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.     

Ovariectomy of adult mice exposed prenatally to diethylstilbestrol

Mice exposed prenatally to diethylstilbestrol (DES) were ovariectomized at 5-10 weeks of age, or at 15 months of age. They were maintained to old age and studied for incidence of DES-related pathologic changes. Uterine metaplasia and adenomyosis were not seen after early ovariectomy, but adenomyosis persisted after late ovariectomy. No adenocarcinomas of the genital tract were found in 30 mice ovariectomized early, or in 18 mice ovariectomized at 15 months of age. This result is significantly different from the 14% frequency of genital tract adenocarcinomas reported previously in non-ovariectomized mice of this strain exposed prenatally to DES.     

Uterine tumors in old female mice exposed prenatally to diethylstilbestrol

Pregnant strain CD-1 mice were treated with diethylstilbestrol (DES) or vehicle. Their female offspring were raised to old age and autopsied when terminally ill. Squamous metaplasia and adenomyosis were more common in uteri of these old mice exposed prenatally to DES than in control mice. Tumors of the uterine horns were seen in 17 of 143 DES-exposed mice and in 3 of 64 control mice. The controls had only leiomyomas, whereas 14 of the DES-exposed mice had adenocarcinomas. There were 5 cervical adenocarcinomas and 1 vaginal adenocarcinoma among treated mice but none in the control mice. Thus the effects of prenatal exposure to DES interacted with the effects of aging to produce a relatively high frequency of uterine adenocarcinoma.     

Tumors of female offspring of mice exposed prenatally to diethylstilbestrol

Strain CD-1 female mice exposed prenatally to diethylstilbestrol (DES) (CAS: 56-53-1; alpha,alpha'-diethyl-4,4'-stilbenediol) were mated to unexposed males. Female offspring of these matings were raised to the stage of terminal illness. They were never exposed to DES and so have been referred to as "DES-lineage mice." Ten uterine adenocarcinomas and 5 ovarian cystadenocarcinomas were found in 40 DES-lineage mice. These findings were significantly different from the absence of such tumors in 24 "vehicle-lineage" mice whose mothers had received injections only of oil and alcohol. The types of tumors that commonly occur spontaneously in the CD-1 strain appeared with comparable frequency in the 2 groups of mice. The DES-lineage mice did not show the increased frequency of adenomyosis and squamous metaplasia of the uterus, nor the reduced frequency of corpora lutea seen in mice exposed prenatally to DES.     

Lesions of the rete testis in mice exposed prenatally to diethylstilbestrol

Adenocarcinoma of the rete testis is an exceptionally rare and malignant testicular neoplasm. Although treatment of pregnant women with diethylstilbestrol (DES) results in reproductive tract abnormalities in their male offspring, increased incidence of testicular tumors has not been verified. However, recently three cases of seminoma have been described in men prenatally exposed to DES, suggesting an association of prenatal DES treatment and the subsequent development of testicular tumors. This report describes the treatment of outbred pregnant CD-1 mice with DES (100 micrograms/kg) on Days 9 through 16 of gestation and its effects on their male offspring. In addition to nonmalignant abnormalities such as retained testes which have been reported in men exposed prenatally to DES, lesions resembling adenocarcinoma of the rete testis were seen in prenatally DES-treated mice at 10 to 18 mo of age (11 of 233; 5%). No comparable lesions were seen in 96 age-matched control male mice. These results suggest an association of prenatal DES exposure and the subsequent development of testicular lesions in the rete testis of mice.     

In vivo formation of N-nitrosomorpholine in CD-1 mice exposed by inhalation to nitrogen dioxide and by gavage to morpholine

Male CD-1 mice were exposed to approximately 20 ppm nitrogen dioxide (NO2) for 5-6 hours, to 1 g morpholine/kg body weight by gavage, or to both. Treatments were repeated daily for 5 consecutive days. N-nitrosomorpholine (NMOR) was found in whole carcasses (16-146 ng NMOR/mouse) in all animals that had been exposed to both NO2 and to morpholine, but NMOR was not found in tissues from animals that had been exposed to either chemical alone. Approximately one-third of the NMOR was found in the gastrointestinal tract, mainly in the stomach. The coadministration of 2 g sodium ascorbate/kg body weight or 1 g alpha-tocopheryl acetate/kg body weight had no effect on the amount of NMOR that was found in any tissue. Another possible product of the interaction of NO2 and morpholine, N-nitromorpholine, was not detected in any tissue. We concluded that the repeated, concurrent exposures of mice to NO2 by inhalation and to morpholine by gavage resulted in the in vivo formation of significant quantities of NMOR. The biological significance of the observation remains unknown.     

Cancer risk in women prenatally exposed to diethylstilbestrol

Prenatal diethylstilbestrol (DES) exposure is associated with excess risks of clear cell adenocarcinoma (CCA), and breast cancer in older women. Whether overall cancer risk is also elevated is unclear. Total and site-specific cancer risks were evaluated in the DES Combined Cohort Follow-up Study using age- and calendar-year specific standardized incidence rate ratios (SIR), and age-adjusted incidence rate ratios (RR) comparing DES exposed and unexposed women. A total of 143 and 49 cancer cases occurred in 97,831 and 34,810 person-years among the exposed and unexposed, respectively. There was no overall excess risk among exposed women when compared with external rates (SIR 1.01; 95% confidence interval [CI] 0.86-1.2). The overall RR comparing exposed with unexposed women was 1.32 (95% CI 0.94-1.8). Breast cancer risk was elevated only among women over 40 years (RR 1.83; 95% CI 1.1-3.2). The CCA SIR among exposed women was nearly 40, and the estimated attack rate through age 39 was 1.6/1,000 women. CCA incidence decreased by over 80% after age 25 when compared with 20-24 years. Excluding CCA and breast cancer, the overall RR was 1.21 (95% CI 0.74-2.0). DES was not associated with excess risks of either endometrial or ovarian cancer. These data suggest that the DES associated increase in CCA incidence remains elevated through the reproductive years. There was no consistent evidence of risk excesses for cancers other than CCA, and breast cancer in older women. Given that the population is still young, continued follow-up is necessary to assess the overall carcinogenic impact of prenatal DES exposure.     

Investigation of the formation and accumulation of liver DNA adducts in mice chronically exposed to tamoxifen

Tamoxifen was administered to three strains of female mice (B6C3F1, C57BL/6 and DBA/2) in short- and long-term studies to determine their ability to activate tamoxifen and cause hepatic DNA damage. 32P- Postlabelling of liver DNA from mice treated for 4 days showed a group of major adducts that increased in a dose-dependent manner and co- chromatographed with the major adducts detected in rat liver. On cessation of dosing, the majority of adducts were cleared within 3 days. Binding of [14C]tamoxifen to DNA nucleotides was demonstrated by the use of accelerator mass spectrometry. In long-term studies of 12 months to 2 years duration, dependent on strain, tamoxifen was administered continuously in the diet to give a daily dose of approximately 40 mg/kg. DNA adducts were detected after 3 months, although the number of adducts decreased with time and by 2 years were not detectable in the tamoxifen treated mice. None of the treated groups showed a significantly increased incidence of liver tumours, with or without phenobarbital promotion and there was no sustained liver cell proliferation. Tamoxifen was detected in the mouse livers, but at levels 50 times lower than those reported in a comparable rat study. These results suggest that, in contrast to the rat, tamoxifen is non-carcinogenic in mice because it does not cause sufficient cumulative DNA damage, or act as a promoter by causing cell proliferation.      

Hexachlorobenzene-induced renal maldevelopment in CD-1 mice and CD rats

Enlarged kidneys and hydronephrosis were observed in day-15 post-partum (pp) CD-1 mouse pups from dams treated with 10 or 50 mg hexachlorobenzene (HCB) per kg body weight (bw) on days 6-16 of gestation. Additional studies showed that enlarged kidneys occurred also on days 1 and 20 pp. CD rat pups from dams exposed to 10 mg HCB per kg bw on days 15-20 of gestation had enlarged kidneys on day 5 pp but not on days 10 or 20. In the CD rat, there was a significant increase in the kidney:bw ratio and the liver:bw ratio for the HCB-exposed pups at all three time periods. Pre- and postnatal exposure to HCB resulted in renal maldevelopment in CD-1 mice and CD rats in terms of enlarged kidneys and hydronephrosis.     

The carcinogenicity of quinoline and benzoquinolines in newborn CD-1 mice

The environmental occurrence and mutagenic activity of quinoline and benzoquinolines are well-documented. In this study, the relative carcinogenic activities of quinoline, benzo[f]quinoline, benzo[h]quinoline, and phenanthridine were evaluated in newborn mice. Mice were injected intraperitoneally on the first, eighth, and fifteenth day of life with 0.25, 0.5, and 1.0 mumol of each of these aza-arenes. Quinoline induced a 71% incidence (P less than 0.005) of hepatic tumors among the male mice sacrificed at 52 weeks of age. None of the female mice treated with these aza-arenes developed hepatomas. Among the female mice treated with quinoline there was a significant development of leukemia or lymphoma (P less than 0.05) which was not evident among the female mice in any of the other experimental groups. Benzo[h]quinoline and phenanthridine were not carcinogenic under these assay conditions. Benzo[f]quinoline did induce an increase in the incidence of hepatomas among male mice (19% as compared to 5.9% among controls). This increase, however, was not statistically significant. These data indicate that quinoline has greater carcinogenic potential than any of these isomeric benzoquinolines in newborn mice.     

Induction of uterine adenocarcinoma in CD-1 mice by catechol estrogens

Catechol estrogens may mediate estrogen-induced carcinogenesis because 4-hydroxyestradiol induces DNA damage and renal tumors in hamsters, and this metabolite is formed in the kidney and estrogen target tissues by a specific estrogen 4-hydroxylase. We examined the carcinogenic potential of catechol estrogen in an experimental model previously reported to result in a high incidence of uterine adenocarcinoma after neonatal exposure to diethylstilbestrol. Outbred female CD-1 mice were treated with 2- or 4-hydroxyestradiol, 17beta-estradiol, or 17alpha-ethinyl estradiol on days 1-5 of neonatal life (2 microg/pup/day) and sacrificed at 12 or 18 months of age. Mice treated with 17beta-estradiol or 17a-ethinyl estradiol had a total uterine tumor incidence of 7% or 43%, respectively. 2-Hydroxyestradiol induced tumors in 12% of the mice, but 4-hydroxyestradiol was the most carcinogenic estrogen, with a 66% incidence of uterine adenocarcinoma. Both 2- and 4-hydroxylated catechols were estrogenic and increased uterine wet weights in these neonates. These data demonstrate that both 2- and 4-hydroxyestradiol are carcinogenic metabolites. The high tumor incidence induced by 4-hydroxyestradiol supports the postulated role of this metabolite in hormone-associated cancers.     

Chemopreventive effects of sarcophine-diol on skin tumor development in CD-1 mice

The objective of this study was to determine the chemopreventive effects of sarcophine-diol (SD) on 7,12-dimethylbenz(a)anthracene initiated and 12-O-tetradecanoylphorbol-13-acetate promoted skin tumor development in mice and its possible mechanisms of action. SD pretreatment significantly (P<0.05) decreased skin papilloma development during promotion phase. SD significantly (P<0.05) increased caspase-3 and decreased cyclooxygenase-2 during initiation phase or promotion phase. SD significantly (P<0.05) increased caspase-8 during promotion phase. SD resulted in a 95% reduction in 12-O-tetradecanoylphorbol-13-acetate-induced DNA synthesis. SD could be an effective chemopreventive agent for skin cancer by enhancing apoptosis and decreasing cell proliferation.     

Inositol and inositol hexaphosphate suppress cell proliferation and tumor formation in CD-1 mice

In previous studies, we have shown that inositol hexaphosphate(InsP₆), a constituent of cereal diet, inhibited azoxymethane-inducedexperimental large intestinal cancer (LIC) in Fischer 344 rats.We now report a similar antineoplastic action of InsP₆ in CD-1mice injected with 1,2-dimethylhydrazine (DMH). We had hypothesizedthat InsP₆ may bring about this effect by undergoing dephosphorylationto lower phosphorylated forms; the ready availability of Ins,to react with phosphates, may increase the total amount of thelower phosphorylated Ins and potentiate the action of InsP₆.LIC induced by DMH (15 mg/kg/week ? 13) in mice given a mixtureof 1% InsP₆ + 1% Ins show a significant reduction (P <0.005)in LIC prevalence over InsP₆ treatment. Surprisingly, Ins, anin vitro growth promoting agent also caused a significant (P< 0.001) suppression of LIC. InsP₆ ? Ins also showed a concomitantreduction in the mitotic rate in the non-neoplastic epithelium.Body weight data did not suggest any overt toxic effect of long-termadministration of InsP₆, Ins or InsP₆ ? Ins. Since InsP₆ isantineoplastic in two species of experimental animals, it should,in combination with Ins, be considered in our strategies forprevention of large intestinal cancer.     

Ethionine carcinogenesis in CD-1, BALB/c and C3H mice

In two separate in vivo studies, ethionine was evaluated forcarcinogenic activity in mice. In the first study, DL-ethioninewas fed in a chow diet at 0 (controls), 0.1 (low dose, LD) and0.25% (high dose, HD) concentrations to the following groupsof mice (30 animals/group): Swiss Webster CD-I females, BALB/cmales, and C3H/HeN males and females. Because of severe toxicity,BALB/c females were fed 0.05% (LD) and 0.1% (HD) ethionine.The Swiss and BALB/c mice were maintained on their respectivediets for up to 105 weeks before killing whereas the C3H micewere killed at 68 weeks because of the high spontaneous incidenceof liver tumors hi this strain. Hie percentages of animals atrisk (surviving the time to the first liver tumor recorded ineach sex and strain) that bore liver tumors were as follows:Swiss female control, 0% (0/29), Swiss female LD, 87% (20/23);Swiss female HD, 89% (16/18); C3H male controls, 35% (8/23);C3H male LD, 55% (16/29); C3H male HD, 58% (15/26); C3H femalecontrols, 5% (1/20); C3H female LD, 60% (12/20); C3H femaleHD, 92% (12/13); BALB/c male controls, 4% (1/23); BALB/c maleLD, 8% (2/24); BALB/c male HD, 31% (5/16); BALB/c female controls,0% (0/30); BALB/c female LD, 52% (14/27); and BALB/c femaleHD, 92% (12/13). The female mice were more responsive than themales hi developing liver tumors. The results of the feedingstudy are compared with those obtained in a second study hiwhich C3H female mice were in-tubated with 0, 150 or 500 mgDL-ethionine/kg body wt three times per week for 30 weeks andkilled at 2 years. Only the LD mice showed a significantly increasedincidence of liver tumors (20/39) as compared to controls (12/41)or HD mice (7/37) hi the latter study. The hepatic levels ofthe major ethionine metabolite and methylase inhibitor, S-adenosyl-ethionine(AdoEt), as well as of the endogenous methyl group donor, S-adenosylmethionine(AdoMet) were determined in Swiss female mice fed either 0.1or 0.3% in the diet for 1–6 weeks. Hepatic AdoEt levelsranged from 37 to 80 /ig/g h'ver hi the LD animals and from61 to 203 /ig/g liver in the HD group; levels of the endogenousmetabolite AdoMet correspondingly dropped to 65% of the normallevels. The present results (0 extend to different strains andto both sexes previous observations demonstrating the hepatocarcinogenkactivity of ethionine hi mice; and (ii) indicate that as hithe rat such activity may be exerted through the formation ofAdoEt.     

Adenomyomatous polyp of the uterus in a patient receiving tamoxifen

We report a case of adenomyomatous polyp that developed during treatment with tamoxifen for breast cancer. A 63-year-old Japanese woman was admitted complaining of atypical genital bleeding. Nine months earlier, she had undergone a modified radical mastectomy for cancer of her right breast, estrogen receptor-positive stage I (T1N0M0). The administration of tamoxifen, 20 mg/day, was started immediately postoperatively. Pelvic examination after tamoxifen administration for 9 months revealed that the uterus was enlarged to the size of a fist. Transvaginal ultrasonography and magnetic resonance imaging revealed a large solid mass with multiple cystic areas in the uterine cavity. The pathological diagnosis of the tumor after total hysterectomy was typical adenomyomatous polyp. It was believed to have developed during tamoxifen administration.      

Chemopreventive effects of alpha-santalol on skin tumor development in CD-1 and SENCAR mice.

Studies from our laboratory have indicated skin cancer chemopreventive effectsof sandalwood oil in CD-1 mice. The purpose of this investigation was to study the skin cancer chemopreventive effects of alpha-santalol, a principal component of sandalwood oil in CD-1 and SENCAR mice. alpha-Santalol was isolated from sandalwood oil by distillation under vacuum and characterized by nuclear magnetic resonance and gas chromatography-mass spectrometry. Chemopreventive effects of alpha-santalol were determined during initiation and promotion phase in female CD-1 and SENCAR mice. Carcinogenesis was initiated with 7,12-dimethylbenz(a)anthracene and promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA). The effects of alpha-santalol treatment on TPA-induced epidermal ornithine decarboxylase (ODC) activity and (3)H-thymidine incorporation in epidermal DNA of CD-1 and SENCAR mice were also investigated. alpha-Santalol treatment during promotion phase delayed the papilloma development by 2 weeks in both CD-1 and SENCAR strains of mice. alpha-Santalol treatment during promotion phase significantly (P < 0.05) decreased the papilloma incidence and multiplicity when compared with control and treatment during initiation phase during 20 weeks of promotion in both CD-1 and SENCAR strains of mice. alpha-Santalol treatment resulted in a significant (P < 0.05) inhibition in TPA-induced ODC activity and incorporation of (3)H-thymidine in DNA in the epidermis of both strains of mice. alpha-Santalol significantly prevents papilloma development during promotion phase of 7,12-dimethylbenz(a)anthracene-TPA carcinogenesis protocol in both CD-1 and SENCAR mice, possibly by inhibiting TPA-induced ODC activity and DNA synthesis. alpha-Santalol could be an effective chemopreventive agent for skin cancer. Additional experimental and clinical studies are needed to investigate the chemopreventive effect of alpha-santalol in skin cancer.     

Immunohistochemical localization and semi-quantitation of hepatic tamoxifen-DNA adducts in rats exposed orally to tamoxifen

Administration of tamoxifen (TAM) has been shown to induce hepatocellular carcinogenesis and TAM-DNA adduct formation in rat liver. Here we present TAM-DNA adduct localization and semi-quantitation in hepatic tissue of rats by immunohistochemical staining followed by image analysis. We have also used a quantitative immunoassay to provide a validation for the immunohistochemical values. Rats were fed diets containing 0, 5, 50, 150 or 500 p.p.m. TAM for 45 weeks. Serial sections of paraffin-embedded liver were stained for TAM-DNA adducts using a polyclonal TAM-DNA antiserum. Subsequently, visualization of TAM-DNA adducts was performed by peroxidase-conjugated secondary antibody-mediated signal amplification using biotinyl tyramide followed by streptavidin-alkaline phosphatase and fast red. Semi-quantitation of nuclear color intensity was achieved with an Automated Cellular Imaging System (ACIS), with a detection limit of 1 TAM-DNA adduct per 10(7) nt for these experiments. In parenchymal cells of liver sections from TAM-exposed animals a dose-dependent increase in nuclear staining was observed by ACIS and the TAM-DNA adduct levels determined by ACIS were validated in liver DNA by quantitative chemiluminescence immunoassay (CIA). Comparison of semi-quantitative values determined by ACIS with quantitative values determined by CIA showed a strong correlation (r = 0.924) between the two methods. At 45 weeks of TAM exposure the liver cytoplasm contained placental glutathione S-transferase (GST-p)-positive foci, as indicated by new fuchsin staining. Staining of serial sections revealed a relative lack of TAM-DNA adducts within these enzyme-altered foci. In addition, some GST-p foci contained islands of cells that did not stain for GST-p but were positive for TAM-DNA adduct formation. This study validates the use of ACIS for TAM-DNA adduct formation and demonstrates that steady-state TAM-DNA adduct levels observed in livers of rats chronically fed TAM for several months increase in relation to dose. In addition, unlike the normal surrounding liver, preneoplastic GST-p-positive foci have virtually no TAM-DNA adducts.     

Proliferative lesions in thorotrast-deposited livers pertaining to the development of primary cancers

We obtained human cadavers livers with thorotrast deposits and examined them light microscopically. The bile ducts of the large portal tract exhibited many foci of intraductal papillary hyperplasia including some severe atypical foci. The proliferation of bile ductules at the periportal zone was more marked in cases with cholangiocarcinoma than in those with other diseases. Micro-peliosis, in which the number of lining endothelial cells was significantly increased, was prominent in angiosarcoma cases. We suggest that advanced micro-peliosis represents a specific proliferative lesion pertaining to the development of angiosarcoma. There were many hyperplastic lesions of hepatocytes with structural deformity that often proceeded to vague nodular growth.     

Dietary factors affecting uterine weights of immature CD-1 mice used in uterotrophic bioassays

A study was conducted to determine the effects of dietary factors in natural ingredient and purified diets on uterine weights of immature CD-1 mice used in uterotrophic bioassays. Factors evaluated included body weight gain, dietary phytoestrogen content, total metabolizable energy, and percent crude fiber. Fifteen to 147 mice per group, housed 5 per cage, were randomly assigned to each of the 20 test diets. The test diets were fed for 7 days to 15-day old immature female CD-1 mice and their body weight gain and uterine weights were determined. Analysis of covariance procedures were used to evaluate differences in uterine weights, after adjusting for body weight and time-related trends. Fisher's least significant difference test was used to compare adjusted uterine weights and weight gains among the test diets. Additionally, multiple linear regression procedures were used to identify those characteristics of the rodent diet that were most predictive of the adjusted uterine weights. Total metabolizable energy was significantly (P < 0.01) correlated with and was predictive of uterine weights. The following dietary variables were not significantly predictive of uterine weights: total daidzein and genistein content, percent protein, fat, N-FE (carbohydrates) or percent crude fiber. We concluded that: (1) total metabolizable energy (ME) in natural ingredient or purified diets has a significant (P < 0.01) effect on the uterine weights of immature mice used in 7-day uterotrophic bioassays; (2) a standardized, estrogen-free diet with a constant level of ME should be used for conducting uterotrophic assays when comparing results between different laboratories or when determining the estrogenic or anti-estrogenic activity of endocrine disruptor compounds; (3) the mouse uterotrophic assay remains a sensitive bioassay for assessing chemicals for estrogenic activity or for the detection of total estrogenic activity in rodent diets that may be contaminated with estrogenic compounds, and (4) chemical assays should be used to detect or measure low levels of the phytoestrogens in rodent diets.