Abstract link added to DES research folder. Reproduced educational purposes only.
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(Journal Title) Toxicology and Applied Pharmacology Volume 199, Issue 2, 1 September 2004, Pages 142-150 Perinatal Carcinogenesis: Growing a Node forEpidemiology, Risk Management, and Animal Studies
Published by Elsevier Inc.
Review:
Lessons learned from perinatal exposure to diethylstilbestrol
Retha R. Newbold,
Developmental Endocrinology Section, Laboratory of Toxicology, Environmental Toxicology Program, Division of Intramural Research, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
Received 27 October 2003; accepted 20 November 2003.
Available online 27 March 2004.
Abstract
The synthetic estrogen diethylstilbestrol (DES) is well documented to be a perinatal carcinogen in both humans and experimental animals. Exposure to DES during critical periods of differentiation permanently alters the programming of estrogen target tissues resulting in benign and malignant abnormalities in the reproductive tract later in life. Using the perinatal DES-exposed rodent model, cellular and molecular mechanisms have been identified that play a role in these carcinogenic effects. Although DES is a potent estrogenic chemical, effects of low doses of the compound are being used to predict health risks of weaker environmental estrogens. Therefore, it is of particular interest that developmental exposure to very low doses of DES has been found to adversely affect fertility and to increase tumor incidence in murine reproductive tract tissues. These adverse effects are seen at environmentally relevant estrogen dose levels. New studies from our lab verify that DES effects are not unique; when numerous environmental chemicals with weak estrogenic activity are tested in the experimental neonatal mouse model, developmental exposure results in an increased incidence of benign and malignant tumors including uterine leiomyomas and adenocarcinomas that are similar to those shown following DES exposure. Finally, growing evidence in experimental animals suggests that some adverse effects can be passed on to subsequent generations, although the mechanisms involved in these trans-generational events remain unknown. Although the complete spectrum of risks to DES-exposed humans are uncertain at this time, the scientific community continues to learn more about cellular and molecular mechanisms by which perinatal carcinogenesis occurs. These advances in knowledge of both genetic and epigenetic mechanisms will be significant in ultimately predicting risks to other environmental estrogens and understanding more about the role of estrogens in normal and abnormal development.
Author Keywords: Cancer; Diethylstilbestrol; Environmental estrogens; Estrogen receptor; Trans-generational carcinogenesis; Transplacental carcinogenesis; Perinatal exposure; Endocrine disruptors
Article Outline
• Introduction
• Historical perspective of DES
• DES rodent models to study human disease
• Prenatal model
• Neonatal model
• Cellular and molecular mechanisms
• Multigenerational carcinogenesis
• DES as a predictor of risks to other environmental estrogens
• Summary and conclusion
• Acknowledgements
• Questions and Answers
• References
Introduction
The developing organism is extremely vulnerable to perturbation by chemicals, especially those with hormone-like activity (Bern, 1992). Rapid cell proliferation and cell differentiation coupled with complex patterns of cell signaling and cell migration occurring during development help contribute to its unique sensitivity. Further, fetuses and neonates have high metabolic rates, undeveloped liver detoxifying mechanisms, and undifferentiated immune systems as compared to adults making them more prone to chemical insult. Unlike adult exposures that can result in reversible alterations, exposure to chemicals during critical stages of development and differentiation may cause irreversible long-lasting consequences. Some of these consequences may not be noticeable or expressed until much later in life. The adverse consequences resulting from prenatal exposure to diethylstilbestrol (DES) is one example.
Ample evidence exists in multiple species including rodents and humans to link prenatal exposure to DES, a synthetic estrogen used for treatment of miscarriage, with numerous detrimental effects including reproductive tract abnormalities and a low but significant increase in vaginal cancer. In fact, DES holds the dubious position of being the only definitely established transplacental chemical carcinogen in humans (Herbst and Bern, 1981 and Tomatis, 1989). Observations in our lab for over three decades and from other laboratories using DES-exposed experimental animal models, combined with similar findings in DES-exposed humans, have added to the substantial literature base documenting the potential adverse consequences of developmental exposure to environmental chemicals with estrogenic activity. These experimental studies provide unique opportunity to study the underlying mechanisms associated with the complex issues related to perinatal carcinogenesis. The aim of this review is to update what is currently known about the consequences of DES exposure in humans and experimental animals, and to discuss the cellular and molecular findings that are contributing to our understanding of perinatal toxicity and carcinogenicity.
Historical perspective of DES
DES, a synthetic nonsteroidal compound with estrogenic activity, was heavily prescribed from the late 1940s through the 1970s to women with high-risk pregnancies with the mistaken belief that it would prevent miscarriage and other complications of pregnancy. In 1971, a landmark report associated prenatal DES exposure with a rare form of reproductive tract cancer, "vaginal clear cell adenocarcinoma", that was detected in a small number (<0.1%) of adolescent daughters of women who had taken the drug while pregnant (Herbst et al., 1971). Subsequently, DES was linked to more frequent benign reproductive tract problems in an estimated >95% of the DES-exposed daughters; reproductive organ malformation and dysfunction, poor pregnancy outcome, and immune system disorders were just some of the reported effects. Similarly, DES-exposed male offspring demonstrated structural, functional, and cellular abnormalities following prenatal exposure; hypospadias, microphallus, retained testes, and increased genital-urinary inflammation were reported to result from prenatal DES exposure (for review, Herbst and Bern, 1981; for update, NIH Publ. No. 00-4722, 1999). Although decreases in male fertility were proposed, subfertility/infertility was not confirmed by an epidemiology survey of a small cohort of DES-exposed men (Wilcox et al., 1995). Further, prostatic and testicular cancers have thus far shown no increase in the DES-exposed population compared to unexposed men but rigorous studies await a definitive conclusion.
DES became one of the first examples of a transplacental toxicant in humans; it was shown to cross the placenta and to induce a direct effect on the developing fetus. Unlike thalidomide that caused immediate observable limb defects, the abnormalities caused by DES were not detectable until later in life. DES resulted in a major medical catastrophe that continues to date to reveal its effects. Although it is no longer used clinically to prevent miscarriage, a major concern remains that, as DES-exposed women age and reach the time at which the incidence of reproductive organ cancers normally increase, they will show a much higher incidence of cancer than unexposed individuals. Also, there is the possibility that other organ systems such as the immune (Baird et al., 1996 and Ways et al., 1987), cardiovascular, brain/nervous, and gastrointestinal systems plus breast and bone will be affected. The uncertainty of effects of additional hormone exposures, for example, oral contraceptives and fertility drugs during reproductive years, or hormone replacement therapy during perimenopause, or tamoxifen for cancer chemotherapy/chemoprevention, are lingering questions that, as of yet, have no clear answers. Further, the possibility of multigeneration effects reported in experimental animal models (Newbold et al., 1998; Newbold et al., 2000; Turusov et al., 1992 and Walker and Haven, 1997) suggests that still another generation may be at risk for developing health problems associated with DES treatment of their grandmothers. The DES episode continues to be a serious health concern and is a salient reminder of the potential toxicity and carcinogenicity that may be caused by developmental exposure to hormonally active chemicals.
Questions of the mechanisms involved in DES-induced teratogenic and carcinogenic effects prompted the development of numerous experimental animal models to study the adverse effects of estrogens on genital tract differentiation. The murine model has been very successful in duplicating and predicting many adverse effects observed in the reproductive tracts of humans with similar DES exposure (McLachlan et al., 1975; Newbold, 1995; Newbold and McLachlan, 1985 and Newbold and McLachlan, 1996).
Similar in-depth comparative effects in other organ systems have not been studied. For breast tissue, this is extremely important because exposure to DES has been associated with increased breast cancer risks in both daughters (Hatch et al., 1998) and their mothers (Calle et al., 1996).
DES rodent models to study human disease
Prenatal model
One extensively described experimental model is the prenatal DES exposure model in which timed pregnant, outbred CD-1 mice are treated subcutaneously with DES dissolved in corn oil on days 9–16 of gestation. This time of in utero DES exposure for the offspring encompasses the major period of organogenesis of the reproductive tract in the mouse (Tuchmann-Duplessis and Haegel, 1982). After the mice are born, they are followed for up to 18 months of age. The doses of DES range from 0.01 to 100 g/kg maternal body weight. The highest dose of DES is equal to or less than that given therapeutically to pregnant women, and the lower DES doses are comparable to exposure to weak estrogenic compounds found in the environment. Exposure to DES during this critical prenatal period of sex differentiation results in alterations in both the female and male reproductive tract. Although significant adverse effects were observed including malformations, decreased fertility, and increased lesions in all regions of the reproductive tract (Table 1), only tumorigenic changes will be further discussed (for review, Newbold, 1995 and Newbold and McLachlan, 1985).
To assess the long-term effects of prenatal DES exposure on the female, mice were euthanized from 12 to 18 months of age, and reproductive tract tissues were studied for histological alterations. Histological examination revealed lesions throughout the reproductive tract (McLachlan et al., 1980). The vagina was characterized by excessive keratinization and female hypospadias (urethra opens into the vagina rather than the vulva), and at the highest DES dose (100 g/kg), 25% of the mice had epidermoid tumors of the vagina. The frequency of vaginal adenocarcinoma was rare in mice as it was in humans (<0.1%); interestingly, when this lesion was seen, it was observed in the 2.5, 5, and 10 g/kg DES dose groups but not in the high-dose group. The cervix of the DES-exposed offspring was also a DES target; it was often enlarged, but the size of cervical lumen was not different from controls. The stromal compartment was responsible for the cervical enlargement, and a low prevalence of benign (leiomyomas) and malignant (stromal cell sarcomas, leiomyosarcomas) tumors was seen in the cervical region. In the uterus, epithelial and stromal hyperplasia were observed in the low dose animals, and cystic endometrial hyperplasia was a common finding; although the high DES dose animals had small hypoplastic uteri, focal pockets of epithelial hyperplasia were seen within the endometrium. As in the cervix, a low incidence of benign (leiomyomas) and malignant (stromal cell sarcomas, leiomyosarcomas) uterine tumors was also observed in all dose groups. The ovaries of prenatal DES-treated females across doses were often more cystic than controls and had a slightly enhanced tumor incidence compared to controls.
To assess the long-term effects of prenatal DES exposure on the male, mice were euthanized from 10 to 18 months of age (Newbold et al., 1987b). Interstitial cell tumors [2/277 (1%)] were found in the aged 18-month-old mice. In addition to the two benign tumors, there were five malignant interstitial cell tumors [5/277 (2%)]. Interstitial cell tumors have been produced experimentally in certain strains of mice after prolonged treatment with various estrogenic compounds; in fact, Huseby (1976) has studied many aspects of interstitial cell tumor formation. The significance of our finding in the prenatal DES model is that there are so many malignant interstitial cell tumors in proportion to the benign tumors. In addition, interstitial cell tumors are not common in this strain of mouse and these mice were treated only during prenatal development, not long term as reported by other investigators (Huseby, 1976).
An association of prenatal DES exposure and the development of testicular seminoma in humans has been previously proposed (Bibbo and Gill, 1981; Conley et al., 1983; Gill et al., 1979 and Rosenfield et al., 1978) so we looked specifically for this lesion in the prenatal DES-exposed mice. Seminomas have been experimentally induced in dogs but such germ cell tumors in rodents are very rare. Cryptorchidism is considered to be a predisposing factor for seminoma in dogs and men, but in spite of the high incidence of retained testis in our mouse model (91%), we were able to demonstrate this particular testicular lesion in only one case in all our historical DES-treated mice.
Additional lesions resembling adenocarcinoma of the rete testis were observed in mice after in utero DES exposure. This lesion is extremely rare and was not found in any control mice; however, it was seen in 5% of the prenatally DES-exposed mice. Although distant metastases were rarely found, the tumors often infiltrated into the seminiferous tubules; the histological pattern of these tumors was suggestive of either papillary adenocarcinoma or tubulopapillary adenocarcinoma (Newbold and McLachlan, 1996; Newbold et al., 1985 and Newbold et al., 1986). To date, no reports of rete adenocarcinoma in humans have been attributed to prenatal DES exposure, but three cases of seminoma reported in DES-exposed men could have been misdiagnosed because seminoma has to be ruled out before a diagnosis of rete adenocarcinoma can be made.
The association of prenatal DES exposure and the development of testicular tumors in men has become a subject of much controversy over the last few years; some reports, specifically addressing factors for cancer of the testis, list prenatal DES exposure as a risk factor, although other studies show no relationship to hormonal treatment during pregnancy. However, the data in our experiment mouse model support the idea that DES-treated males are at a greater risk for testicular tumors than unexposed males.
In addition to the testes, tumors were observed in the retained Müllerian remnants in 8% of the DES-exposed mice (Newbold et al., 1987a); tumors in Wolffian-derived tissue were rare other than in the rete testis. Prostatic inflammation and squamous metaplasia of the prostatic ventricle were seen in our DES-exposed mice and have been reported in similarly exposed humans.
Taken together, these data in female and male mice suggest that in utero exposure to DES results in reproductive tract abnormalities including a low, but significant, increase in reproductive tract tumors across all doses tested. Some of these tumors are very rare including vaginal adenocarcinoma in females and rete testis adenocarcinoma in males.
Neonatal model
Because data from the literature suggested that developmental exposure to DES just during neonatal life resulted in a high incidence of abnormalities (Iguchi et al., 1986; Kalland et al., 1980 and Plapinger and Bern, 1979), prenatal and neonatal DES exposures were compared and resulting lesions determined. With the same rodent species as for prenatal exposure, outbred CD-1 mice were treated neonatally with DES (2 g/pup/day) on days 1–5. For females, neonatal exposure resulted in a high incidence (90–95%) of uterine cancer when they aged to 18 months (Newbold et al., 1990). Other species including rats (Arai et al., 1978 and Rothschild et al., 1988) and hamsters (Leavitt et al., 1981) neonatally exposed to DES also have a high incidence of reproductive tract abnormalities including uterine tumors. Thus, the neonatal mouse model duplicates tumors seen in other experimental rodent models and possibly is predictive of the carcinogenic potential of estrogens in the uterus of women as they age. The murine uterine tumors associated with neonatal DES treatment rarely metastasize, but, in aged animals (24 months of age or older), the lesions sometimes show spread to paraaortic lymph nodes or direct extension to contiguous organs. It is significant that these mouse tumors progress through the same morphological and biological continuum of hyperplasia to atypical hyperplasia to neoplasia as seen in women. Uterine carcinomas have not been observed in the uterus of untreated control CD-1 mice at corresponding ages, nor at various stages of the estrous cycle, nor after similar adult short-term exposure to estrogens suggesting that the developmental stage of uterine differentiation and the time of estrogen exposure are important factors in the development of the lesions.
In summary, perinatal exposure (prenatal or neonatal) to DES results in increased incidences of reproductive tract tumors (benign and malignant). In general, prenatal treatment causes a high incidence of malformation and a low, but significant increase in reproductive tract tumors. In contrast, neonatal treatment causes a low incidence of malformation but a high incidence of reproductive tract neoplasia. Because developmental events in the murine reproductive tract that occur during prenatal and neonatal differentiation occur entirely prenatally in humans, the prenatal plus neonatal mouse model can be used to predict what happens prenatally in humans. With both the prenatal and neonatal rodent models, it is apparent that the timing of exposure and the stage of tissue differentiation determine the subsequent resulting abnormalities. In humans, the timing of exposure during gestation was also shown to be an important factor for cancer risk in DES daughters; research showed that exposure early in pregnancy was associated with a greater risk for vaginal adenocarcinoma than exposure later in pregnancy (Herbst et al., 1986).
Cellular and molecular mechanisms
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. Studies by Taylor et al. (1997) report that HOX genes are involved in the structural differentiation of the reproductive tract. Further studies from Ma et al. (1998) show that prenatal DES delays the expression of these genes. Thus, this molecular "misprogrammimg" is responsible for the structural alterations observed in the DES reproductive tract (Newbold et al., 1983). Cellular changes may also be closely linked to these structural alterations. Studies with the Wnt genes also suggest DES is working through multiple gene pathways (Miller et al., 1998 and Pavlova et al., 1994).
Permanent abnormal gene imprinting has also been described (Li et al., 1997; Li et al., 2001 and Li et al., 2003) in which neonatal exposure to DES causes demethylation of the estrogen-responsive gene, lactoferrin, in the mouse uterus. The relationship of this finding to tumor induction continues to be investigated.
The role of the estrogen receptor (ER) in the induction of abnormalities and tumors following developmental exposure to DES has also been studied by using transgenic mice which over-express ER alpha
(MT-mER). Transgenic ER mice were treated with DES during neonatal life and followed as they aged. It was hypothesized that because of the abnormal expression of the ER alpha, the reproductive tract tissues of the MT-mER mice may be more susceptible to tumors after neonatal exposure to DES. It is interesting that mice over-expressing ER alpha were at a higher risk of developing abnormalities including uterine adenocarcinoma in response to neonatal DES as compared to DES-treated wild-type mice. At 8 months, 73% of the DES-treated MT-mER mice compared to 46% of the DES-treated wild-type mice had uterine adenocarcinoma. Further, these abnormalities occurred at an earlier age as compared to wild-type DES mice (Couse et al., 1997). Similar findings of increased tumors and earlier occurrence were observed in the male littermates of these MT-mER mice (Newbold et al., submitted for publication). These transgenic mouse studies suggest that the level of ER alpha present in tissue may be a determining factor in the development of estrogen-related tumors. Additional transgenic mouse models that express variant forms of ER alpha, and the lack of tumors in DES-treated ER knockout mice (Couse et al., 2001) also suggest that ER alpha is playing a role in the development of reproductive tract lesions. ER beta protein has not been detected in the murine uterus; thus, its role is unclear and requires further study (Jefferson et al.,2000).
The role of metabolism in DES-induced lesions has long been an area of investigation. Recent data suggest that catechol estrogens, particularly 4-hydroxyestradiol, are effective at tumor induction, if exposure occurs during neonatal life (Newbold and Liehr, 2000). Although both 2- and 4-hydroxyestradiol were carcinogenic, the later induced a 9-fold higher tumor incidence compared to the parent hormone estradiol. These data suggest that estrogens may be operating through multiple mechanisms to induce tumors. In addition to hormone-associated cell proliferation that may be associated with DNA damage, 4-hydroxyestradiol may 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 the proliferation of cells damaged by such processes (Liehr, 2000).
Multigenerational carcinogenesis
Ongoing mechanistic studies continue to provide support that estrogens cause both genetic and epigenetic alterations in developing target tissues (Li et al., 2003). This then raises the possibility that changes seen following prenatal or neonatal DES treatment may be transmitted to subsequent generations. In fact, when the granddaughters and grandsons of DES-treated female mice were examined late in life at 18 months of age, they had an increased incidence of tumors in reproductive tract tissues (Newbold et al., 1998 and Newbold et al., 2000). Multigenerational effects of DES have been reported by other laboratories (Marselos and Tomatis, 1992; Turusov et al., 1992 and Walker and Haven, 1997). The mechanisms involved in these trans-generational events are unknown but may involve alterations or damage to germ cells. Whether other estrogenic chemicals have trans-generational potential is uncertain at the current time.
DES as a predictor of risks to other environmental estrogens
There has been increasing concern that environmental and dietary chemicals with estrogenic activity are causing adverse reproductive health consequences by altering normal developmental processes (Colborn and Clement, 1992; Colborn et al., 1993; Colborn et al., 1996 and McLachlan, 1985). Initial attention focused only on chemicals with estrogenic activity, but now concern has broadened to include numerous chemicals that mimic or interfere with the normal actions of all endocrine hormones. These chemicals are collectively called "endocrine disrupters". With over 80,000 chemicals in use in the United States alone, concern is certainly warranted because few of these chemicals have been tested for endocrine disrupter activity. It is generally assumed that most of these chemicals are not likely to pose a significant health risk at the levels of environmental exposures that exist, but the full extent of the health consequences of these chemicals is unknown. Although adult exposure is important, the focus on exposure of the fetus is of primary concern because exposure to DES proved that the developing organism is extremely sensitive to perturbation by chemicals with hormone-like activity (Bern, 1992). As reviewed, DES has been linked with numerous benign and malignant abnormalities following exposure during critical stages of differentiation. Using the neonatal DES exposed animal model, mice were treated on days 1–5 with a variety of estrogenic chemicals over a wide dose range of exposure to determine if any of the chemicals are tumorigenic or if DES was a unique estrogen in causing carcinogenic effects. The estrogenic potency of various environmental chemicals has been compared over a large dose range (Jefferson et al., 2000; Jefferson et al., 2002 and Shelby et al., 1996). As summarized in Table 2, other estrogenic chemicals also possess the ability to induce uterine tumors if exposure occurs during critical stages of development. This summary points out the carcinogenic potential of other chemicals but does not address the issue of effective dose. Methoxychlor (pure grade, not technical grade) was the only compound tested thus far that did not cause uterine cancer. Because reports in the literature suggest it is a metabolite of methoxychlor, not methoxychlor itself that is estrogenic (Kupfer and Bulger, 1987), and because the neonatal liver is not fully functional during the time of treatment, the most likely explanation for lack of tumors is that methoxychlor was not metabolized to an active estrogen form. Other compounds are currently being tested in the neonatal rodent model and future studies will attempt to rank them more precisely for carcinogenic potential based on amount of chemical to target tissue at specific critical windows of differentiation.
Female pups were treated by subcutaneous injections of compounds dissolved in corn oil on days 1–5 of neonatal life. Dose range was from 0.1 to 200 g/pup/day. Mice were followed until 18 months of age and uterine tumors were determined. This summary demonstrates carcinogenic potential of the compounds but does not address the effective dose for each compound.
Currently, the use of tamoxifen and other selective estrogen receptor modulators (SERMs) in chemoprevention trials of young women at high risks of developing breast cancer is of particular concern and warrants careful attention in light of animal studies Newbold et al., 1997). Another population receiving developmental estrogen exposure that needs follow-up study is infants fed soy infant formula. Soy formula contains estrogenic substances such as genistein and daidzein that have caused reproductive abnormalities and cancer in experimental models including our mouse model (Jefferson et al., 2002 and Newbold et al., 2001).
Summary and conclusion
Sufficient evidence has been accumulated through the years by many laboratories to show that exposure of the developing fetus to exogenous estrogens adversely affects the differentiation of the genital tract. Experimental data demonstrate that reproductive tract structure and function are altered, and long-term cellular changes occur including both benign and malignant abnormalities. Cellular changes were seen in aged DES-exposed mice at all doses examined; the severity of specific lesions was related to dose, as was the type of lesions observed.
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 (such as those found in soy products), synthetic or environmental contaminants (such as pesticides and industrial byproducts), or from pharmaceutical agents (such as tamoxifen and other SERMs). Further, mature animals may experience adverse effects to these chemicals but developing organisms are particularly sensitive to perturbation by these compounds and often experience permanent long-lasting consequences. Hopefully, mechanistic studies will identify potential reproductive toxicants and will help better access the risks of exposure to other hormonally active chemicals in the environment if chemical exposures occur during critical stages of development.
Acknowledgements
The author is greatly indebted to Ms. Wendy Jefferson and Elizabeth Padilla-Banks of NIEHS for skillful technical expertise in the conduct of the DES experiments and their critical editorial comments in the preparation of this paper. Also, the insightful diagnosis of the pathological lesions by Dr. Bill Bullock, Wake Forest University Medical School, Winston Salem, NC, is greatly appreciated. Finally, the long-standing association with Dr. John McLachlan, Tulane University, New Orleans, LA (formerly of NIEHS, Research Triangle Park, NC) and his invaluable contribution to the field of environmental estrogens is graciously acknowledged.
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Questions and Answers
Q: Can you speculate as to why vaginal adenocarcinoma was seen after the low, but not the high, DES transplacental doses in mice?
A: During differentiation of the mammalian reproductive tract, the columnar epithelium lining the vaginal lumen is derived from the fetal Mullerian duct(MD). As differentiation progresses, these columnar cells are replaced by up-growing urogenital sinus (UGS) epithelium. These UGS cells then differentiate into squamous cells that line the differentiated vagina. Exposure to DES during this critical stage of differentiation interferes with the replacement of the original MD epithelium leaving foci of columnar cells in the vagina. These columnar cells grow into benign "gland-like" structures called "vaginal adenosis". Most of these foci of adenosis will be eventually covered over and replaced by squamous cells but a few foci may remain after DES treatment. Vaginal adenocarcinoma occurs in these foci of adenosis. Our animal data suggests that adenocarcinoma does not originate from adenosis but rather both lesions occur in the same foci of columnar cells. In all of my experience, vaginal adenocarcinoma has only been observed to coexist with adenosis. Thus, more foci of columnar cells increase the probability of developing adenocarcinoma. Adenosis and adenocarcinoma were only seen in the lower DES dose mice, not in the high dose. High dosed mice had excessive vaginal keratinization, so that any remaining columnar cells were completely replaced and covered over by squamous cells (squamous metaplasia). In mice, these squamous cells are also susceptible to neoplasia (squamous cell carcinoma). Thus, the high dosed animals have a different tumor manifestation due to different cellular phenotypes lining the vagina. Further, the high dosed mice often had structural malformations of the vagina that interfered with subsequent cellular differentiation.
Q: The issue of possible DES involvement in testicular tumors in males is important. Is it true that incidence of testicular cancer is increasing in the U.S.? You note that some studies have been positive,
others negative, with regard to prenatal DES exposure as a risk factor. Can you comment about these studies further?
A: Epidemiology reports continue to suggest that testicular cancer is on the rise in the United States. Whether this increase is real continues to be debated among investigators; early and better detection methods, and changes in diagnostic criteria over the years are reasons quoted that make it difficult to detect a true increase. With regard to DES exposure, DES was known to cause a high incidence of retained testes. Since cryptorchid testes are a known risk factor for testicular cancer, it was speculated that DES exposed men would have a higher cancer risk. However, the boys with retained testis were treated for this abnormality by surgical or hormone intervention, and to date, these men do not appear to have an increased incidence of testicular cancer. NCI epidemiology studies are still following these DES exposed men as they age. DES animal models suggest that there will be an increase in genital tract inflammation and cancer with age. Interestingly, an epidemiology report from Wilcox and colleagues (The New England Journal of Medicine 332(21): 1411–1416, 1995) reported that high doses of DES did not lead to impairment of fertility or sexual function in adult men who had been exposed to the drug in utero; although genital tract malformation including retained testes were higher in men exposed to DES, it did not seem to affect any measure of fertility studied.
Q: What is special about the neonatal uterus in mice that makes it uniquely sensitive to carcinogenesis by DES? How does the effective dose of DES transplacentally to the fetus verses directly to the neonates compare, in your studies? Have you tried prenatal plus postnatal exposure?
A: We have studied this question for over 30 years. We know that the amount of estrogen receptor in the uterine epithelium plays a role in tumor development; mice that over-express ER alpha, have a higher incidence and early latency period for DES tumors; and ER knockout mice do not develop uterine adenocarcinoma in response to DES during neonatal life. We also know that numerous genes are abnormally expressed in neonatal life following exposure to DES and other estrogenic compounds. We have also shown that permanent gene imprinting results from neonatal DES treatment; DES causes demethylation of the estrogen-regulated gene, lactoferrin, in the mouse uterus. This is a permanent alteration that does not occur after adult treatment. So the stage of cellular differentiation appears to be related to the sensitivity of the tissue and guides the response pattern to estrogens. Comparing effective prenatal and neonatal doses is difficult; the stage of differentiation of the tissue is different, different metabolic pathways occur in the fetal verses the neonatal liver, maternal contribution, binding proteins etc. However, our pharmacokinetic studies have shown that DES injected into the mom reaches the fetal uterus and accumulates there relative to fetal plasma levels. This suggests that even small amounts of DES are capable of altering reproductive tract differentiation since the uterus is acting as a
"sponge" and concentrating DES. Neonatal doses have been tested in very low ranges of exposure and found to result in uterine adenocarcinoma in a dose dependent manner. We have tried dosing mice prenatal and neonatally but this dosing regime is highly toxicity so our studies focus on dosing during specific different developmental periods rather than dosing throughout prenatal and neonatal life.
Q: Does over-expression of estrogen receptor alpha in transgenic mice cause increased susceptibility to other types of carcinogens, as it does for DES?
A: We have not looked at any non-estrogenic carcinogens but this would be an interesting experiment. Other estrogenic compounds cause similar
results as DES but known carcinogens without estrogenic activity would be interesting to investigate. We are currently looking at the tumor
incidence in extra-genital estrogenic target tissues as well as changes in males treated with DES.
Q: You mention several possible mechanisms of action of DES, including misprogramming of the reproductive tract, alteration in imprinting, and
metabolism-generated, DNA-damaging, radical production. Which of these is most likely, in your opinion? This seems an important issue in view of the ongoing concern about environmental endocrine-disrupting chemicals. Are there studies in progress that could shed more light on mechanism?
A: It is not likely that there is one mechanism that is responsible for DES carcinogenic effects. It is probably a series of multiple mechanisms. Likewise, I think different estrogens will have a different series of mechanisms. Currently, we are focusing on altered methylation patterns of some estrogen target genes.
Q: The multigenerational effect is particularly interesting and potentially worrisome, especially since the increased risk is transmitted without diminution. Can you comment further on the characteristics of this effect, and its possible mechanisms?
A: Currently we are conducting microarray studies to determine if specific gene pathways are altered in each generation. In addition, we are focusing on altered methylation in some estrogen responsive genes
to determine if that characteristic is passed from one generation to another. With regard to uterine adenocarcinoma, the increased cancer risk is transmitted with diminution to subsequent generations.
Q: The data in Table 2, summarizing uterine tumors after various environmental and dietary estrogens, will get the attention of many readers. Evidently experiments to establish effective dose for these
chemicals in still in progress.
A: Correct. These studies are still underway. Thus far, structure does not seem to predict estrogenicity or carcinogenicity but estrogenicity seems to predict carcinogenicity if exposure occurs during development.
Corresponding author. Developmental Endocrinology Section, Laboratory of Molecular Toxicology, Environmental Toxicology Program, Division of Intramural Research, National Institute of Environmental Health Sciences, Mail Drop E4-02, Research Triangle Park, NC 27709. Fax:
+1-919-541-4634.
