David L. Greenman

Commercial laboratory animal diets: toxicant and nutrient variability.

A commercial rodent feed was analyzed for a series of nutrients and potential contaminants during a 5-yr period. Annual average Cu and vitamin A concentrations were generally at least 12% lower than the approximate concentrations listed by the manufacturer, whereas Ca, protein, and vitamin B1 were within +/- 5% and fat and Zn within +/- 8% of the manufacturers specifications. Frequently, Se was found at concentrations at which it has been shown to interact with the process of chemical carcinogenesis. DDT, dieldrin, Cd, and Pb were occasionally close to concentrations known to have biological effects.

Strain differences in the response of the mouse to diethylstilbestrol

BALB/c StCrlfC3Hf/Nctr, C57BL/6/, C57BL/6 X BALB/c F1 hybrid (B6CF1), and monohybrid-cross offspring from the breeding of B6CF1 mice were examined with respect to uterine, vaginal, and thymus responses to diethylstilbestrol (DES). About 400 mice of each genetic population were used. Weanling mice were fed DES at dietary concentrations of 2.5 to 1,000 ppb (microgram/kg feed) for 6 days and were killed by cervical dislocation about 20 hr after removal of the feed. C57BL/6, B6CF1, and the monohybrid-cross offspring did not differ in the uterine-weight response to DES, but the slope of the dose-response line was shallower for the BALB/c than for the other strains. Dietary DES concentrations of 250 ppb or more inhibited the uterotrophic response in all populations. Vaginal cornification occurred at lower concentrations of DES in the C57BL/6 strain than in the B6CF1 animals. BALB/c and monohybrid-cross offspring were indistinguishable from each other in their vaginal response to Des and were less sensitive to DES than the other mouse populations. The use of ethanol or corn oil as the solvent for mixing DES into the diet had no apparent effect on the uterine weight or vaginal response in any of the mice. DES depressed thymus weight in a dose-related fashion at dietary concentrations of 100 ppb and above in all genetic populations.

Nuclear interaction of Fusarium mycotoxins with estradiol binding sites in the mouse uterus

By using cell-free preparations of uteri obtained from immature BALB/c mice, it was demonstrated that zearalenone and zearalanol, Fusarium mycotoxins, inhibited [3H]estradiol-17 beta binding to specific sites in cytosol. Significant inhibition was noted from zearalenone at 4 x 10(-6) M and from zearalanol at 4 x 10(-7) M. Unlabeled mycotoxins (5 x 10(-6) M) incubated with intact uteri caused translocation of specific estrogen binding sites into nuclei that were exchangeable with [3H]estradiol-17 beta. Zearalanol was more effective in this regard than zearalenone. Ability of the mycotoxins to compete with estradiol-17 beta for the cytosol receptor and to cause translocation of the receptor to the nucleus in general is correlated with their biological activity. These data suggest that the uterotrophic effects of Fusarium mycotoxins are mediated through their association with estrogen receptors in the uterus.

Cause-of-Death Assignment at the National Center for Toxicological Research

The system for assigning cause of death in animal studies of carcinogenicity at the National Center for Toxicological Research (NCTR) is described. An empirical study of the NCTRs experience with its current cause-of-death assignment system based on selected representative experiments is reported. Issues investigated include the degree of confidence associated with histologic cause-of-death assignment, potential age-, dose-, and sex-related differences in assigned grades of certainty of cause of death, and frequencies of identification of various organ-specific and systemic diagnoses as the cause of death. Implications for age-adjusted statistical tests of carcinogenicity that require cause-of-death data are discussed.

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.

Increased incidence of spontaneous and 2-acetylaminofluorene-induced liver and bladder tumors in B6C3F1 mice fed AIN-76A diet versus NIH-07 diet

Diet is a major influence on the responses of experimental animals to drugs, toxins, and carcinogens. Two diets used widely in toxicological and carcinogenic studies, and considered to be nutritionally adequate, were compared with respect to neoplastic responses to 2-acetylaminofluorene (2-AAF). Both sexes of weanling B6C3F1 mice were fed either AIN-76A (a purified diet) or NIH-07 (a natural ingredient diet), with or without 2-AAF, for up to 2 years. Dosages of 2-AAF were administered to males at 0, 40, 60, or 80 ppm in each diet and to females at 0, 150, 200, or 250 ppm. Each group consisted of 96 mice, except the groups of females dosed at 0 and 150 ppm, which consisted of 120 and 72 mice, respectively. The incidence of malignant liver tumors was significantly greater in all AIN-fed groups compared to corresponding NIH-fed groups, as was the total incidence of tumors (malignant + benign). Similarly, the incidence of malignant and total bladder tumors was greater in AIN-fed groups of mice administered the two high doses of 2-AAF for each sex compared to NIH-fed groups. No malignant bladder tumors were observed among any of the groups of mice receiving control diets. This was also true for the males on low dosages (40 ppm) of 2-AAF. The AIN-fed group of low dose females (150 ppm) developed 4% incidence of malignant bladder tumors. This study dramatically showed the importance of diet selection in chronic carcinogenic studies and suggests that results obtained with the use of a purified diet may differ both qualitatively and quantitatively from results obtained with a natural ingredient diet.

Effects of Diet Type on Incidence of Spontaneous and 2-Acetylaminofluorene-Induced Liver and Bladder Tumors in BALB/c Mice Fed AIN-76A Diet versus NIH-07 Diet

Diet is a major influence on the responses of experimental animals to drugs, toxins, and carcinogens. Two diets used widely in toxicological and/or nutritional studies, and considered to be nutritionally adequate, were compared with respect to their influence on growth, body weight, lifespan, spontaneous neoplasia, and neoplastic responses to 2-acetylaminofluorene (2-AAF). Both sexes of weanling BALB/c mice were fed either a purified diet (AIN-76A) or a nonpurified, natural ingredient diet (NIH-07), with or without 2-AAF for up to 2 years. Dosages of 2-AAF were administered to males at 0, 20, 40, or 60 ppm in each diet and to females at 0, 100, 125, or 150 ppm. Each group consisted of 96 mice. In most instances, males and females fed purified diet (AIN-fed) gained weight more rapidly, attained higher maximum body weights, and died earlier than their non-purified diet (NIH-fed) counterparts. 2-AAF inhibited weight gain significantly only in AIN-fed females. Thus, females receiving 150 ppm 2-AAF gained little more than their NIH-fed counterparts. At the dosages used in males, 2-AAF did not induce liver neoplasia but the AIN diet was clearly associated with a higher spontaneous frequency of liver neoplasia than the NIH diet. Although 2-AAF induced liver tumors in females fed either diet at all dosages, a higher frequency and earlier appearance of liver tumors among AIN-fed females than their NIH-fed counterparts was apparent mainly at the lowest dosage. 2-AAF induced bladder neoplasia in both sexes.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Variability of selected nutrients and contaminants monitored in rodent diets: a 6-year study.

The results are given from monitoring a commercial closed-formula cereal-based rodent diet (Purina 5010), two open-formula cereal-based diets (NIH-31 and NIH-07), and one purified diet (AIN-76) for selected nutrients and contaminants. The observed concentrations of nutrients (protein, fat, vitamin A, and thiamine) approximated the manufacturer specifications for closed-formula cereal diet, while the average concentrations of nutrients found in the open-formula cereal diets were well above the nominal concentrations. Nominal concentrations for these open-formula diets tended to be close to the minimum values that were observed. Except for protein levels, greater variation in nutrient concentrations was found in the purified diet than in the cereal diets, but the variation of contaminants was about equal in the two types of diets. Open- and closed-formula cereal diets appear to be very similar to each other in the degree of variation of nutrients and contaminants. Cadmium, lead, and selenium are the constituents of greatest concern in assuring the quality of the rodent diets that were evaluated.

Comparison of histological responses of balb/C and B6C3F1 female mice to estradiol when fed purified or natural‐ingredient diets

Female BALB/c and B6C3F1 mice were examined after a 3-wk exposure to dietary estradiol (0, 400, 800, 1600, and 3200 ppb) in a purified diet (AIN-76A) or a natural-ingredient diet (NIH-07). Histological findings, which became more prevalent with increasing estradiol dosage in both mouse genotypes, included vaginal hyperkeratosis and mucoid stroma, uterine inflammation, hydrometra and glandular hyperplasia, and ovarian corpora lutea depletion. At the two lower doses of estradiol, responses were generally more prevalent or severe in mice fed the purified diet than in those fed the natural-ingredient diet. However, in BALB/c mice, several responses to the two higher estradiol doses were greater when estradiol was given in the natural-ingredient diet rather than in the purified diet. These responses included corpora lutea depletion, vaginal hyperkeratosis, and uterine inflammation and hydrometra. In B6C3F1 mice, most responses to estradiol at concentrations of 400, 800, and 1600 ppm were more prevalent or severe in mice fed the purified diet than in those fed the natural-ingredient diet. It can be concluded that several responses to estradiol in mice maintained for a 3-wk period on a purified diet differed significantly from mice maintained on a natural-ingredient diet.