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.

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.

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.

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.

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.

Osteosarcomas were found in 16 of 24,192 (0.066%) BALB/c female mice, 14 were in 21,816 (0.064%) 2-acetylaminofluorene-treated mice, and two were in 2,376 (0.084%) untreated controls. The osteosarcomas were classified into osteoblastic, fibroblastic, and mixed types. Seven of the osteosarcomas metastasized to the lungs. The osteosarcomas in control and 2-acetylaminofluorene-treated mice showed little or no difference in the incidence, type, size, or site of origin, indicating that 2-acetylaminofluorene did not affect the development of osteosarcomas in BALB/c female mice.U

In a study on the long-term effects of dietary diethylstilbestrol or 17 beta-estradiol on C3H mice, estrogens induced a proliferation of osseous trabeculae and increased the incidence and hastened the development of osteofibrotic areas in the sterna. There were 6 osteosarcomas, 2 having metastases, in 1,242 mice fed dietary estrogens over 360 days, but none in 356 untreated controls. These tumors were reviewed along with 4 early sternal osteosarcomas selected from 17 osteosarcomas (only 1 in a control) found thus in two other ongoing comparable studies. In at least 1 case, and possibly in 2 other early cases, tumors were associated with areas of osteofibrosis, and 1 tumor was probably associated with proliferation of bony trabeculae in the medullary cavity.

To study the long term effects of estrogenic diets, 2160 virgin female C3H/Hel mice, having a high titer to the mammary tumor virus factor (MMTV), were fed diets containing 0, 10, 100, 500, or 1000 ppb diethylstilbestrol (DES) or 100, 1000, or 5000 ppb 17 beta-estradiol (E2) from 6 to 110 weeks of age; 1368 virgin female C3HeB/FeJ mice, having a low titer to the MMTV, were fed diets containing 0, 10, 1000, or 500 ppb DES from 6 to 136 weeks. In estrogen-treated mice, the incidence of cervical adenosis and of mammary hyperplastic alveolar nodules was increased and the time to development of mammary adenocarcinomas was shortened. These changes tended to increase with dose and time and appeared earlier in the C3H/HeJ mice. Other tumors observed included 32 cervical and 20 endometrial adenocarcinomas, 16 cervical granular cell myoblastomas, 12 peritoneal mesotheliomas involving the uterus, 2 cervical and 4 vaginal squamous cell carcinomas, 2 ovarian teratomas, 6 osteosarcomas, 25 pheochromocytomas and 3 thyroid carcinomas. Of these tumors, 1 cervical and 2 endometrial adenocarcinomas, and 4 pheochromocytomas occurred in C3HeB/FeJ control mice at 104-130 weeks; none occurred in C3H/HeJ controls. This study indicates that the MMTV facilitates the development of mammary lesions in C3H mice, that estrogens predispose C3H mice to endometrial and cervical adenocarcinomas, and that cervical adenosis may be a precursor of cervical adenocarcinoma in C3H mice and serve as an early indicator of the potential uterine carcinogenicity of a test compound. It supports the view that the C3H mouse may serve as an animal model for uterine adenocarcinomas and adenosis in women exposed to estrogens.

In an earlier study, maternal mice were given by gavage 60–120 mg/kg 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on days 6—14 of pregnancy and sacrificed on day 17. The Gomori stain revealed diminished alkaline phosphatase in the renal proximal tubules of fetuses exposed to 2,4,5-T during gestation indicating retarded renal functional and, probably, morphological development. Spare sections of the fetal kidneys from the earlier study were stained by the periodic acid-Schiff (PAS) procedure. This revealed, in exposed fetuses, a reduction of renal tubules with PAS-positive material in the brush borders comparable in incidence and distribution to those with alkaline phosphatase in the earlier study. These findings indicate that PAS can replace the Gomori stain as a screening procedure when retarded fetal renal development by a toxic agent is suspected. Unlike alkaline phosphatase, the PAS procedure is effective even after prolonged fixation in Bouin's solution which is commonly used in fetal studies.

To study the long term effects of estrogen administration in mice, virgin female C3H/HeJ mice are being fed diets containing 0, 10, 100 or 500 ppb of diethylstilbestrol (DES) or 0, 100, 1000, or 5000 pph of 17beta-estradiol (E2) from 6 to 110 weeks of age. C3HeB/FeJ mice are being fed diets containing 08 10, 100, or 500 ppb DES FROM 6 TO 136 WEEKS OF AGE. Pathologic studies were conducted on 396 such mice sacrificed at 52 weeks and on over 500 others sacrificed at various intervals. After 52 weeks on 500 ppb DES or 5000 ppb E2, the cervix of both populations often showed stromal mucoid changes and adenosis characterized by focal replacement of squamous by columnar epithelium lining the cervical canal assoicated with glandular downgrowths into the subjacent stroma. The uterine horns showed hyperplastic glands, which often penetrated the muscularis, and focal endometrial and perivascular hyalin deposits. The ovaries showed atrophy with absence of corpora lutea. Ceroid deposits were increased in the ovaries and adrenals. Sternal bony trabeculae were increased. The incidence of uterine cervical adenosis and of mammary hyperplastic alveolar nodules and tumors (mainly type B, Dunn's classification), was higher in C3H/HeJ than in C3HeB/FeJ mice. Mice on lower doses of DES or E2 had less frequent and severe similar changes. Tumors observed to date only in estrogen-treated mice included 4 endometrial adenocarcinomas and an adenoacanthoma of a uterine horn, 14 cervical adenocarcinomas often appearing to arise from areas of adenosis, a vaginal squamous cell carcinoma, a cervical granular cell myoblastoma, 1 sternal and 3 cranial osteosarcomas, and a mesothelioma. The majority of the malignancies occurred in C3H/HeJ mice. These findings indicate that the mammary tumor virus factor facilitates DES-induced mammary tumorigenesis in C3H mice and may contribute to other DES-induced malignant and premalignant lesions.

Histologic study of the fetal offspring of maternal mice given 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) suggested that the previously reported fetal "cystic kidneys" were due to a retardation in fetal renal development and downgrowth of the renal papilla into the pelvis. To determine a possible retardation in renal alkaline phosphatase or functional development, maternal mice received by gavage 60-120 mg/kg, 2,4,5-T on days 6-14 of pregnancy. At necropsy on day 17, the fetal kidneys were excised and fixed 24 hr in cold 65% ethanol. Paraffin sections stained by Gomori's method revealed alkaline phosphatase mainly in tubules in the inner renal cortex. Fetal kidneys showing diminished or no alkaline phosphatase were designated subnormal. There was a statistically significant greater incidence of subnormal fetal kidneys in the 2,4,5-T-treated mice than in the untreated controls. In three experiments, some mice were also sacrificed on day 18, and the incidence of subnormal fetal kidneys was significantly lower than on day 17. This retardation in renal alkaline phosphatase development indicates a retardation in renal functional development and indirectly supports the view that 2,4,5-T also retards the morphological development of the fetal kidney and is not a renal teratogen in mice. It also illustrates that selected histochemical studies may be helpful in a teratologic investigation.

Maternal mice were given 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on days 6 through 14 of pregnancy in a tetratologic study at the National Center for Toxicological Research. Sick or moribund mice sacrificed after 4-8 doses of 120 mg/kg 2,4,5-T often showed severe myocardial lesions, hypocellularity of the bone marrow, and depletion of lymphocytes in the thymus, spleen, or lymph nodes. Healthy mice sacrificed on day 17, 11 days after treatment began, showed few or no severe lesions. To determine if lesions earlier in gestation contributed significantly to an increase in fetal abnormalities in the healthy 17-day survivors, dihybrid croos F2 pregnant and nonpregnant mice received by gavage 0, 60, or 120 mg/kg 2,4,5-T on days 6 through 14 of pregnancy. One group received a technical preparation containing 97.9 +/- 0.4% 2,4,5-T; another received a purified preparation containing 99 +/- 0.3% 2,4,5-T. Mice were sacrificed when they became moribund and at 6, 24, and 30 hr, as well as at 4, 6, 8, and 11 days after beginning treatment. Almost all mice given 60 mg/kg and many given 120 mg/kg 2,4,5-T appeared normal at sacrifice either early or late in pregnancy and showed little or no pathologic changes. Mice that became ill or moribund often showed severe lesions; few survived 11 days. Severe myocardial lesions were seen in 26 of 70 moribund mice fiven the technical 2,4,5-T and 24 of 33 given the purified preparation of 2,4,5-T. The moribund mice, particularly those given the purified compound, also showed a high incidence of lesions in other organs and marked hematological and blood chemistry changes. These findings indicate that the lesions are primarily due to 2,4,5-T rather than to impurities in the technical preparation; also impaired maternal health is not the primary cause of the increase in fetal abnormalities.

Cold-acclimated and unacclimated rats were exposed for 4 hours to 6.6 or 4.9% O2 in N2 at 1.7°C. Unacclimated rats tolerated both degrees of hypoxia better than cold-acclimated rats. Cold-acclimated rats showed relatively greater increases in serum concentrations of GOT and GPT, aldolase and LDH immediately after exposure to 4.9% O2 and a greater concentration of GPT at 6.6% O2 than unacclimated rats. In both groups serum urea nitrogen and plasma corticosterone were elevated. Serum glucose was increased above air flow controls (20.9% O2) in both groups by 50% at 6.6% O2 and 100% at 4.9% O2. Blood lactic acid levels were elevated by about 130%. The cold-acclimated rats had a higher incidence of renal tubular dilatation, fatty changes in striated muscles, and severe hepatic glycogen depletion. All rats in both groups showed myocardial inflammatory foci one day after exposure which persisted for 4 days. Cold-acclimated rats maintained higher body temperatures. The reduced tolerance of the cold-acclimated rats is attributed to an increased metabolism which increased tissue hypoxia.

ARTICLESReduced hypoxic tolerance of cold-acclimated rats: serum enzyme and tissue changesPD Altland, B Highman, and MP DieterPD Altland, B Highman, and MP DieterPublished Online:01 Jun 1972https://doi.org/10.1152/ajplegacy.1972.222.6.1441MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByTranscriptional events co-regulated by hypoxia and cold stresses in Zebrafish larvae15 May 2015 | BMC Genomics, Vol. 16, No. 1'Cross-adaptation': habituation to short repeated cold-water immersions affects the response to acute hypoxia in humans16 September 2010 | The Journal of Physiology, Vol. 588, No. 18Ultrastructural changes in V79 hamster lung fibroblasts during hypoxic exposureVirchows Archiv B Cell Pathology Including Molecular Pathology, Vol. 49, No. 1Myocardial LDH Isoenzyme Patterns in Rats Exposed to Cold and/or Hypobaric Hypoxia24 April 2009 | Acta Medica Scandinavica, Vol. 212, No. S668Interaction between hypoxic and thermal stress with particular reference to thermoregulatory mechanismTolerance of cold-acclimated and unacclimated rats to hypoxia at 1.7�CInternational Journal of Biometeorology, Vol. 17, No. 1 More from this issue > Volume 222Issue 6June 1972Pages 1441-1445 Copyright & PermissionsCopyright © 1972 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1972.222.6.1441PubMed5057714History Published online 1 June 1972 Published in print 1 June 1972 Metrics

Unacclimated and cold-acclimated rats were exercised for 3 h, 5 h, or 9 h in a cold room maintained at 1.7 °C. The cold-acclimated rats tolerated these exercise periods, but two-thirds of the unacclimated rats died during 9 h exercise. In red and white muscle the intermediate exercise interval (5 h) induced significantly greater increases in the activities in muscle of creatinephosphokinase and glycolytic enzymes of unacclimated rats, while during 9 h exercise enzyme activity declined in muscles of unacclimated rats and increased in cold-acclimated ones. The rise in serum enzyme activity during exercise was consistently greater in unacclimated than in cold-acclimated rats. Apparently the reduction in exercise tolerance was associated with and may have been in part due to loss of enzyme content and activity in muscles. Collectively, these and other biochemical responses suggested that homeostatic mechanisms had been exhausted in the rats dually stressed by cold exposure and exercise. Except for the activities of aldolase and the ratio of lactic dehydrogenase to alpha-glycerophosphate dehydrogenase, those enzymes associated with "aerobic" function (transaminases) showed the predominant changes in red muscle, and those associated with "anaerobic" function (glycolytic enzymes) the predominant changes in white muscle. The greater responses of the glycolytic enzymes in a predominantly "aerobic" tissue suggest that the biochemical adaptability of red muscle is greater than that of white muscle.