B. Highman

Neoplastic and nonneoplastic responses to chronic feeding of diethylstilbestrol in C3H mice

Over 3000 female C3H/Hen-MTV+ mice continuously received graded dietary levels (0, 2.5, 5, 10, 20, 40, 80, 160, 320, and 640 ppb) of diethylstilbestrol (DES) beginning at weaning. Mice were scheduled to be killed after 3 or 26 wk of exposure and were palpated weekly and removed for histological evaluation when subcutaneous masses reached 1 cm diameter. Mammary tumors were more prevalent than in controls only at 320 and 640 ppb DES. However, palpable mammary tumors appeared significantly earlier than in controls in mice fed 80 ppb and above. Mice killed at 3 wk and later showed a dose-response for several nonneoplastic endpoints. At 3 wk, moderate to severe uterine glandular hyperplasia, lack of corpora lutea, and vaginal keratinization were more prevalent than in controls at 80 ppb; cervical adenosis was more prevalent than in controls at 160 ppb and above. Generally, the prevalence of other nonneoplastic responses such as uterine fibrosis, stromal mucoid changes, and bony trabecular proliferation were increased above control levels only later than 3 wk at 160 ppb and greater. This study demonstrated neoplastic and nonneoplastic responses to DES at and above 80 ppb, but gave no clear evidence of either type of response below this level. Conclusions are, (1) dietary levels of DES causing nonneoplastic effects also cause neoplastic effects when fed chronically, and (2) neoplastic levels of DES may be predicted from a 3 wk feeding study in C3H/HeN-MTV+ female mice based on nonneoplastic responses.

Histology and ultrastructure of the adult mouse ovary following a single prenatal exposure to diethylstilbestrol

SummaryHistology, selective histochemistry and electron microscopy were used to examine the ovarian structure of offspring from mice administered DES (10 μg/kg in 0.1 cc of corn oil, subcutaneously) or corn oil alone on Day 15 of gestation. Offspring were sacrificed at 7 months of age. Ovarian changes in DES exposed offspring included the absence of distinguishable corpora lutea but the presence of follicles in various stages of growth and atresia. Large accumulations of pigmented cells and numerous enlarged, pale vacuolated interstitial cells were observed. Interstitial cells contained membrane bound vacuoles, lipid droplets and clumped pigmented material. A concentric pattern of membranes was often observed within the pigment. The results indicate that a single exposure to DES on Day 15 of gestation had a dramatic influence on ovarian morphology and function in 7 month old offspring. The ovarian morphology is consistent with tonic release of FSH and LH and failure in ovulation.

Estrogen‐induced thyroid follicular cell adenomas in C57BL/6 mice

Diethylstilbestrol (DES) was fed chronically to C57BL/6 mice at concentrations of 0, 5, 10, 20, 40, 160, 320, or 640 ppb in order to define the dose-response curve for neoplastic responses. The incidence of thyroid follicular cell adenomas was higher in control females than in males and was increased at mid-level doses of DES, especially in males. None were found in mice fed 640 ppb DES, probably because these mice died from other causes before follicular cell adenomas had developed. In both sexes, DES fed at 160 or 320 ppb significantly shortened time-to-onset of these tumors, and 40 ppb increased their probability late in life. It is concluded that DES has a causal relationship to thyroid neoplasia in C57BL/6 mice, and similarities between this and the human disease suggest that C57BL/6 mice may be an appropriate model for human thyroid neoplasia.

Nonneoplastic changes induced in female c3h mice by chronic exposure to diethylstilbestrol or 17β‐estradiol

The long-term nonneoplastic effects of estrogenic diets were studied in female C3H/HeJ and C3HeB/FeJ mice. C3H/HeJ mice received diets containing 0, 10, 100, or 500 ppb diethylstilbestrol (DES) or 100, 1000, or 5000 ppb 17 beta-estradiol (E2) from 6 to 110 wk of age. C3HeB/FeJ females were fed diets containing nominal concentrations of 0, 10, 100, or 500 ppb DES from 6 to 136 wk of age. Responses of both strains to DES were qualitatively identical. Histological changes in the reproductive tract induced or increased by DES in both strains and by E2 in C3H/HeJ mice included stromal mucoid changes in the vagina and cervix, epithelial keratinization in the vagina, and glandular hyperplasia in the uterine horns. Increasing doses above 10 ppb DES or 100 ppb E2 increased the prevalence and, in some cases, severity of these responses. Dose-responses to DES for these endpoints were virtually indistinguishable in the two strains. At 10 ppb DES or 100 ppb E2 there were minimal or no observable effects. When the nonneoplastic dose-response data were compared with neoplastic dose-response data previously reported, no consistent relation between doses causing neoplastic and nonneoplastic responses was seen for the two estrogens.

Strain differences in histopathologic, hematologic, and blood chemistry changes induced in mice by a technical and a purified preparation of 2,4,5‐trichlorophenoxyacetic acid

Including controls, 978 mice were studied. On days corresponding to days 6 through 14 of pregnancy, groups of pregnant and nonpregnant CD-1 mice and male and nonpregnant female dihybrid cross F2 mice received by gavage 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) ranging in dosage from 30 to 140 mg/kg. Some groups received a technical preparation containing 97.9 +/- 0.4% 2,4,5-T and some a purified preparation containing 99 +/- 0.3% 2,4,5-T. Mice were sacrificed when they became moribund and at 1, 2, 4, 6, 8, and 11 days after beginning treatment. Sick or moribund mice sacrificed after 2-9 doses of 2,4,5-T often showed severe myocardial lesions, hypocellularlity of the bone marrow, and depletion of lymphocytes in the thymus, spleen or lymph nodes. They also showed marked hematologic and blood chemistry changes. Treated mice remaining healthy showed few or no lesions or blood chemistry changes, but often developed a mild anemia attributable to a hemolytic effect of 2,4,5-T. The incidence of animals becoming moribund was less than 1% in the CD-1 mice, including those given 140 mg/kg, and 53-82% in groups of male and female F2 mice receiving 120 mg/kg 2,4,5-T. The incidence of moribund mice tended to be higher in male than in female F2 mice and in those given the purified compound. These findings indicate that impairment of maternal health by severe lesions early in gestation is not the primary cause of an increase in incidence of fetal abnormalities observed in mice given 2,4,5-t. they also indicate that the lesions are due primarily to 2,4,5-T, rather than contaminants in the technical preparation, and illustrate the importance of using more than one strain of mouse in a toxicologic or teratologic study.

Influence of strain and age on the induction of mammary tumours by diethylstilboestrol in C3H mice

C3H/HeJ and C3H/HeN female mice were fed diets containing targeted concentrations of 320 or 640 ppb diethylstilboestrol (DES) starting at 7 or 11 wk of age and continuing throughout their remaining lifespan. Regardless of the DES concentration there was a faster rate of development and higher final incidence of mammary adenocarcinomas among the C3H/HeN mice than among the C3H/HeJ mice. In C3H/HeN mice started on DES when 11 wk old, mammary tumours developed more rapidly than when treatment was started at 7 wk of age. This was also true for C3H/HeJ mice given 320 ppb DES but not for those treated with 640 ppb DES. Both age at the start of treatment and strain of C3H mice are important factors to be considered in designing experiments to study the tumorigenic activity of oestrogens such as DES.

Mammary tumorigenesis in C3H/HEN-MTV + mice treated with diethylstilboestrol for varying periods

C3H/HeN-MTV+ female mice were fed diets containing 320 or 640 ppb diethylstilboestrol (DES). DES feeding was started at 3 wk of age and was either continued throughout life or discontinued after 4, 8 or 26 wk of administration. A control group consisted of mice fed the same diet without DES, for the duration of the experiment. Mice were killed when palpable body masses (presumed to be mammary adenocarcinomas) reached a diameter of 1 cm. Adenocarcinomas developed in 79% of control mice and 96% of the mice exposed to DES for 26 wk, irrespective of the dose. The frequency and rate of removal of tumour-bearing mice were not increased further with lifetime exposure at a given dose. The time at which the first tumour occurred was largely dependent on the duration of exposure, not dose. The rate of occurrence of subsequent tumours was dependent on dose and duration of exposure; the rate of removal of mice with mammary adenocarcinomas was significantly greater at 640 ppb than at 320 ppb DES. Tumour frequency was 83% in mice exposed to 320 ppb DES for 8 wk and in those exposed for 4 wk; however, tumours developed at a faster rate in mice exposed for 8 wk. Tumour frequency was 94-96% in mice exposed to 640 ppb DES for 4 wk and 8 wk, and tumours developed more rapidly in mice exposed for 8 wk than in those exposed for 4 wk. When data were plotted as log-dose v. log-t50 (time to a probability that half the mice would be removed with mammary tumours) linear extrapolation to the control log-t50 gave an estimate of the no-effect level of exposure to DES. This estimate was remarkably consistent for all data sets (40-93 ppb) and was independent of the duration of exposure.

Significance of Neoplastic and Nonneoplastic Changes in Female C3H/HeN-MTV—Mice Fed Diethylstilbestrol Continuously

Female C3H/HeN-MTV—mice were fed DES (0, 10, 40, 160, 640, or 1280 ppb) continuously. They were palpated weekly for mammary tumors and killed when masses reached a 1 cm diameter or at scheduled periods of 6, 12, 18, 24, or 30 months of exposure. Dead and moribund mice were examined histologically. In scheduled-sacrifice animals, mammary tumors were more prevalent than in controls at 640 and 1280 ppb. Time-to-removal with palpable tumors was reduced at and above 40 ppb. Pituitary adenomas, endometrial and cervical adenocarcinomas, and peritoneal mesotheliomas were more frequent and occurred earlier than in controls at 160 ppb or above. In scheduled-sacrifice animals fed 40 ppb, prevalences of several nonneoplastic findings were increased, including uterine grandular hyperplasia, cervical adenosis, splenic hypererythropoiesis, osseous trabecular proliferation, and mammary hyperplastic alveolar nodules. Corpora luteal depletion, pituitary cystoid degeneration, and sternal osteofibrosis were more prevalent at or above 160 ppb than in controls. Among mice removed at unscheduled periods, mammary tumors and nonneoplastic changes tended to be more frequent than in controls, even at 10 ppb DES. This study shows that exposure of mice to DES levels causing nonneoplastic alterations is also likely to increase neoplastic effects with time and suggests that any efficacious use of DES as a human drug increases the probability of cancer to an extent related to the drug-induced increase in estrogenic body burden.

Influence of age and environment on the induction of mammary tumours by diethylstilboestrol in C3H/HeN-MTV+ mice

C3H/HeN-MTV+ female mice were fed diets containing targeted concentrations of 320 or 640 ppb diethylstilboestrol (DES) starting at 3, 5, 7 or 11 wk of age and continuing throughout their remaining lifespan. Mice were housed in either a single-corridor conventional animal room or in a double-corridor barrier-type animal room. Mice housed in the conventional animal room and started on DES at 7 or 11 week of age developed palpable mammary tumours somewhat sooner than the corresponding groups of mice kept in the barrier animal room. In mice housed in the barrier animal room and exposed to a given DES concentration, there was very little difference between mice started on DES at 3, 5 or 7 wk of age in the exposure time required for the development of palpable mammary tumours. There was a striking difference, however, between mice started on DES at 7 wk and those started at 11 wk of age in the exposure time needed before mammary tumours appeared. Mice started at 11 wk of age developed tumours with, on average, about 4 wk less exposure than did those started at 7 wk. This suggests that treatment between 7 and 11 wk of age had little or no effect on mammary tumour development. In conclusion, both animal-room environment and age at the start of DES treatment influenced the mammary tumour response in female C3H/HeN-MTV+ mice.