Adverse effects of the model environmental estrogen diethylstilbestrol are transmitted to subsequent generations
2006 Study Abstract
The synthetic estrogen diethylstilbestrol (DES) is a potent perinatal endocrine disruptor. In humans and experimental animals, exposure to DES during critical periods of reproductive tract differentiation permanently alters estrogen target tissues and results in long-term abnormalities such as uterine neoplasia that are not manifested until later in life.
Using the developmentally exposed DES mouse, multiple mechanisms have been identified that play a role in its carcinogenic and toxic effects. Analysis of the DES murine uterus reveals altered gene expression pathways that include an estrogen-regulated component. Thus, perinatal DES exposure, especially at low doses, offers the opportunity to study effects caused by weaker environmental estrogens and provides an example of the emerging scientific field termed the developmental origin of adult disease. As a model endocrine disruptor, it is of particular interest that even low doses of DES increase uterine tumor incidence.
Additional studies have verified that DES is not unique; when other environmental estrogens are tested at equal estrogenic doses, developmental exposure results in increased incidence of uterine neoplasia similar to that caused by DES.
Interestingly, our data suggest that this increased susceptibility for tumors is passed on from the maternal lineage to subsequent generations of male and female descendants; the mechanisms involved in these transgenerational events include genetic and epigenetic events. Together, our data point out the unique sensitivity of the developing organism to endocrine-disrupting chemicals, the occurrence of long-term effects after developmental exposure, and the possibility for adverse effects to be transmitted to subsequent generations.
Numerous studies have demonstrated that developmental exposure to DES interferes with normal differentiation of the Müllerian duct and regression of the Wolffian duct. Although the mechanisms are not completely understood, a molecular component in the malformation of the tissues and perhaps in the cellular changes may be responsible. Developmental studies have reported that HOX genes are involved in the structural differentiation of the reproductive tract and that prenatal DES delays the expression of these genes. Thus, this molecular misprogramming is apparently responsible for the structural alterations observed in the DES reproductive tract. Additional investigations with Wnt genes also suggest DES is working through multiple gene pathways to cause structural changes.
We have also described permanent abnormal gene imprinting, which may be involved in tumor induction and other cellular alterations in the reproductive tract; neonatal exposure to DES caused demethylation of the estrogen-responsive gene LF in the mouse uterus. Studies to determine altered methylation patterns in other estrogen-responsive genes continue.
Similar to tumors described in mice, the neonatal estrogen-exposed hamster developed uterine carcinoma at a high frequency after developmental exposure to DES. Molecular studies with the hamster concluded that imbalances in the estrogen-regulated uterine expression of c-jun, c-fos, c-myc, bax, bcl-2, and bcl-x protooncogenes probably played a role in the molecular mechanisms by which neonatal DES treatment ultimately induced epithelial neoplasia in the rodent uterus. Microarray studies with the murine uterus in our laboratory revealed similar altered gene expression pathways that included an estrogen-regulated component
The role of estrogen receptor (ER) in the induction of abnormalities and tumors after developmental DES exposure has been studied using transgenic mice that overexpressed ERα (MT-mER). MT-mER mice were treated with DES during neonatal life and followed as they aged. It was hypothesized that because of abnormal overexpression of ERα, reproductive tract tissues of the MT-mER mice might be more susceptible to tumors after neonatal DES treatment. This was indeed the case because mice overexpressing ERα were at a higher risk of developing abnormalities including uterine carcinoma in response to neonatal DES compared with DES-treated wild-type mice. At 8 months, 73% of the DES-treated MT-mER mice compared with 46% of the DES-treated wild-type mice had uterine tumors. Furthermore, these lesions occurred at an earlier age compared with wild-type DES mice. These transgenic mouse studies suggested that ERα levels present in a tissue may be a determining factor in the development of estrogen-related tumors. Additional transgenic mouse models that expressed variant forms of ERα, and DES-treated ER knockout mice that lacked uterine tumors, also suggested that ERα played a role in the development of reproductive tract lesions. ERβ was not identified in the murine uterus ; thus, its role in uterine abnormalities is unclear and requires additional study.
The role of metabolism in DES-induced lesions has long been an area of investigation. Using the DES mouse model, catechol estrogens, in particular 4-hydroxyestradiol, were very effective at uterine tumor induction. Although both 2- and 4-hydroxyestradiol were carcinogenic, the latter induced a 9-fold higher tumor incidence compared with the parent hormone estradiol. In addition to hormone-related cell proliferation that may be associated with DNA damage, 4-hydroxyestradiol can be further oxidized to a quinone reactive intermediate. Metabolic redox cycling between this quinone and the hydroquinone (4-hydroxyestradiol) may then produce mutagenic free radicals. Thus, estrogenic compounds may induce tumors in target tissues by inducing DNA damage and genetic lesions and by stimulating proliferation of cells damaged by such processes. Together, these data suggested that estrogens may be operating through multiple mechanisms to induce tumors.
Because mechanistic studies provided support that estrogens caused both genetic and epigenetic alterations in developing target tissues, the possibility was raised that abnormalities seen after prenatal or neonatal DES treatment could be transmitted to subsequent generations. In fact, studies from our laboratory showed that prenatal or neonatal treatment with DES led to tumors in the female and male genital tract, and in addition, the susceptibility for tumors was transmitted to the descendants through the maternal germ cell lineage; transmission via the DES-exposed male was not studied. Mice were treated with DES prenatally (2.5, 5, or 10 μg/kg·d) on d 9–16 of gestation, or neonatally (0.002 μg/pup·d) on d 1–5, which were the highest doses that did not drastically interfere with fertility later in life. When female mice (F1) reached sexual maturity, they were bred to control untreated males. Female and male offspring (DES lineage or F2) from these matings were aged to 17–24 months and examined for genital tract abnormalities. An increased incidence of proliferative lesions of the rete testis (an estrogen target tissue in the male) and tumors of the reproductive tract was observed in DES-lineage males. Furthermore, in DES-lineage females, an increased incidence of uterine adenocarcinoma was seen. The incidence was lower in DES descendants than in their parents; uterine tumor incidence in DES F1 at 18 months was 31% at the neonatal dose of 0.002, whereas it was 11% in their DES descendants. These data suggest that alterations occurred in germ cells and were passed to subsequent generations. Interestingly, multigenerational effects of DES have been reported by other laboratories, and some of these report transmission through the paternal lineage.
The mechanisms involved in these transgenerational events are unknown, but altered methylation patterns can be transmitted to subsequent generations. We have shown altered methylation patterns in several uterine genes that are permanently dysregulated after developmental DES treatment. Although the consequences of these types of alterations are unclear, studies suggested that methylation patterns can be passed to subsequent generations. A recent report supports this theory because prenatal exposure to vinclozolin or methoxychlor caused adverse effects on testis morphology and male fertility, and these effects were transmitted to subsequent generations. In addition, this report showed that these two chemicals caused epigenetic alterations in the DNA, specifically hyper- and hypomethylation, and that these alterations were also observed in subsequent generations. Because the response of estrogen-regulated genes is set during development, altered hormone response may be transmitted to subsequent generations.
Transgenerational effects may also be associated with alterations in specific estrogen-responsive genes. For example, LF induction in prepubertal females that were exposed neonatally to DES showed that this gene continued to be overexpressed even after treatment was completed. Furthermore, this gene was also overexpressed in uterine tissues from DES-lineage females, although these mice never received DES. Other estrogen-responsive genes are being similarly studied in DES-lineage mice, and thus far, these data suggest involvement of an altered gene expression pathway that includes an estrogen-regulated component.
Future study into transgenerational effects of other environmental chemicals and the mechanisms that govern these effects is a newly emerging research focus that deserves serious attention.
Summary and Recommendations
Sufficient evidence has been accumulated through the years in experimental animals and humans to show that the developing fetus and neonate are uniquely sensitive to exogenous estrogen exposure. If exposure occurs during critical periods of differentiation, permanent adverse effects are well documented to result. Some of these effects, such as reproductive tract abnormalities and uterine tumors, may not be observed until much later in life, long after exposure occurs. Most importantly, evidence with experimental animals suggests that adverse effects may be transmitted to subsequent generations; however, more studies are needed to determine whether this transmission of tumor potential occurs in humans. An important cohort to follow that would answer many of the unresolved questions for humans is the grandchildren of DES-exposed women. Furthermore, additional studies in both experimental animals and humans are needed to identify and understand the mechanisms involved in the transmission of disease and to detect early markers of subsequent disease.
Although animal studies must be considered carefully before extrapolation to humans follows, the DES-exposed mouse model has provided some interesting comparisons to similarly exposed humans. The model has duplicated and predicted many of the lesions observed in DES-exposed women. Although DES is a potent estrogen, it continues to provide markers of the adverse effects of exposure to estrogenic and other endocrine-disrupting substances during development, whether these exposures come from naturally occurring chemicals, from synthetic or environmental contaminants, or from pharmaceutical agents. Ongoing mechanistic studies will help identify other potential reproductive toxicants and will help better access the risks of exposure to other endocrine-disrupting chemicals in the environment if chemical exposures occur during critical stages of development.
- Full text (free access) : Adverse effects of the model environmental estrogen diethylstilbestrol are transmitted to subsequent generations, Endocrinology, NCBI PubMed, PMID: 16690809, 2006 Jun.
- Featured image credit Liv Bruce.