Physiological effects and mechanisms of action of endocrine disrupting chemicals that alter estrogen signaling
Diethylstilbestrol (DES) is a nonsteroidal synthetic estrogen developed by Sir Charles Dodds and colleagues in 1938. During the late 1940’s physicians began using DES to treat pregnant women in an attempt to maintain normal placental steroid levels and to prevent miscarriages and premature births. It was not until 1953 that a randomized controlled clinical trial evaluating the effectiveness of DES in preventing miscarriage and premature birth was reported. Results from this study showed that DES offered no protective effects with respect to miscarriage or premature birth. Despite the outcome of this clinical trial, DES continued to be prescribed to pregnant women until 1971, when it was reported that in utero DES exposure was associated with a rare reproductive tract cancer, termed vaginal clear cell adenocarcinoma, in approximately 0.1% of daughters of women who had taken the drug. As a result of this association the FDA advised against using DES in pregnant women in 1971. Subsequent studies have reported multiple adverse effects in males and females as a result of prenatal DES exposure that occur with greater frequency than clear cell adenocarcinoma. In females, these include anatomical malformations of the cervix, vagina, and uterus, decreased fertility, and fewer successful pregnancies. In contrast to females, no increased incidence of cancer has been reported in males prenatally exposed to DES. However, adverse effects have been observed in their reproductive tracts, including testicular hypoplasia, cryptorchidism, and epididymal cysts.
Epidemiological analyses continue for those men and women exposed to DES in utero as physicians and scientists strive to better understand the long-term effects in humans. These efforts have been accompanied by intensive laboratory-based research to elucidate the mechanisms by which DES elicits its carcinogenic and teratogenic effects and to better predict and prevent the untoward effects of in utero DES exposure in humans. Toward this end, a variety of rodent models have been developed to study the mechanism of action and effects of DES based primarily on different potentials as a chemical substance, pharmaceutical or hormonal agent. One well characterized model for DES exposure is the neonatal mouse model in which female and male pups are treated with DES (2μg/day) for the first five days of life and aged up to 18 months. DES treatment of female mice in this model produces a high incidence of reproductive tract tumors. The neonatal mouse model and other, previously utilized, rodent models were limited in that they could not elucidate the role of the estrogen receptor (ER) or DES as a hormonal substance in DES-induced toxicity.
The generation of mice lacking either estrogen receptor α (ERα) or estrogen receptor β (ERβ), i.e. ER knockout (ERKO) mice, and their incorporation into the neonatal mouse model of DES exposure allowed for examination of the role of the ER subtypes in mediating the effects of DES in vivo. Wild type (WT) female mice treated neonatally with DES displayed characteristic DES-induced reproductive tract lesions.
Treatment resulted in atrophy, smooth muscle disorganization, hyalinization, squamous metaplasia of the glandular epithelium, and endometrial hyperplasia. In the vagina, persistent epithelial cornification and vaginal adenosis were observed in a small percentage of animals following DES treatment. Finally, DES induced progressive proliferative lesions of the epithelium in the oviduct. None of these reproductive tract lesions were observed in identically treated αERKO female mice, indicating that DES toxicity is mediated via an ERα-dependent signaling pathway.
Similar experiments utilizing the neonatal mouse model of DES exposure were conducted with WT,αERKO, and βERKO male mice to determine if DES-induced effects in the male reproductive tract are ER-mediated. When WT males are exposed neonatally to DES, they exhibit significant decreases in seminal vesicle size at all ages, while no changes in seminal vesicle size are observed in identically treated αERKO males and males treated with oil alone.
With respect to the prostate gland which expresses clearly measureable amounts of both ERα and ERβ, DES treatment of neonatal WT andβERKO mice produced histological changes in the aged ventral and dorsolateral prostates indicative of the phenotype of an estrogenized prostate gland, including increased stromal mass, epithelial hyperplasia and dysplasia, and interstitial lymphocyte infiltration. In addition, DES induced abnormal epithelial cell differentiation indicative of estrogen imprinting in the dorsolateral prostate of WT andβERKO mice, including the presence of a continuous layer of basal cells lining the epithelial ducts and acini, luminal cell hyperplasia, and loss of expression of the functional differentiation marker dorsolateral prostate-2 protein in the dorsolateral prostate. None of these effects were observed in the prostate glands of identically treated αERKO mice, indicating that a functional ERα is required for DES-mediated activity. Collectively, these data suggest that DES elicits its toxic effects in the male reproductive tract through an ERα-mediated mechanism.
Having confirmed the necessary role of ERα in mediating the DES-induced phenotypic changes in the reproductive tracts of female and male mice, studies have focused on elucidating the mechanisms underlying these changes. One area of focus in this regard has been the examination of DES-induced changes in the expression of genes in the Hox and Wnt families that are known to be critical for the normal differentiation and organization of the reproductive tract. Several lines of evidence suggest that altered gene expression in the Hox and Wnt families may be central to the DES-induced effects on the reproductive tract.
- First, their expression in the paramesonephric duct is essential for the development and organization of the female reproductive tract.
- Second, null mouse models of the Hox and Wnt gene families exhibit reproductive tract phenotypes similar to those of DES-treated mice.
- Finally, DES treatment of neonatal mice reduces the expression of Hoxa9, Hoxa10, Hoxa11, and Wnt7a in the female reproductive tract.
Based on this evidence, studies were conducted with αERKO mice in the neonatal mouse model of DES exposure to determine the role of ERα in mediating DES-induced decreases in Hox and Wnt gene expression. Results from these studies showed that the uteri of 5-day old WT mice neonatally exposed to DES had an approximately 50% reduction in Hoxa10 and Hoxa11 mRNA levels, and a greater than 80% reduction in Wnt7a mRNA levels when compared to their expression levels in the uteri of control animals. In the uteri of DES treated αERKO mice, the expression levels of Hoxa10, Hoxa11, and Wnt7a were similar to those measured in the uteri of control mice, indicating that ERα mediates the DES-induced reduction in expression of these genes.
These results substantiate the central role that ERα plays in mediating the in vivo effects of DES and provide further support for the argument that DES-induced alterations in Hox and Wnt gene expression are at least partially responsible for the reproductive tract abnormalities observed in DES-treated mice.
The molecular mechanisms underlying DES-induced decreases in Hox and Wnt gene expression have yet to be fully characterized. One proposed mechanism put forth involved epigenetic modifications such as DNA methylation, since DES increases lactoferrin and c-fos gene expression in the mouse uterus through demethylation of the lactoferrin promoter and hypomethylation of exon 4 of the c-fos gene. Subsequent studies showed that DES had no effect on promoter methylation of the Hoxa10 and Hoxa11 genes, indicating that DES utilizes a different mechanism to decrease expression of these genes. Subsequent studies examining the Hoxa10 promoter identified a nonconsensus estrogen response element (ERE) that is differentially regulated by estradiol and DES. Results from these studies showed that the maximal estradiol-induced luciferase activity was four times greater than that induced by DES in cells transfected with a luciferase reporter plasmid containing the Hoxa10 nonconsensus ERE. These results suggest that the nonconsensus Hoxa10 ERE is induced in a ligand-specific manner, which may be due to distinct conformational changes in the ER as a result of the unique ligand structures and/or the Hoxa10 nonconsensus ERE. Furthermore, these results may provide the mechanistic explanation for decreased Hoxa10 expression and the subsequent distinct phenotypes observed in the female reproductive tract following neonatal DES treatment. Whether a similar mechanism is responsible for DES-induced downregulation of Hoxa11 and Wnt7a expression remains to be determined.
Sources and more information
- Full text (free access) : Physiological effects and mechanisms of action of endocrine disrupting chemicals that alter estrogen signaling, Hormones (Athens), Journal List, Author Manuscripts, NCBI PubMed PMC4782146, 2010 Jul-Sep.
- Estrogen synthesis and signaling pathways featured image credit cell.