Biochemical,pathologic and morphometric alterations induced in male B6C3F1 mouse liver by short-term exposure to dichloroacetic acid

Dichloroacetic acid (DCA) is a complete hepatocarcinogen and tumor promoter in the male B6C3F1 mouse. Published reports indicate that the compound is non-genotoxic. This study examines possible non-genotoxic (epigenetic) mechanisms by which DCA elicits its carcinogenic response. Correlative biochemical, pathologic and morphometric techniques are used to characterize and quantify the acute, short-term response of hepatocytes in the male B6C3F1 mouse to drinking water containing DCA. Cellularity, [ ³H]ithymidine incorporation, DNA concentration, nuclear size, and binuclearity are evaluated in terms of level of exposure (0, 0.5 and 5 g/l) and length of exposure to DCA. The dose-related alterations in hepatocytes of animals exposed to DCA for 30 days or less indicate that shortterm exposure to DCA results in inhibition of mitoses, alterations in cellular metabolism and a shift in ploidy class. Thus, DCA carcinogenesis may involve cellular adaptations, development of drug resistance and selection of phenotypically altered cells with a growth advantage.     

The induction of hepatocellular neoplasia by trichloroacetic acid administered in the drinking water of the male B6C3F1 mouse

The prevalence (percent of animals with a tumor) and multiplicity (number of tumors per animal) of hepatocellular neoplasia in the male B6C3F1 mouse exposed to trichloroacetic acid (TCA) in the drinking water were determined. Male mice were exposed to 0.05, 0.5, and 5 g/L TCA for 60 wk (Study 1), to 4.5 g/L TCA for 104 wk (Study 2) and to 0.05 and 0.5 g/L TCA for 104 wk (Study 3). Time-weighted mean daily doses measured for the low, medium, and high dose groups were consistent over the three studies, 6-8, 58-68, and 572-602 mg/kg-d for the 0.05, 0.5, and the 4.5-5 g/L treatment groups, respectively. No significant changes in animal survival were noted across the studies. A significant increase in the prevalence and multiplicity of hepatocellular tumors was found in the 58-68 and 572-602 mg/kg/d TCA dose groups. Nonhepatoproliferative changes (cytoplasmic alterations, inflammation, and necrosis) in mice treated with TCA were mild and dose related. A TCA-induced increase in liver palmitoyl CoA oxidase activity, a marker of peroxisome proliferation, correlated with tumor induction. A linear association was found between peroxisome proliferation and tumor induction. Sporadic increases in the labeling index of nuclei outside of proliferative lesions were observed at carcinogenic doses throughout the studies. Given that there are no compelling data demonstrating genotoxic activity of either TCA or any metabolite, data are consistent with an epigenetic mode of action. The studies provide dose-response data on the development of hepatocellular neoplasia in male mice over a lifetime exposure to TCA. A no-observed-effect-level (NOEL) of 6 mg/kg/d was calculated for neoplastic and nonproliferative liver pathology.     

Comparison of the effects of hexavalent chromium in the alimentary canal of F344 rats and B6C3F1 mice following exposure in drinking water: implications for carcinogenic modes of action

Exposure to high concentrations of hexavalent chromium (Cr[VI]) in drinking water is reported to induce oral mucosa tumors in F344 rats and intestinal tumors in B6C3F1 mice. To investigate the modes of action underlying these tumors, 90-day drinking water studies (with interim necropsy at day 8) were conducted with concentrations of 0.1-182 mg/l Cr(VI), administered as 0.3-520 mg/l sodium dichromate dihydrate. Blood and tissue samples were analyzed for chromium content, oxidative stress, iron levels, and gross and microscopic lesions. Results for the F344 rats are described herein and compared with results from B6C3F1 mice published previously. After 90 days of exposure, total chromium concentrations in the rat and mouse oral mucosae were comparable, yet significant dose-dependent decreases in the reduced-to-oxidized glutathione ratio (GSH/GSSG) were observed only in rats. In the duodenum, changes in GSH/GSSG were only observed in mice. Levels of 8-hydroxydeoxyguanosine were not increased in the oral or duodenal mucosae of either species. Glutathione levels were increased in the duodenum but decreased in the jejunum of both species, indicating potential differential responses in the intestinal segments. Histiocytic infiltration was observed in the duodenum of both species, yet duodenal cytokines were repressed in mice but increased in rats. Serum and bone marrow iron levels were more decreased in rats than mice. Collectively, these data suggest that Cr(VI)-induced carcinogenesis in the rodent alimentary canal involves oxidative stress; however, differences in histopathology, cytokines, and iron status suggest potential contributions from other factors as well.     

Macrocytic-megaloblastic anemia in male B6C3F1 mice following chronic exposure to 1,3-butadiene

In the present study exposure to 1,3-butadiene (BD) resulted in a macrocytic-megaloblastic anemia in male B6C3F1 mice following chronic inhalation of 1250 ppm for 6 to 24 weeks. Treatment-related changes evident after 6 weeks of exposure included a decrease in circulating erythrocytes, total hemoglobin, and hematocrit and an increase in mean corpuscular volume. A leukopenia, due primarily to a decrease in segmented neutrophils, and a five- to sixfold increase in circulating micronuclei were observed after 6 and 24 weeks of exposure. These changes were not accompanied by a significant alteration in mean corpuscular hemoglobin concentration, an increase in circulating reticulocytes, or circulating nucleated erythrocytes. A consistent treatment-related alteration in bone marrow cellularity was not found. However, flow cytofluorometric analysis of bone marrow DNA cell cycle kinetics revealed a 44% increase in proliferative index relative to controls, due primarily to an increase in the proportion of cells in S phase. These findings are consistent with a treatment-related macrocytic-megaloblastic anemia and indicate the bone marrow to be an important target organ for BD toxicity.     

Genome-wide gene expression effects in B6C3F1 mouse intestinal epithelia following 7 and 90days of exposure to hexavalent chromium in drinking water

Chronic administration of high doses of hexavalent chromium [Cr(VI)] as sodium dichromate dihydrate (SDD) elicits alimentary cancers in mice. To further elucidate key events underlying tumor formation, a 90-day drinking water study was conducted in B6C3F1 mice. Differential gene expression was examined in duodenal and jejunal epithelial samples following 7 or 90 days of exposure to 0, 0.3, 4, 14, 60, 170 or 520 mg/L SDD in drinking water. Genome-wide microarray analyses identified 6562 duodenal and 4448 jejunal unique differentially expressed genes at day 8, and 4630 and 4845 unique changes, respectively, in the duodenum and jejunum at day 91. Comparative analysis identified significant overlap in duodenal and jejunal differential gene expression. Automated dose-response modeling identified > 80% of the differentially expressed genes exhibited sigmoidal dose-response curves with EC₅₀ values ranging from 10 to 100 mg/L SDD. Only 16 genes satisfying the dose-dependent differential expression criteria had EC₅₀ values < 10 mg/L SDD, 3 of which were regulated by Nrf2, suggesting oxidative stress in response to SDD at low concentrations. Analyses of differentially expressed genes identified over-represented functions associated with oxidative stress, cell cycle, lipid metabolism, and immune responses consistent with the reported effects on redox status and histopathology at corresponding SDD drinking water concentrations. Collectively, these data are consistent with a mode of action involving oxidative stress and cytotoxicity as early key events. This suggests that the tumorigenic effects of chronic Cr(VI) oral exposure likely require chronic tissue damage and compensatory epithelial cell proliferation.     

Carcinogenicity of glycidamide in B6C3F1 mice and F344/N rats from a two-year drinking water exposure

Acrylamide is a contaminant in baked and fried starchy foods, roasted coffee, and cigarette smoke. Previously we reported that acrylamide is a multi-organ carcinogen in B6C3F₁ mice and F344/N rats, and hypothesized that acrylamide is activated to an ultimate carcinogen through metabolism to the epoxide glycidamide. We have now examined the carcinogenic effects of glycidamide administered at 0, 0.0875, 0.175, 0.35 and 0.70 mM in drinking water to the same strains of rodents for two years. In male and female mice, there were significant increases in tumors of the Harderian gland, lung, forestomach, and skin. Female mice also had an increased incidence of tumors of the mammary gland and ovary. In male and female rats, there were significant increases in thyroid gland and oral cavity neoplasms and mononuclear cell leukemia. Male rats also had increases in tumors of the epididymis/testes and heart, while female rats demonstrated increases in tumors of the mammary gland, clitoral gland, and forestomach. A similar spectrum of tumors was obtained in mice and rats administered acrylamide. These data indicate that, under the conditions of these bioassays, acrylamide is efficiently metabolized to glycidamide and that the carcinogenic activity of acrylamide is due to its conversion into glycidamide.     

Carcinogenicity of acrylamide in B6C3F1 mice and F344/N rats from a 2-year drinking water exposure

Acrylamide is a component of roasted coffee and certain baked and fried carbohydrate-rich foods prepared at high temperatures. We have assessed the carcinogenicity of acrylamide in male and female B6C3F₁ mice and F344/N rats administered 0, 0.0875, 0.175, 0.35, or 0.70 mM acrylamide in the drinking water ad libitum for 2 years. Acrylamide caused significant dose-related decreasing trends in the body weights of F344/N rats. Acrylamide administration resulted in significant dose-related decreasing trends in survival in both sexes of B6C3F₁ mice and in female F344/N rats. Histopathological analyses indicated significant dose-related increases in Harderian gland and lung tumors in male and female B6C3F₁ mice. Male B6C3F₁ mice also had a significantly increased incidence of forestomach tumors, while female B6C3F₁ mice had significant dose-related increases in mammary gland, ovary, and skin tumors. In male and female F344/N rats, there were significant increases in thyroid tumors. Male F344/N rats also had significant dose-related increases in testes, heart, and pancreas tumors, while female F344 rats demonstrated significant increases in clitoral gland, mammary gland, oral cavity, and skin tumors. These results, combined with previous mechanistic studies, provide strong support for the concept that acrylamide is activated to a carcinogen through metabolism to glycidamide.     

Immunotoxicity of sodium bromate in female B6C3F1 mice: a 28-day drinking water study

Bromate is one of the water disinfection by-products (DBPs) produced during the process of ozonation. The purpose of this study was to evaluate the immunotoxic potential of sodium bromate (SB) in female B6C3F1 mice. SB was administered in the drinking water for 28 days at doses of 80-800 mg/l. There was no difference in drinking water consumption between the animals exposed to SB and the tap water controls. Exposure to SB did not produce any signs of overt toxicity. Furthermore, no significant differences were observed in body weight, body weight gain, or the weights of thymus, liver, kidneys or lungs. No gross pathological lesions were observed in SB-treated animals. However, animals exposed to SB had a significant increase in absolute (28%) and relative (26%) spleen weights. The erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume (MCV), platelet count, total leukocyte count, and counts of differential leukocytes were unaffected by SB. A dose-related increase in reticulocytes was observed following exposure to SB with the greatest increase (78%) observed at the highest dose level. Overall, there were no changes in the absolute number of total T cells, CD4+CD8- T cells, CD4-CD8+ T cells, natural killer (NK) cells and macrophages. Exposure to SB did not affect the percentage of B cells, although a slight increase in absolute number of B cells at the dose of 600 mg/l was observed. There was no alteration in IgM antibody-forming cell (AFC) response, mixed leukocyte reaction (MLR) and NK cell activity after exposure to SB. When the activity of peritoneal macrophages, unstimulated or stimulated with IFN-gamma and LPS, was evaluated using the cytotoxic/cytostatic assay of B16F10 tumor cells, the suppressive effect of macrophages on the proliferation of B16F10 tumor cells was decreased after exposure to SB. In conclusion, SB, when administered in the drinking water at doses from 80 mg/l to 800 mg/l, produced minimal toxicological and immunotoxic effects in female B6C3F1 mice.     

Determination of lovastatin hydroxy acid in female B6C3F1 mouse serum

A liquid chromatographic-mass spectrometricmethod for the determination of lovastatin hydroxy acid in female B6C3F(1) mouse serum was developed for use in supporting toxicokinetic studies of animals dosed with the cholesterol lowering agent lovastatin. The method does not require an extensive sample cleanup and shows good correlation between serum matrix standards and solvent standards. The method was validated and used to analyze serum samples from a preliminary dose level range-finding study. The method was validated for a concentration range of approximatel 1.0 to 100 ng/mL in serum, and linearity was verified to ~2000 ng/mL. The stability of sample extracts was determined under various storage conditions and the stability of serum samples stored frozen was determined over a period of seven weeks. During the course of analyzing the animal samples, the serum was monitored for the presence of lovastatin not hydrolyzed to the hydroxy acid, but no attempt was made to quantify lovastatin. No unhydrolyzed lovastatin was noted in any of the serum samples from animals dosed with lovastatin.     

Tumor induction in F344/N rats and B6C3F1 mice following inhalation exposure to ethylbenzene

Carcinogenesis studies of ethylbenzene were conducted because of its extensive use as a solvent and because it is structurally similar to the known carcinogen benzene. Groups of 50 male and 50 female Fischer rats and B6C3F1 mice were exposed to ethylbenzene by inhalation at 0, 75, 250, and 750 ppm 6 h per day, 5 days per week, for 2 years. The dose levels were selected based on the results of 13-week studies. In the 750 ppm group of male and female rats, body weights were slightly lower and incidences of renal hyperplasia and tubular neoplasms were significantly increased compared with controls. Incidence of testicular tumors was also significantly increased in male rats. Survival and body weights of the exposed groups of male and female mice and controls were comparable. Incidences of alveolar epithelium metaplasia, alveolar/bronchiolar adenoma, and hepatocyte hypertrophy and necrosis were significantly increased in the 750 ppm male mice and incidences of liver eosinophilic foci and hepatocellular neoplasms were significantly increased in the 750 ppm female mice compared with controls. Ethylbenzene is carcinogenic inducing neoplasms in kidneys and testes in Fischer rats and in lungs in male and liver in female B6C3F1 mice.     

Carcinogenicity of bromodichloromethane administered in drinking water to Male F344/N Rats and B6C3F1 mice

A life-time exposure study was conducted to assess the carcinogenicity of bromodichloromethane (BDCM) administered in the drinking water to male F344/N rats and B6C3F(1) mice. In mouse, the calculated mean daily BDCM concentrations (measured concentrations corrected for on-cage loss of chemical) were 0.06, 0.28 and 0.49 g/l. Time-weighted water consumption of 135, 97, and 89 ml/kg/day resulted in mean daily doses of 8.1, 27.2, and 43.4 mg BDCM/kg/day. No changes in feed consumption, final body weight, or survival were observed. Kidney weights were significantly depressed at 27.2 and 43.4 mg BDCM/kg/day. There was no increase in neoplasia in the liver, kidney, spleen, testis, bladder, sections along the alimentary tract, excised lesions, or at any other organ site. In rat, the corrected mean daily BDCM concentrations were 0.06, 0.33, and 0.62 g/l. Time-weighted water consumption of 65, 63, and 59 ml/kg/day yielded 3.9, 20.6 and 36.3 mg BDCM/kg/day. No alterations in feed consumption, body weight gain, and survival were seen. Kidney weight was significantly depressed in the 36.3-mg/kg/day treatment group. There was a significantly enhanced prevalence and multiplicity of hepatocellular adenomas at 3.9 mg BDCM/kg/day (15.5% and 0.16/animal vs. 2.2% and 0.02/animal for the control). Hepatocellular carcinomas increased from 2.2% and 0.02/animal for the control and 3.9 mg BDCM/kg/day to 8.3% and 0.10/animal at 20.6 mg BDCM/kg/day. The combined neoplasms were enhanced at 3.9 and 20.6 mg BDCM/kg/day. Liver neoplasia was depressed to the control value at 36.3 mg BDCM/kg. The prevalence of basophilic and clear cell, but not eosinophilic cells, altered foci of cells declined with increasing dose. BDCM did not increase cancer in the large bowel, renal tubules, or in any of the other tissues examined. Renal tubular hyperplasia was observed at 36.3 mg BDCM/kg (15.8% vs. 8.7% for the control group). Under the conditions of the study, BDCM in the drinking water was not carcinogenic in the male B6C3F(1) mouse, but was carcinogenic in the male F344/N rat based on an increased hepatocellular neoplasia.     

Immunotoxicological profile of chloroform in female B6C3F1 mice when administered in drinking water

Chloroform can be formed as a disinfection by-product during water chlorination, one of the primary modalities for purifying municipal water supplies for human consumption. The aim of this study was to characterize the immunotoxic effects of chloroform in female B6C3F1 mice when exposure occurred via the drinking water. Consistent with human exposure, female B6C3F1 mice were exposed to chloroform-containing drinking water at 2.5, 10, 25, 100, and 250 ppm for 28 days. The examined endpoints included the effects of chloroform on body and organ weights, water consumption, hematology, innate immunity, humoral immunity, and cell-mediated immunity. The functions of natural killer, B-, and T-cells were not altered by chloroform in drinking water at the concentrations tested, except that an increase in splenocyte basal proliferation was observed at chloroform levels of 100 and 250 ppm. Following chloroform administration, there was a decreased number of circulating neutrophils in the blood in all treatment groups, but neutrophil function in lung homogenates, as evaluated using an assay for myeloperoxidase activity following lipopolysaccharide and N-Formyl-Met-Leu-Phe stimulation, was not compromised. Further, the results of host resistance to Listeria monocytogenes infection also suggested that neutrophil function was normal. At the highest treatment level of chloroform (250 ppm), erythrocyte number and hemoglobin levels were significantly decreased. Some significant changes were also observed for body weights, water consumption, and organ weights; however, most of these effects were only observed at the highest treatment level of chloroform (250 ppm). Taken together, the results demonstrate that while chloroform administered via the drinking water affects body weight and selected hematological parameters at high dose levels, overall immune responses, as measured in several tests for immune function, are not compromised.     

Immunological studies in B6C3F1 mice following exposure to ethylene glycol monomethyl ether and its principal metabolite methoxyacetic acid

Exposure to glycol ethers has been associated with adverse effects in laboratory animals including thymus atrophy and mild leukopenia. These effects may involve depletion of immunoresponsive cells. This study examined possible alterations in immune function and host resistance of B6C3F1 mice following exposure to ethylene glycol monomethyl ether (EGME) or its principal metabolite, methoxyacetic acid (MOAA). EGME and MOAA were administered by gavage to mice in 10 doses over a 2-week period at total dosages of 250, 500, and 1000 micrograms/g of body weight. Following exposure, immunopathology, humoral immunity, cell-mediated immunity, macrophage function, and host resistance to Listeria monocytogenes bacterial challenge were examined. A 48% reduction in thymus weight was observed at the intermediate and high doses of both chemicals. No significant alterations in immune function or host resistance to L. monocytogenes were observed in animals exposed to either EGME or MOAA.     

Genetic determinants of hepatocarcinogenesis in the B6C3F1 mouse

The B6C3F1 mouse is highly susceptible to the induction of liver tumors because of the contribution of a specific gene, an allele of the Hcs (Hepatocarcinogen sensitivity) locus, inherited from its C3H inbred parent. This gene affects the rate of growth of preneoplastic hepatic lesions and results in the more rapid appearance of hepatic neoplasms in mice carrying the C3H allele in comparison to mice homozygous for the resistant C57BL/6 allele. The Hcs locus also acts synergistically with at least one class of chemical tumor promoters, the halogenated aromatic hydrocarbons. Because of this genetic promotion of hepatocarcinogenesis, B6C3F1 mice are more sensitive to liver tumor induction by both genotoxic and non-genotoxic carcinogens.     

Subchronic toxicity study of 3-monochloropropane-1,2-diol administered by drinking water to B6C3F1 mice.

3-Monochloropropane-1,2-diol (3-MCPD) is a food processing contaminant in a wide range of foods and ingredients and is a suspected cause of cancer. In this study, the 13-week toxicity of 3-MCPD was examined in B6C3F1 mice (10/sex/group) administered 3-MCPD doses of 0, 5, 25, 100, 200 and 400 ppm dissolved in their drinking water over a 13-week period. All the mice survived to the end of study. The mean body weight gains in the males and females given 400 ppm were significantly lower than those of the controls. The relative kidney weights of the males and females given 200 and 400 ppm were significantly higher than those of the controls without any corresponding histopathological changes. The sperm motility was lower in the 400 ppm group than the control, and there was a significant increase in the incidence of germinal epithelium degeneration in the 200 and 400 ppm groups. A delayed total estrus cycle length was observed in the 400 ppm group without any histopathological changes. Based on these results, the target organ was determined to be kidney, testis, and ovary. The no-observed-adverse-effect level (NOAEL) was found to be 100 ppm (18.05 mg/kg/day for males and 15.02 mg/kg/day for females).     

Hepatocarcinogenicity of Chioral Hydrate, 2-Chloroacetaldehyde, and Dichioroacetic Acid in the Male B6C3F1 Mouse

The chlorinated acetaldehydes, chloral hydrate (CH) and 2-chloroacetaldehyde(CAA), have been identified as chlorination by-products in finisheddrinking water supplies. Although both chemicals are genotoxic,their potential for carcinogenicity had not been adequatelyexplored. The studies reported here are chronic bioassays conductedwith male B6C3F1 mice exposed to levels of 1 g/liter CH and0.1 g/liter CAA via the drinking water for 104 weeks. Distilledwater (H₂O) served as the untreated control and dichloroaceticacid (DCA; 0.5 g/liter), another chlorine disinfection by-product,was included. The mean daily ingested doses were approximately166 mg/kg/day for CH, 17 mg/kg/day for CAA, and 93 mg/kg/dayfor DCA. Evaluations included mortality, body weight, organweights, gross pathology, and histopathology. The primary targetorgan was the liver as the organ weights and pathological changesin the other organs (spleen, kidneys, and testes) were comparablebetween the treated groups and the H₂O control group. Liverweights were increased for all three test chemicals at the terminaleuthanasia with the greatest increase seen in the CH and DCAgroups. Hepatocellular necrosis was induced by all three testchemicals, and it was also most prevalent and severe in theCH and DCA groups. A significant increase in the prevalenceof liver tumors was seen for all three chemicals. The strongestresponse was with DCA, in which 63% of the 104-week survivorshad hepatocellular carcinomas (carcinomas) and 42% possessedhepatocellular adenomas (adenomas) and the combined prevalencefor carcinomas plus adenoma was 75%. The corresponding prevalencerate for carcinomas, adenomas, and combined tumors were 46,29, and 71% 31, 8, and 38% and 10, 5, and 15% for CH, CAA, andH₂O, respectively. In addition to the tumors we evaluated theprevalence of a possible preneoplastic lesion, the hepatocellularhyperplastic nodule (nodules), a lesion which occurred in allthree treated groups but not in the H₂O group.     

Decreased feed consumption and body-weight gain in the B6C3F1 mouse after dietary exposure to 15-acetyldeoxynivalenol

15-Acetyldeoxynivalenol (15-ADON), a biosynthetic precursor of deoxynivalenol (DON), was extracted from rice cultures of Fusarium graminearum R6576 and purified. Growing female B6C3F₁ mice were fed semi-purified diets containing 0, 0.5, 2.0 and 5.0 ppm 15-ADON over 56 days and assessed for effects on feed intake, body-weight gain, terminal organ weights and blood clotting function. A significant reduction in feed intake was observed at the 5.0-ppm level after 44 days, whereas reduced rates of weight gain were found to occur at the 5.0-ppm level after only 16 days. Terminal liver, kidney and spleen weights were significantly lower in mice consuming the 5.0-ppm diet when compared with controls. Dietary 15-ADON at the 0.5- and 2.0-ppm levels did not show significant effects on weight gain, feed intake or organ weights. Although mice treated with 15-ADON had significantly decreased bleeding times, other measurements of clotting function indicated no differences between the control and treated groups. Results indicated that 15-ADON was only slightly less toxic than DON and that chronic manifestations of dietary 15-ADON were similar to those found previously for DON. Future risk assessments for DON should therefore include consideration of 15-ADON occurrence and toxicity.     

Oxymetholone modulates cell-mediated immunity in male B6C3F1 mice

Oxymetholone is a synthetic androgen, structurally related to testosterone. It is currently used to treat anemias, but has also been abused as a performance enhancing anabolic steroid by the sport community. Concern about its suspected immunomodulatory properties provided the incentive for a detailed investigation into its effects on the mammalian immune system. In this study, male B6C3F1 mice were treated for 14 d with oxymetholone (0, 50, 150, and 300 mg/kg) by gastric intubation, then evaluated for immunotoxicity using a panel of immunotoxicity assays. Except for an increasing trend in kidney and liver weights, and a dose-dependent increase in serum blood urea nitrogen levels, no other signs of systemic toxicity were observed. Bone marrow DNA synthesis was reduced, though this did not translate into alterations in myeloid or monocyte colony forming units. Spleen B and T cell numbers, antibody response to sheep red blood cells, proliferative response to both mitogen and immunoglobulin receptor immunogens, and NK cell activity were all unaltered in mice treated with oxymetholone. Peritoneal macrophage activity was also unaffected by oxymetholone treatment. A 38% decrease in the spleen cell mixed leukocyte response, and a 15% decrease in cytotoxic T cell activity, measured in the highest oxymetholone treatment group, indicate that cell-mediated immunity was impaired following exposure. This immunomodulation did not however, translate into a change in host resistance to Listeria monocytogenes.     

Carcinogenesis studies of 4,4'-methylenedianiline dihydrochloride given in drinking water to F344/N rats and B6C3F1 mice

Carcinogenesis studies of 4,4'-methylenedianiline dihydrochloride (98.6% pure) were conducted by administering this chemical in the drinking water of F344/N rats and B6C3F1 mice. Groups of 50 rats and 50 mice of each sex received drinking water containing 150 or 300 ppm 4,4'-methylenedianiline dihydrochloride (dosage expressed as the free base) for 103 wk. Groups of 50 rats and 50 mice of each sex, given drinking water adjusted with 0.1 N HCl to the pH (3.7) of the 300-ppm formulation, served as controls. Survival was comparable among groups except for male mice receiving the 300-ppm dose of 4,4'-methylenedianiline dihydrochloride; survival in that group was lower than that in controls. Mean body weight was reduced in 300-ppm-dose female rats and 300-ppm-dose male and female mice compared to controls. Water consumption was reduced in a dose-related manner in both sexes of rats. No compound-related clinical effects were observed. Under the conditions of these studies, there was clear evidence of carcinogenicity for F344/N rats and for B6C3F1 mice in that 4,4'-methylenedianiline dihydrochloride caused increased incidences of (1) follicular-cell carcinomas of the thyroid gland (controls, 0/49; low dose, 0/47; high dose, 7/48, 15%; p less than or equal to 0.012) and neoplastic nodules of the liver (controls, 1/50, 2%; low dose, 12/50, 24%; high dose, 25/50, 50%; p less than or equal to 0.001) in male rats, (2) follicular-cell adenomas (controls, 0/47; low dose, 2/47, 4%; high dose, 17/48, 35%; p less than or equal to 0.001) and C-cell adenomas (controls, 0/47; low dose, 3/47, 6%; high dose, 6/48, 13% p less than or equal to 0.029) of the thyroid gland in female rats, (3) follicular-cell adenomas of the thyroid gland (controls, 0/47; low dose, 3/49, 6%; high dose, 16/49, 33%; p less than or equal to 0.001), carcinomas of the liver (controls, 10/49, 20%; low dose, 33/50, 66%; high dose, 29/50, 58%; p less than or equal to 0.001), and pheochromocytomas of the adrenal gland in male mice (controls, 2/48, 4%; low dose, 12/49, 24%; high dose, 14/49, 29%; p less than or equal to 0.001), and (4) follicular-cell adenomas of the thyroid gland (controls, 0/50; low dose, 1/47, 2%; high dose, 13/50, 26%; p less than or equal to 0.001), carcinomas (controls, 1/50, 2%; low dose, 6/50, 12%; high dose, 11/50, 22%; p less than or equal to 0.002) and adenomas (controls, 3/50, 6%; low dose, 9/50, 18%; high dose, 12/50, 24%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Toxicokinetics of chloral hydrate in ad libitum-fed, dietary-controlled, and calorically restricted male B6C3F1 mice following short-term exposure

Chloral hydrate is widely used as a sedative in pediatric medicine and is a by-product of water chlorination and a metabolic intermediate in the biotransformation of trichloroethylene. Chloral hydrate and its major metabolite, trichloroacetic acid, induce liver tumors in B6C3F1 mice, a strain that can exhibit high rates of background liver tumor incidence, which is associated with increased body weight. This report describes the influence of diet and body weight on the acute toxicity, hepatic enzyme response, and toxickinetics of chloral hydrate as part of a larger study investigating the carcinogenicity of chloral hydrate in ad libitum-fed and dietary controlled mice. Dietary control involves moderate food restriction to maintain the test animals at an idealized body weight. Mice were dosed with chloral hydrate at 0, 50, 100, 250, 500, and 1000 mg/kg daily, 5 days/week, by aqueous gavage for 2 weekly dosing cycles. Three diet groups were used: ad libitum, dietary control, and 40% caloric restriction. Both dietary control and caloric restriction slightly reduced acute toxicity of high doses of chloral hydrate and potentiated the induction of hepatic enzymes associated with peroxisome proliferation. Chloral hydrate toxicokinetics were investigated using blood samples obtained by sequential tail clipping and a microscale gas chromatography technique. It was rapidly cleared from serum within 3 h of dosing. Trichloroacetate was the major metabolite in serum in all three diet groups. Although the area under the curve values for serum trichloroacetate were slightly greater in the dietary controlled and calorically restricted groups than in the ad libitum-fed groups, this increase did not appear to completely account for the potentiation of hepatic enzyme induction by dietary restriction.