Uterine adenocarcinoma in mice treated neonatally with genistein.

The developing fetus is uniquely sensitive to perturbation with estrogenic chemicals. The carcinogenic effect of prenatal exposure to diethylstilbestrol (DES) is the classic example. Because phytoestrogen use in nutritional and pharmaceutical applications for infants and children is increasing, we investigated the carcinogenic potential of genistein, a naturally occurring plant estrogen in soy, in an experimental animal model previously reported to result in a high incidence of uterine adenocarcinoma after neonatal DES exposure. Outbred female CD-1 mice were treated on days 1-5 with equivalent estrogenic doses of DES (0.001 mg/kg/day) or genistein (50 mg/kg/day). At 18 months, the incidence of uterine adenocarcinoma was 35% for genistein and 31% for DES. These data suggest that genistein is carcinogenic if exposure occurs during critical periods of differentiation. Thus, the use of soy-based infant formulas in the absence of medical necessity and the marketing of soy products designed to appeal to children should be closely examined.

The immature mouse is a suitable model for detection of estrogenicity in the uterotropic bioassay.

The traditional rodent uterotropic response assay has been incorporated into the U.S. Environmental Protection Agency's screening and testing program for environmental endocrine-disrupting chemicals (EDCs). While much effort continues to focus on determining protocol variables, few studies compare uterotropic responses in rats, a species commonly used in toxicologic testing, with other rodent species. In this study, we compared uterine responses in immature outbred CD-1 mice and Sprague-Dawley rats. After three daily subcutaneous injections with 17beta-estradiol (0.1-500 microg/kg/day), immature mice and rats demonstrated a similar dose-response increase in absolute uterine wet weight and uterine weight:body weight ratio. Further, morphologic and biochemical parameters of estrogenicity, including uterine epithelial cell height and number, gland number, and induction of estrogen-responsive proteins lactoferrin and complement C3, mirror wet weight increases. We conclude that mice are as well suited as rats for the uterotropic bioassay. Because of the advantages of using mice, including lower costs, less space required, and smaller amounts of compound needed for tests, mice should be given appropriate consideration in testing paradigms for EDCs.

Lactoferrin is an estrogen responsive protein in the uterus of mice and rats.

To identify lactoferrin (LF) and determine its estrogen-responsiveness in the rat uterus, immature Sprague-Dawley rats were untreated or subcutaneously injected with 17beta-estradiol (500 microg/kg) for 3 days and uterine tissues collected. Outbred immature CD-1 mice, treated with 17beta-estradiol, provided the positive control. By using a polyclonal antibody raised against mouse LF, minimal detectable protein was immunolocalized in uterine epithelial cells of untreated immature rats and mice. After estrogen treatment, LF was localized in all uterine epithelial cells of both species, although staining was more intense in mice than rats. In mice, LF was evenly distributed throughout the cytoplasm with intense staining in some cells, while in rats, it was seen mainly in the apical cytoplasm. For comparison to another well-known estrogen responsive protein in rats, complement C3 was immunolocalized within epithelial cells and it showed a different staining pattern than LF. Uterine tissue homogenates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots showed cross-reactivity with the mouse LF antibody. These findings indicate that LF is present in the rat uterus, and is induced by estrogens as reported in other species. Thus, LF is an important marker of estrogenic activity across species and will, therefore, have utility in screening for effects of environmental estrogenic compounds.

Cell response endpoints enhance sensitivity of the immature mouse uterotropic assay.

Outbred immature CD-1 mice were subcutaneously (s.c.) injected once on postnatal day 17 or on postnatal days 17, 18, and 19 with 17beta-estradiol, diethylstilbestrol, tamoxifen, 4-hydroxytamoxifen, methoxychlor, the methoxychlor metabolite HPTE, nonylphenol, o,p'-DDT, endosulfan, or kepone over a wide dose range (0.1 to 1,000,000 microg/kg). On the day following the last injection, uterine weight/body weight ratios were determined and uterine tissues processed for histologic examination. All compounds except endosulfan and kepone increased uterine wet weight compared to vehicle controls; however, the dose response curve and magnitude of response varied depending on the compound. Choosing the maximum wet weight dose for each compound, uterine tissue was evaluated for epithelial cell height, epithelial and stromal cell proliferation, endometrial gland number, and induction of estrogen-inducible proteins lactoferrin and complement C3. All compounds elicited estrogen-responsive changes in these endpoints that were individually more sensitive than uterine weight alone. We conclude that these endpoints enhance the sensitivity of the uterotropic bioassay.

Immature mouse uterine tissue in organ culture: estrogen-induced growth, morphology and biochemical parameters.

Although estrogens have been shown to stimulate a variety of morphologic and biochemical changes in the uterus in vivo, no clear consistent demonstration of similar responses in vitro have been made; thus, a defined organ culture system using the immature mouse uterus was established to study the possibility of demonstrating estrogenic responses in vitro. Uterine tissue from immature outbred mice (17 to 24 days of age) were cut crosswise in 1-mm3 coins and cultured in a defined medium in the absence of serum, phenol red, or growth factor supplements. Diethylstilbestrol (DES), a synthetic estrogen, was added to the media at doses ranging from 1 to 100 ng/ml. The effect of DES on uterine cell proliferation was assessed by morphologic changes in uterine epithelial and stromal cells, increase in number of epithelial cells per unit basement membrane, increase in height of luminal epithelial cells, and [3H]thymidine incorporation. Functional changes were determined by measuring the amounts of the estrogen-inducible uterine protein, lactoferrin, that was localized in the epithelial cells and secreted into the media, and the localization of the estrogen receptor in the cultured tissues. Results indicate that under the described conditions of culture, estrogens like DES can induce morphologic and biochemical responses in the uterus that are similar to those seen in vivo. This organ culture system will aid in the investigation of various mechanisms involved in the hormonal regulation of growth and differentiation of estrogen target tissues.

Immature mouse uterine tissue in organ culture: Estrogen-induced growth, morphology and biochemical parameters.

Although estrogens have been shown to stimulate a variety of morphologic and biochemical changes in the uterus in vivo, no clear consistent demonstration of similar responses in vitro have been made; thus, a defined organ culture system using the immature mouse uterus was established to study the possibility of demonstrating estrogenic responses in vitro. Uterine tissue from immature outbred mice (17 to 24 days of age) were cut crosswise in 1-mm(3) coins and cultured in a defined medium in the absence of serum, phenol red, or growth factor supplements. Diethylstilbestrol (DES), a synthetic estrogen, was added to the media at doses ranging from 1 to 100 ng/ml. The effect of DES on uterine cell proliferation was assessed by morphologic changes in uterine epithelial and stromal cells, increase in number of epithelial cells per unit basement membrane, increase in height of luminal epithelial cells, and [(3)H]thymidine incorporation. Functional changes were determined by measuring the amounts of the estrogen-inducible uterine protein, lactoferrin, that was localized in the epithelial cells and secreted into the media, and the localization of the estrogen receptor in the cultured tissues. Results indicate that under the described conditions of culture, estrogens like DES can induce morphologic and biochemical responses in the uterus that are similar to those seen in vivo. This organ culture system will aid in the investigation of various mechanisms involved in the hormonal regulation of growth and differentiation of estrogen target tissues.

Ontogeny of lactoferrin in the developing mouse uterus: a marker of early hormone response.

Lactoferrin (LF) was mapped during organogenesis of the murine reproductive tract, starting on fetal Day 12, as a marker of estrogen responsiveness. To induce LF expression, pregnant outbred CD-1 mice were injected s.c. with diethylstilbestrol (DES; 100 microg/kg maternal body weight), and fetal genital tract tissues were removed; neonatal and immature mice received s.c. injections of DES (2 microg/pup per day). Corn oil-treated and untreated mice at corresponding ages provided the controls. Immunocytochemical techniques using a polyclonal antibody showed no detectable LF in control genital tract tissues until late gestation. However, after DES treatment, LF was localized in uterine epithelial cells as early as fetal Day 14; the intensity of LF staining increased with age and number of DES treatments. Control uterine tissues responded to the rise of circulating estrogens at parturition (fetal Day 19) by producing LF, although the magnitude of response was lower than that of DES-treated tissues. Uterine tissue homogenates from control and DES mice were analyzed by SDS-PAGE and Western blots, verifying the protein to be LF. Isolation of mRNA and Northern blot analysis further showed that LF mRNA was present in the developing Mullerian duct and that DES stimulated early induction of the LF gene. The early appearance of LF suggests that it may play an important role in the hormonal regulation of growth and differentiation of developing uterine tissues.

Uterine carcinoma in mice treated neonatally with tamoxifen.

The induction of preneoplastic and neoplastic lesions by the widely used antiestrogen Tamoxifen was studied in female mice. Outbred CD-1 mice were treated with Tamoxifen (1, 2, 5, 10, 25 or 50 microg/pup/day) for the first 5 days after birth. At 14-17 months, reproductive tract tissues were examined for pathological changes. In the ovary, corpora lutea were lacking while cysts were quite common in Tamoxifen-exposed mice at all doses; cystadenomas were seen in two mice. Structural malformations and epithelial hyperplasia of the oviduct were seen in 100% of the treated mice. Malformations of the uterus, cervix, and vagina were also seen. Excessive vaginal keratinization was not a common feature although vaginal adenosis was observed more often after Tamoxifen treatment than previously reported after similar treatment with diethylstilbestrol (DES). The most striking histological features, however, were seen in the uterus. One hundred percent of the Tamoxifen-treated mice at all doses exhibited uterine hypoplasia with focal areas of basal cell hyperplasia in the lining endometrium. Progressive cellular atypias were seen in the lining endometrium ranging from atypical hyperplasia to uterine adenocarcinoma; the highest incidence of uterine adenocarcinoma was 7/14 (50%) observed in the Tamoxifen 10 microg/pup/day dose group. No similar tumors were observed in corresponding control mice. The induction of atypical uterine hyperplasia and adenocarcinoma combined with other abnormalities observed in genital tract structure following neonatal treatment with Tamoxifen suggests the developing reproductive tract is exquisitely sensitive to perturbation by compounds with hormonal activity. These studies provide the basis for future investigation into the mechanisms of Tamoxifen's carcinogenic effects in experimental animals, and to the risk benefit analysis for the prophylactic use of Tamoxifen in healthy women who are at risk of developing breast cancer.

Proliferative lesions and reproductive tract tumors in male descendants of mice exposed developmentally to diethylstilbestrol.

Prenatal exposure to diethylstilbestrol (DES) is associated with reproductive tract abnormalities, subfertility and neoplasia in experimental animals and humans. Studies using experimental animals suggest that the carcinogenic effects of DES may be transmitted to succeeding generations. To further evaluate this possibility and to determine if there is a sensitive window of exposure, outbred CD-1 mice were treated with DES during three developmental stages: group 1 was treated on days 9-16 of gestation (2.5, 5 or 10 microg/kg maternal body weight) during major organogenesis; group II was treated once on day 18 of gestation (1000 microg/kg maternal body weight) just prior to birth; and group III was treated on days 1-5 of neonatal life (0.002 microg/pup/day). DES-exposed female mice (F(1)) were raised to maturity and bred to control males to generate DES-lineage (F(2)) descendants. The F(2) males obtained from these matings are the subjects of this report; results in F(2) females have been reported previously [Newbold et al. (1998) CARCINOGENESIS:, 19, 1655-1663]. Reproductive performance of F(2) males when bred to control females was not different from control males. However, in DES F(2) males killed at 17-24 months, an increased incidence of proliferative lesions of the rete testis and tumors of the reproductive tract was observed. Since these increases were seen in all DES treatment groups, all exposure periods were considered susceptible to perturbation by DES. These data suggest that, while fertility of the DES F(2) mice appeared unaltered, increased susceptibility for tumors is transmitted from the DES 'grandmothers' to subsequent generations.

Expression of estrogen receptor beta is developmentally regulated in reproductive tissues of male and female mice.

By the use of ribonuclease protection assay (RPA) combined with immunohistochemical techniques, the expression of estrogen receptor (ER) alpha and ERbeta was mapped in the developing gonads and reproductive tracts of male and female mice from fetal day 14 to postnatal day 26 (PND 26). This study was designed to determine the pattern of expression of both ER subtypes in specific tissue compartments during development. In ovaries, ERalpha mRNA was detected at all ages examined; ERbeta mRNA was seen as early as PND 1, and its expression increased with age. Immunolocalization showed ERbeta in differentiating granulosa cells of the ovary, whereas ERalpha was predominantly seen in interstitial cells. The remainder of the female reproductive tract showed ERalpha mRNA at all ages examined with little or no significant levels of ERbeta, except on PND 1 when a low level of message appeared. In males, ERalpha and ERbeta mRNA were detected in the fetal testis; however, ERbeta gradually increased until PND 5 and subsequently diminished to undetectable levels by PND 26. Immunolocalization showed ERalpha in the interstitial compartment of the testis, whereas ERbeta was seen predominantly in developing spermatogonia. The remainder of the male reproductive tract showed varying amounts of both receptors by RPA and immunostaining throughout development. These studies provide information useful in studying the role of both ER subtypes in normal differentiation, and they provide indications of differential tissue expression during development.

In vitro estrogenicity of the catechol metabolites of selected polychlorinated biphenyls.

A considerable body of work has demonstrated that phenolic polychlorinated biphenyl (PCB) metabolites, structural analogues to estradiol, bind to the soluble estrogen receptor (ER) and that hydroxy PCB-ER complexes will translocate into the nucleus and bind to ER response elements in cultured cells. Although catechol estrogens exhibit weak estrogenic activity, the catechol PCB metabolites which are structurally similar to these ER agonists have gone untested for potential estrogenicity. In the present work we have assessed the estrogenicity of this second group of PCB metabolites, the catechols. The test compounds used in the present study were chosen to elucidate the effects of chlorine and catechol position on in vitro estrogenicity. Cultured HeLa cells, transfected with the estrogen reporter gene ERET81CAT and mouse ER cDNA, were incubated with PCB catechols. The cells were harvested at 28 h posttransfection and assayed for chloramphenicol acetyl transferase (CAT) activity. The responses elicited by the PCB catechols tested fell within the range of effect measured for the catechol estrogens and phenolic PCBs, and were within the range previously reported for other "environmental estrogens" such as nonylphenol and o,p'-DDT. Maximal measured responses were achieved at concentrations approximately two to three orders of magnitude higher than that of 17-beta-estradiol, indicating that PCB catechols have estrogenic activity in vitro. The extent of chlorination and the position of the catechol (3,4 vs 2,3 substitution) were important in determining estrogenicity in the compounds tested. The 2,3-catechol showed no detectable activity in this system, while activity of the 3, 4-catechols increased with the degree of chlorination. The observed estrogenicity of PCB catechols suggests that further oxidative metabolism of estrogenic PCB phenolic metabolites would not necessarily result in lowering the total estrogenic burden of a PCB-exposed organism. The present results imply that if estrogenic activity is assigned to an individual phenol, the potential contribution of its catechol metabolites to the total estrogenic burden should also be taken into consideration.

Increased tumors but uncompromised fertility in the female descendants of mice exposed developmentally to diethylstilbestrol.

Prenatal exposure to diethylstilbestrol (DES) has been associated with the subsequent development of reproductive tract abnormalities, including poor reproductive outcome and neoplasia, in experimental animals and humans. Experimental animal studies with chemical carcinogens have raised the possibility that adverse effects of DES may be transmitted to succeeding generations. To evaluate this possibility and to determine if there is a sensitive window of developmental exposure, outbred CD-1 mice were treated with DES during three stages of development: group 1 was treated on days 9-16 of gestation (2.5, 5 or 10 microg/kg maternal body wt), the time of major organogenesis; group II was treated once on day 18 of gestation (1000 microg/kg maternal body wt) just prior to birth; group III was treated on days 1-5 of neonatal life (0.002 microg/pup/day). Female mice (F1) in each group were raised to sexual maturity and bred to control males. As previously reported, fertility of the F1 DES-exposed females was decreased in all groups. Female offspring (DES lineage or F2) from these matings were raised to maturity and housed with control males for 20 weeks. The fertility of these DES lineage female mice was not affected by DES exposure of their 'grandmothers'. DES lineage mice were killed at 17-19 and 22-24 months of age. An increased incidence of malignant reproductive tract tumors, including uterine adenocarcinoma, was seen in DES lineage mice but not in corresponding controls; the range and prevalence of tumors increased with age. Because uterine adenocarcinomas were seen in all three DES groups, all developmental exposure periods were considered susceptible to the adverse effects of DES. These data suggest that the reduced fertility observed in the DES F1 female mice was not transmitted to their descendants; however, increased susceptibility to tumor formation is apparently transmitted to subsequent generations.

Fluctuations of lactoferrin protein and messenger ribonucleic acid in the reproductive tract of the mouse during the estrous cycle.

The physiological role of lactoferrin (LF), the major estrogen-inducible protein in the murine uterus, is unclear; however, LF may be a useful marker for the study of estrogen action in the uterus. Thus, the expression of LF mRNA and the localization of the protein in genital tract tissues and secretions of female mice (6-8 wk old) at different stages of the estrous cycle were investigated. Uterine luminal fluid (ULF) was analyzed for LF by means of gel electrophoresis and Western blot techniques; LF mRNA and protein were identified in reproductive tract tissues through in situ hybridization and immunocytochemistry. At diestrus, the level of LF mRNA was low, and staining for the protein was very light in uterine epithelial cells; LF was undetectable in ULF. At proestrus, LF mRNA and protein increased in the uterine epithelium and LF was readily detectable in ULF. LF mRNA and protein reached the highest levels at estrus. At early metestrus as compared to estrus, LF mRNA and protein were detected in decreasing amounts in uterine epithelial cells; the protein was undetected in ULF. By late metestrus and diestrus, LF mRNA and protein returned to a low level, and the protein was undetectable in ULF. LF protein was also demonstrated by immunocytochemistry in the epithelium of the oviduct, cervix, and vagina. LF protein fluctuation similar to that observed in the uterus was seen in these tissues; however, the uterus demonstrated the most dramatic changes in the number of epithelial cells involved in LF production during the estrous cycle. In summary, LF mRNA and its expression in uterine epithelial cells of the mouse varied with the stage of the estrous cycle. These results, combined with previously reported findings that LF is a major constituent of mouse ULF under the influence of estrogen, suggest that LF may play an important role in normal reproductive processes.

Accelerated onset of uterine tumors in transgenic mice with aberrant expression of the estrogen receptor after neonatal exposure to diethylstilbestrol.

The role of estrogen and the estrogen receptor (ER) in the induction and promotion of tumors was investigated by using transgenic MT-mER mice, which overexpress the ER. It was hypothesized that because of this abnormal expression of the ER, the reproductive-tract tissues of the MT-mER mice may be more susceptible to tumors after neonatal exposure to the potent synthetic estrogen diethylstilbestrol (DES). Normally non-estrogen responsive tissues that may have expressed ER as a result of the transgene were also studied for DES-induced tumors. Wild-type and MT-mER littermates were treated with 2 micrograms/pup/d DES 1-5 d after birth and then killed at 4, 8, 12, and 18 mo of age. The DES-treated MT-mER mice demonstrated a significantly higher incidence of uterine adenocarcinoma at 8 mo (73%) than the DES-treated wild-type mice (46%). The tumors of the MT-mER mice were often more aggressive than those in the wild-type animals. These tumors were also preceeded at 4 mo by a significantly higher incidence of the preneoplastic lesion atypical hyperplasia in the MT-mER mice (26% compared with 0% in the wild-type mice). Other DES-induced abnormalities were observed at equal rates in the wild-type and MT-mER mice. Although no tumors were observed in untreated wild-type females, a single untreated MT-mER female had uterine adenocarcinoma at 18 mo. These data indicate that the level of ER present in a tissue may also be a determining factor in development of estrogen-responsive tumors.