exposure does not appear to significantly increase genomic instability in breast epithelium, as measured by MI and AI. Breast tissue may respond differently from that of the reproductive tract to in utero DES exposure. Consequences of in utero DES exposure on the breast may be mediated by proliferative effects of estrogen.
Sources and more information
In utero exposure to diethylstilbestrol (DES) does not increase genomic instability in normal or neoplastic breast epithelium, Cancer, NCBI PubMed PMID: 16998936, 2006 Nov.
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:
Group 1 (n=180): firstborn children without DES treatment,
Group 2 (n=740): exposed children,
and Group 3 (n=262): children born after a previous pregnancy treated by DES and/or EE.
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):
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.
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?
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.
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.
Executive Summary to EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals, The Endocrine Society, dx.doi.org/10.1210/er.2015-1093, September 28, 2015. Full study: EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals, The Endocrine Society, dx.doi.org/10.1210/er.2015-1010, November 06, 2015.
Five years later, a substantially larger body of literature has solidified our understanding of plausible mechanisms underlying EDC actions and how exposures in animals and humans—especially during development—may lay the foundations for disease later in life. At this point in history, we have much stronger knowledge about how EDCs alter gene-environment interactions via physiological, cellular, molecular, and epigenetic changes, thereby producing effects in exposed individuals as well as their descendants. Causal links between exposure and manifestation of disease are substantiated by experimental animal models and are consistent with correlative epidemiological data in humans. There are several caveats because differences in how experimental animal work is conducted can lead to difficulties in drawing broad conclusions, and we must continue to be cautious about inferring causality in humans.
In this second Scientific Statement, we reviewed the literature on a subset of topics for which the translational evidence is strongest:
obesity and diabetes;
hormone-sensitive cancers in females;
and neurodevelopment and neuroendocrine systems.
Our inclusion criteria for studies were those conducted predominantly in the past 5 years deemed to be of high quality based on appropriate negative and positive control groups or populations, adequate sample size and experimental design, and mammalian animal studies with exposure levels in a range that was relevant to humans. We also focused on studies using the developmental origins of health and disease model. No report was excluded based on a positive or negative effect of the EDC exposure. The bulk of the results across the board strengthen the evidence for endocrine health-related actions of EDCs. Based on this much more complete understanding of the endocrine principles by which EDCs act, including nonmonotonic dose-responses, low-dose effects, and developmental vulnerability, these findings can be much better translated to human health.
Armed with this information, researchers, physicians, and other healthcare providers can guide regulators and policymakers as they make responsible decisions.
Discussion (DES and Fertility-specific)
Diethylstilbestrol and beyond: perhaps the best-studied endocrine-based example is in utero exposure to diethylstilbestrol (DES), a potent synthetic nonsteroidal estrogen taken by pregnant women from the 1940s to 1975 to prevent miscarriage and other complications. DES was prescribed at doses from less than 100 mg (in most cases) upward to 47 000 mg, with a median dose of 3650 to 4000 mg in the United States (IARC 2012). Most women received low doses (ie, 5 mg) and increased their intake (up to 125 mg) as symptoms or pregnancy progressed, translating to doses of about 100 μg/kg to 2 mg/kg DES per day. In 1953, a study proved DES was ineffective. Its use was discontinued when a subset of exposed daughters presented with early-onset vaginal clear-cell adenocarcinoma, with a 40-fold increase in risk compared to unexposed individuals. A highly significant incidence ratio for clear-cell adenocarcinoma was also found in the Dutch DES cohort, a population that may have had lower exposures than US women. It was subsequently determined that exposed offspring of both sexes had increased risk for multiple reproductive disorders, certain cancers, cryptorchidism (boys), and other diseases, although the risk for sons is more controversial. New data are emerging to implicate increased disease risk in grandchildren. Not surprisingly, a plethora of examples is emerging for increased disease susceptibility later in life as a function of developmental exposures to EDCs that include BPA, phthalates, PCBs, pesticides, dioxins, and tributyltin (TBT), among others.
Epigenetics and transgenerational effects of EDCs: EDC-induced epigenetic changes are also influenced by dose of exposure, and they are tissue specific. Thus, it is important to consider both dose of EDC and the tissue before making firm conclusions about the epigenetic effects of EDCs. DNA methylation changes are the best-studied mechanism in this regard. For example, 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.
Little is known about the ability of EDCs to cause histone modifications and whether this leads to transgenerational effects in animals or humans. The herbicides paraquat and dieldrin caused histone modifications in immortalized rat mesencephalic dopaminergic cells, and the insecticide propoxur causes histone modifications in gastric cells in vitro. DES caused histone deacetylation in the promoter region of the cytochrome P450 side chain cleavage (P450scc) gene. Further studies, however, need to be conducted to identify other EDCs causing histone modifications in animals and humans and to determine whether such modifications lead to transgenerational effects.
Female Reproductive Health: in the past 5 years, no new information became available on the effects of DES on the postnatal human ovary. Recent animal studies indicate that DES adversely affected the postnatal ovary. Neonatal exposure to DES inhibited germ cell nest breakdown (408) and caused the formation of polyovular follicles in mice, likely by interfering with the ERβ pathway and inhibiting programmed oocyte death and germ cell loss. It also reduced the primordial follicle pool and increased atresia in prepubertal lambs, and it caused polyovular ovaries in hamsters. Although these previous studies provide solid evidence that DES adversely affects ovarian structure in a variety of species, studies are needed to determine whether other synthetic estrogens adversely affect the ovary.
Effects of EDCs on uterine structure and function: synthetic estrogens are well known disruptors of uterine structure and function in humans and animals. Consistent with previous studies, recent data indicate that neonatal DES exposure caused endometrial hyperplasia/dysplasia in hamsters and increased uterine adenocarcinoma and uterine abnormalities in Donryu rats. 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.
Effects of EDCs on the vagina: only a limited number of studies assessed the effects of EDCs on the vagina, and of these, all but one on phthalates focused on DES. A recent study of women showed an association between in utero exposure to DES and clear cell carcinoma of the vagina, confirming previous findings. Furthermore, DES disrupted the expression of transformation-related protein 63, which makes cell fate decisions of Müllerian duct epithelium and induces adenosis lesions in the cervix and vagina in women.
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 (520); 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 the one study in the previous 5 years that did not focus on DES, polypropylene and polyethylene terephthalate did not increase vaginal weight in Sprague-Dawley rats. Although a few studies have been conducted during the previous 5 years on the effects of EDCs on the vagina, such studies are very few in number, small in scope, and focused on DES. Thus, future studies are needed in this largely understudied area before we fully appreciate whether other EDCs impair the vagina.
Premature ovarian failure/early menopause: combined data from three studies on DES indicated that in utero exposure was associated with an increased lifetime risk of early menopause in women (602). However, animal studies have not determined whether DES exposure causes premature ovarian failure. Thus, future studies should focus on this issue.
Fibroids: a few recent studies confirmed the known association between DES exposure and fibroids. In the Sister Study, in utero exposure to DES was positively associated with early-onset fibroids. Similarly, in the Nurses’ Health Study II, prenatal DES exposure was associated with uterine fibroids, with the strongest risk being for women exposed to DES in the first trimester. Given the consistency in findings, future studies should be focused on determining the mechanism by which DES exposure increases the risk of fibroids.
High-dose estrogen treatment to reduce final height of tall girls increases their risk for infertility in later life.
The aim was to study the effect of estrogen dose on fertility outcome of these women.
We conducted a retrospective cohort study of university hospital patients.
We studied 125 tall women aged 20-42 yr, of whom 52 women had been treated with 100 ?g and 43 with 200 ?g of ethinyl estradiol (EE) in adolescence.
Time to first pregnancy, treatment for infertility, and live birth rate were measured.
The time to first pregnancy was increased in treated women. Of untreated women, 80% conceived within 1 yr vs. 69% of women treated with 100 ?g EE and 59% of women treated with 200 ?g EE. This trend of increased time to pregnancy with increasing estrogen dose was significant (log rank trend test, P = 0.01). Compared with untreated women, fecundability was reduced in women treated with both 100 ?g EE [hazard ratio = 0.42; 95% confidence interval (CI), 0.19-0.95] and 200 ?g EE (hazard ratio = 0.30; 95% CI, 0.13-0.72). We also observed a significant trend in the incidence of treatment for infertility with increased estrogen dose (P = 0.04). Fecundity was affected in women treated with 200 ?g EE who had reduced odds of achieving at least one live birth (odds ratio = 0.13; 95% CI, 0.02-0.81), but not in women treated with 100 ?g EE.
We report a dose-response relationship between fertility in later life and estrogen dose used for the treatment of tall stature in adolescent girls; a higher estrogen dose is associated with increased infertility.
It has been shown that high-dose estrogen treatment to reduce final height of tall girls increases their risk for infertility in later life (3, 4). Here, we studied the effect of estrogen dose on fertility outcome of these women. We compared women who received no treatment to women who received either 100 ?g EE or 200 ?g EE. Our study confirms that tall women treated with high-dose estrogen have an increased time to pregnancy and experience more fertility problems compared with untreated women. We demonstrate for the first time that the association between estrogen treatment and the observed infertility is dose-dependent.
Although human studies on the effects of treatment with estrogens have mostly focused on OCP users, animal studies have focused on environmental exposure to EE as an endocrine-disruptor and on the effects of diethylstilbestrol (DES). In rodents, both in utero and postnatal exposure to EE or DES produces permanent adverse effects on the developing female reproductive system. Animal studies on in utero exposure to DES have shown disruption at the follicle level. In DES-exposed mice, reduced numbers of primordial follicles and of oocytes after ovulation induction have been found. Neonatal exposure to DES in lambs reduces the primordial follicle pool by stimulating their initial recruitment, resulting in increased numbers of atretic follicles. Finally, DES induces transient changes in gene expression during gestation; these changes could be involved in follicle development, rate of atresia, or patterns of secretion or metabolism of steroid hormones. These animal studies suggest that pharmacological doses of estrogens may influence fertility in many ways and at various time points. This knowledge, although difficult to extrapolate, may help in better understanding the mechanism behind the observed infertility in tall women treated with high-dose estrogen.
Previously, it has been shown that a considerable number of tall women treated with high-dose estrogen in adolescence suffer from primary ovarian insufficiency with concomitant early follicle pool depletion diagnosed by increased serum FSH levels, decreased serum anti-Müllerian hormone levels, and low antral follicle counts. Although the mechanism behind this accelerated follicle loss observed in these women remains unknown, based on our results we conclude that estrogen may play a key dose-dependent role. This is supported by a study on in utero exposure of women to DES, who reported an earlier age at menopause with cumulating doses of DES.
There is growing interest in the possible health threat posed by endocrine-disrupting chemicals (EDCs), which are substances in our environment, food, and consumer products that interfere with hormone biosynthesis, metabolism, or action resulting in a deviation from normal homeostatic control or reproduction.
In this first Scientific Statement of The Endocrine Society, we present the evidence that endocrine disruptors have effects on male and female reproduction, breast development and cancer, prostate cancer, neuroendocrinology, thyroid, metabolism and obesity, and cardiovascular endocrinology.
Results from animal models, human clinical observations, and epidemiological studies converge to implicate EDCs as a significant concern to public health. The mechanisms of EDCs involve divergent pathways including (but not limited to) estrogenic, antiandrogenic, thyroid, peroxisome proliferator-activated receptor ?, retinoid, and actions through other nuclear receptors; steroidogenic enzymes; neurotransmitter receptors and systems; and many other pathways that are highly conserved in wildlife and humans, and which can be modeled in laboratory in vitro and in vivo models. Furthermore, EDCs represent a broad class of molecules such as organochlorinated pesticides and industrial chemicals, plastics and plasticizers, fuels, and many other chemicals that are present in the environment or are in widespread use.
We make a number of recommendations to increase understanding of effects of EDCs, including enhancing increased basic and clinical research, invoking the precautionary principle, and advocating involvement of individual and scientific society stakeholders in communicating and implementing changes in public policy and awareness.
Discussion (DES and Fertility-specific)
General Introduction: the group of molecules identified as endocrine disruptors is highly heterogeneous and includes synthetic chemicals used as industrial solvents/lubricants and their byproducts [polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs), dioxins], plastics [bisphenol A (BPA)], plasticizers (phthalates), pesticides [methoxychlor, chlorpyrifos, dichlorodiphenyltrichloroethane (DDT)], fungicides (vinclozolin), and pharmaceutical agents [diethylstilbestrol (DES)].
Reproduction: in the adult female, the first evidence of endocrine disruption was provided almost 40 yr ago through observations of uncommon vaginal adenocarcinoma in daughters born 15–22 yr earlier to women treated with the potent synthetic estrogen DES during pregnancy. Subsequently, DES effects and mechanisms have been substantiated in animal models. Thus, robust clinical observations together with experimental data support the causal role of DES in female reproductive disorders. However, the link between disorders such as premature pubarche and EDCs is so far indirect and weak, based on epidemiological association with both IUGR and ovulatory disorders. The implications of EDCs have been proposed in other disorders of the female reproductive system, including disorders of ovulation and lactation, benign breast disease, breast cancer, endometriosis, and uterine fibroids.
In the case of DES, there are both human and experimental observations indicating heritability.
Premature ovarian failure, decreased ovarian reserve, aneuploidy, granulosa steroidogenesis: interestingly, mice exposed in utero to DES, between d 9–16 gestation, have a dose-dependent decrease in reproductive capacity, including decreased numbers of litters and litter size and decreased numbers of oocytes (30%) ovulated in response to gonadotropin stimulation with all oocytes degenerating in the DES-exposed group, as well as numerous reproductive tract anatomic abnormalities. In women with in utero exposure to DES, Hatch et al reported an earlier age of menopause between the 43–55 yr olds, and the average age of menopause was 52.2 yr in unexposed women and 51.5 yr in exposed women. The effect of DES increased with cumulative doses and was highest in a cohort of highest in utero exposure during the 1950s. These observations are consistent with a smaller follicle pool and fewer oocytes ovulated, as in DES-exposed mice after ovulation induction.
Reproductive tract anomalies: disruption of female reproductive tract development by the EDC DES is well documented. A characteristic T-shaped uterus, abnormal oviductal anatomy and function, and abnormal cervical anatomy are characteristic of thisin utero exposure, observed in adulthood, as well as in female fetuses and neonates exposed in utero to DES. Some of these effects are believed to occur through ER? and abnormal regulation of Hox genes. Clinically, an increased risk of ectopic pregnancy, preterm delivery, miscarriage, and infertility all point to the devastating effect an endocrine disruptor may have on female fertility and reproductive health. It is certainly plausible that other EDCs with similar actions as DES could result in some cases of unexplained infertility, ectopic pregnancies, miscarriages, and premature deliveries. Although another major health consequence of DES exposurein utero was development of rare vaginal cancer in DES daughters, this may be an extreme response to the dosage of DES or specific to pathways activated by DES itself. Other EDCs may not result in these effects, although they may contribute to the fertility and pregnancy compromises cited above. Of utmost importance clinically is the awareness of DES exposure (and perhaps other EDC exposures) and appropriate physical exam, possible colposcopy of the vagina/cervix, cervical and vaginal cytology annually, and careful monitoring for fertility potential and during pregnancy for ectopic gestation and preterm delivery.
Endometriosis is an estrogen-dependent gynecological disorder associated with pelvic pain and infertility. There are suggestive animal data of adult exposure to EDCs and development of or exacerbation of existing disease, and there is evidence that in utero exposure in humans to DES results in an increased relative risk = 1.9 (95% confidence interval, 1.2–2.8).
Environmental estrogens effects on the prostate: DES exposure is an important model of endocrine disruption and provides proof-of-principle for exogenous estrogenic agents altering the function and pathology of various end-organs. Maternal usage of DES during pregnancy resulted in more extensive prostatic squamous metaplasia in human male offspring than observed with maternal estradiol alone. Although this prostatic metaplasia eventually resolved during postnatal life, ectasia and persistent distortion of ductal architecture remained. These findings have led to the postulation that men exposed in utero to DES may be at increased risk for prostatic disease later in life, although the limited population studies conducted to date have not identified an association. Nonetheless, several studies with DES in mouse and rat models have demonstrated significant abnormalities in the adult prostate, including increased susceptibility to adult-onset carcinogenesis after early DES exposures. It is important to note that developmental exposure to DES, as with other environmental estrogens, has been shown to exhibit a biphasic dose- response curve with regard to several end-organ responses, and this has been shown to be true for prostatic responses as well. Low-dose fetal exposure to DES or BPA (see full study) resulted in larger prostate size in adulthood compared with controls, an effect associated with increased levels of prostatic ARs. This contrasts with smaller prostate sizes, dysplasia, and aging- associated increases in carcinogenesis found after perinatal high-dose DES exposures as noted above. This differential prostatic response to low vs. high doses of DES and other EDCs must be kept in mind when evaluating human exposures to EDCs because the lack of a response at high doses may not translate into a lack of negative effects at low, environmentally relevant doses of EDCs.
Linking basic research to clinical practice: it should be clear from this Scientific Statement that there is considerable work to be done. A reconciliation of the basic experimental data with observations in humans needs to be achieved through translation in both directions, from bench to bedside and from bedside (and populations) to bench. An example of how human observation and basic research have successfully converged was provided by DES exposure in humans, which revealed that the human syndrome is faithfully replicated in rodent models. Furthermore, we now know that DES exposure in key developmental life stages can have a spectrum of effects spanning female reproduction, male reproduction, obesity, and breast cancer. It is interesting that in the case of breast cancer, an increased incidence is being reported now that the DES human cohort is reaching the age of breast cancer prevalence. The mouse model predicted this outcome 25 yr before the human data became available. In the case of reproductive cancers, the human and mouse data have since been confirmed in rats, hamsters, and monkeys. This is a compelling story from the perspective of both animal models and human exposures on the developmental basis of adult endocrine disease.
Prevention and the “precautionary principle”: although more experiments are being performed to find the hows and whys, what should be done to protect humans? The key to minimizing morbidity is preventing the disorders in the first place. However, recommendations for prevention are difficult to make because exposure to one chemical at a given time rarely reflects the current exposure history or ongoing risks of humans during development or at other life stages, and we usually do not know what exposures an individual has had in utero or in other life stages.
In the absence of direct information regarding cause and effect, the precautionary principle is critical to enhancing reproductive and endocrine health. As endocrinologists, we suggest that The Endocrine Society actively engages in lobbying for regulation seeking to decrease human exposure to the many endocrine-disrupting agents. Scientific societies should also partner to pool their intellectual resources and to increase the ranks of experts with knowledge about EDCs who can communicate to other researchers, clinicians, community advocates, and politicians.