Carcinogenicity of the anticancer topoisomerase inhibitor, amsacrine, in Wistar rats.

Amsacrine is an antineoplastic drug used in the treatment of acute adult leukemias. To assess its carcinogenic potential, groups of 50 male and 50 female rats were administered amsacrine by lateral tail vein injection at 0 (vehicle control), 0.25, 1, or 3 mg/kg once daily for 5 days, followed by a 23-day recovery period. This cycle of dosing and recovery was repeated a total of six times. The animals were then maintained without dosing for an 18-month observation period. During the dosing phase, signs of toxicity were limited to the 3 mg/kg animals and included alopecia, diarrhea, injection site lesions, and skin and subcutaneous nodules. Statistically significant reductions in body weight gain and food consumption also occurred at 3 mg/kg during each 5-day dosing period followed by recovery during the latter 3 weeks of each cycle. Except for skin and subcutaneous nodules, signs of toxicity in the 3 mg/kg animals ultimately disappeared during the 18-month observation phase. Survival at study termination for the vehicle control, 0.25, 1, and 3 mg/kg groups was 56, 52, 34, and 0%, respectively, in males, and 64, 48, 54, and 4%, respectively, in females. Mortality was primarily due to bone marrow suppression during the dosing phase, chronic progressive nephropathy, or development of tumors. Incidences of the following tumors were significantly increased in the 3 mg/kg groups of both sexes (Fisher exact test, two-tailed, p < 0.01): all malignancies; all tumors of the small intestine, adenocarcinoma and adenoma of the small intestine, all tumors of the skin, and squamous cell papilloma. Other tumor incidences that were significantly increased in the 3 mg/kg males were thymoma and multiple neoplastic histotypes of the skin and adnexa including basal cell tumor, fibroma, sebaceous gland adenoma, and squamous cell carcinoma. A disproportionate number of the skin tumors were located on the tail, suggesting a localized tissue concentration effect. In the 3 mg/kg females, significantly increased tumor incidences also included all tumors of the mammary gland, adenocarcinoma of the mammary gland, all tumors of the uterine horn, and endometrial stromal polyps of the uterine horn. The 1 mg/kg males had significantly increased incidences of all tumors of the small intestine and skin, adenocarcinoma of the small intestine, and fibroma of the skin. Fibroma of the skin was also significantly increased in the 0.25 mg/kg males. Incidences of all tumors and all benign tumors were significantly increased in the 1 mg/kg females. There were no significantly increased tumor incidences in the 0.25 mg/kg females. The results of this study show that amsacrine is carcinogenic in Wistar rats. Target organs for tumorigenicity include small intestine, skin, mammary gland, thymus, and uterus.

Neurotoxicity of misonidazole in rats following intravenous administration.

Misonidazole is a hypoxic cell radiosensitizer that induces a peripheral neuropathy in humans after exceeding a schedule-dependent cumulative threshold dose. Clinical studies of misonidazole have been conducted using oral administration, whereas most other radiosensitizers have been administered intravenously. Since route of exposure can potentially influence the toxicity of xenobiotics, the objective of this study was to assess the neurotoxicity of misonidazole in rats following intravenous dosing using a battery of routine clinical, neurofunctional, biochemical, and histopathologic screening methods. Male Sprague-Dawley rats were administered intravenous doses of misonidazole at 0 (vehicle control), 100, 200, 300, or 400 mg kg-1 once per day, 5 days per week, for 2 weeks. Animals were evaluated for neurofunctional and pathological changes following termination of treatment (Days 15-17) and at the end of a 4 week observation period (Days 43-45). During the dosing phase, hypoactivity, salivation, rhinorrhea, chromodacryorrhea, rough pelage and ataxia were observed at 400 mg kg-1, and body weight gain of the 300 and 400 mg kg-1 groups was significantly decreased relative to the vehicle controls by 24% and 49%, respectively. Corresponding reductions in food consumption were 8% and 23%, respectively. Although most 400 mg kg-1 animals appeared normal on Day 15 prior to the neurofunctional evaluations, rotorod testing precipitated a number of clinical signs including: ataxia, impaired righting reflex, excessive rearing, tremors, vocalization, circling, head jerking, excessive sniffing and hyperactivity. All of these animals recovered and appeared normal from Day 17 through study termination. There were no treatment-related effects on motor activity, acoustic startle response, rotorod performance, forelimb group strength, toe and tail pinch reflexes, tibial nerve beta-glucuronidase activity or tail nerve conduction velocity. Although hindlimb grip strength of the 400 mg kg-1 group was significantly decreased by 17% relative to the vehicle controls on Day 15, this finding appeared related to the reduced food consumption and body weight gain in these animals. No microscopic changes were detected in peripheral nerves. Necrosis and proliferation of fibrillary astrocytes (gliosis) were seen in the cerebellum and medulla of the 400 mg kg-1 animals on Day 16. Gliosis in these same brain regions was observed in the 300 and 400 mg kg-1 groups on Day 44. The results show that intravenous administration of misonidazole to rats causes dose-limiting central nervous system toxicity without effects on peripheral nervous tissue. The lack of peripheral neurotoxicity was most likely due to a combination of several interrelated factors including route of administration, duration and intensity of the dosing regimen, and total cumulative dose.

Toxicology and carcinogenicity studies of diuretics in F344 rats and B6C3F1 mice. 1. Hydrochlorothiazide.

Toxicology and carcinogenesis studies of hydrochlorothiazide, a benzothiadiazide diuretic, were conducted by administering diets containing the drug to both sexes of F344 rats and B6C3F1 mice in 15-day, 13-week and 2-year studies. No rats died during the 15-day or 13-week studies at dietary concentrations of up to 50,000 ppm. Deaths of male mice in the top dose group in the 13-week study were likely to be related to chemical administration. In the prechronic studies, increased nephrosis and mineralization at the kidney corticomedullary junction were the primary toxic effects of hydrochlorothiazide observed in rats. In mice, chemical-related effects included nephrosis and calculi, inflammation and epithelial hyperplasia in the urinary bladder. In 2-year studies using dietary concentrations of 0, 250, 500 and 2000 ppm in rats and 0, 2500 and 5000 ppm in mice, survival of dosed and control groups of rats and mice was similar, as were body weights of mice. Dosed groups of male and female rats were uniformly lighter than controls (up to 25%) throughout the studies. Severe chronic renal disease with secondary parathyroid hyperplasia and fibrous osteodystrophy of the bone were attributed to chemical administration in rats. No neoplasms in rats or female mice or non-neoplastic lesions in mice were associated with hydrochlorothiazide. In high-dose male mice, liver neoplasms were increased but were not considered to be related to hydrochlorothiazide administration because of an unusually low incidence in the control group relative to historical controls.

Toxicology and carcinogenicity studies of diuretics in F344 rats and B6C3F1 mice. 2. Furosemide.

Toxicology and carcinogenesis studies of furosemide, a widely used diuretic, were conducted by administering diets containing the drug to both sexes of F344 rats and B6C3F1 mice in 14-day, 13-week and 2-year studies. Deaths occurred among rats and mice receiving diets containing 46,000 ppm furosemide in 14-day studies, and animals given diets containing lower concentrations lost weight. No deaths were seen in 13-week studies using top concentrations ranging from 10,000 to 20,000 ppm, but animals at higher concentrations had lower weight gains than controls. Nephrosis in rats and mice was the only significant compound-related lesion observed in the prechronic studies. In 2-year studies, rats received diets containing 0, 350 or 700 ppm furosemide and mice received diets containing 0, 700 or 1400 ppm furosemide. Survival of dosed and control rats of both sexes and male mice was similar; survival of high-dose female mice was lower than controls. Nephropathy was increased in male rats and in male and female mice. In female mice, increased malignant tumors of the mammary gland were associated with furosemide administration. In male rats, marginal increases in tubular cell neoplasms of the kidney and in meningiomas of the brain were observed in dosed animals, but these were not considered to be related clearly to exposure to furosemide.

Examination of genotoxicity, toxicity and morphologic alterations in hepatocytes following in vivo or in vitro exposure to methapyrilene.

The antihistamine methapyrilene (MP) was widely used as a component of cold, allergy and sleep-aid medications in the 1970s until it was identified as a potent rat liver carcinogen. MP does not induce positive responses in most short-term genotoxicity assays, which suggests that it is carcinogenic by a non-genotoxic mechanism. We have evaluated the potential of MP to induce unscheduled DNA synthesis (UDS), a genetic end point and S-phase synthesis (SPS), and indicator of cell proliferation, in Fischer-344 (F344) rat and B6C3F1 mouse liver. We also examined the response of MP in hepatocytes from two species treated in vitro. MP failed to induce UDS in rat or mouse liver following in vivo treatment, or in hepatocytes from rat and adult human treated in vitro. Control rats and mice yielded less than 0.3% of cells in S-phase (%S). In contrast, MP induced significant elevations in SPS both in male F344 rat (6.3%S) and female B6C3F1 mice (1.4%S). In the male rat, sorbitol dehydrogenase (SDH), bilirubin, serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvate transaminase (SGPT) showed elevations of 9-, 10-. 17- and 28-fold over controls respectively, indicating that significant hepatotoxicity was induced by MP. This was confirmed by histopathologic examination, which revealed significant periportal and focal necrosis followed by an increased presence of mitotic figures. These results indicate that MP is not genotoxic in rat liver, but is a potent inducer of hepatic cell proliferation by inducing toxicity and subsequent regeneration, which may be an important mechanism of hepatocarcinogenesis.

Differences in rat liver enzyme-altered foci produced by chlorinated aliphatics and phenobarbital.

Nine chlorinated aliphatics (CAs)--1,1-dichloroethane, 1,2-dichloroethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane, trichloroethylene, tetrachloroethylene, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane, and hexachloroethane--were examined in a rat liver foci assay for evidence of initiating and promoting potential. Young adult male Osborne-Mendel rats (ten/group) were given partial hepatectomies, followed 24 hr later by a single i.p. dose of either diethylnitrosamine (30 mg/kg body weight) or CA, 1 wk later either a diet containing 0.05% (w/w) phenobarbital or daily oral gavage (5 X/wk) of CA in corn oil for 7 weeks, and sacrificed 1 wk later. Putative preneoplastic markers monitored were foci with increased gamma-glutamyltranspeptidase activity [GGT(+)]. CAs were without significant effect in the initiation protocol at the maximum tolerated dose. In the promotion protocol, 1,1-dichloroethane, 1,1,2-trichloroethane, tetrachloroethylene, 1,1,2,2-tetrachloroethane, and hexachloroethane induced significant increases in GGT(+) foci above control levels. Two variants of GGT(+) foci were distinguishable, one associated predominantly with phenobarbital promotion, resembling preneoplastic foci in other models, and the other associated with CA promotion, which was less intensely stained and exhibited branching, resembling foci undergoing redifferentiation. The marked differences in response may relate to differences in cytotoxic potential or mechanism of action of the two types of agents.

Carcinogenicity of chloroform in drinking water to male Osborne-Mendel rats and female B6C3F1 mice.

The carcinogenic activity of chloroform administered at 0, 200, 400, 900, and 1800 mg/liter in drinking water was studied in male Osborne-Mendel rats and female B6C3F1 mice. A second control group was included in the study and was restricted to the water consumption of the high-dose group. Animals were maintained on study for 104 weeks. Group sizes were adjusted at low doses such that a detectable tumor response would result at the lowest dose if there was a linear relationship with dose, and the higher doses produced responses similar to previous carcinogenesis bioassays of chloroform. The primary finding was that chloroform increased the yield of renal tubular adenomas and adenocarcinomas in male rats in a dose-related manner. For the high-dose group, which corresponded to a time-weighted average dose of 160 mg/kg per day for 104 weeks, there was a 14% incidence of renal tubular adenomas and adenocarcinomas, vs 1% in the control group. This compares to a 24% incidence observed when 180 mg/kg per day of chloroform was administered for 78 weeks in earlier studies. In contrast, chloroform in the drinking water of mice failed to increase the incidence of hepatocellular carcinomas in female B6C3F1 mice. The highest dose group received a time-weighted average dose of 263 mg/kg for 104 weeks, resulting in a 5% combined incidence of hepatocellular adenoma and carcinoma relative to a 6% incidence in the control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Irreversibility of liver tumors in C3H mice.

The present study was designed to test the possibility that spontaneous regression of hepatocellular tumors might be observed in mice. This problem was studied by sequential liver biopsies in C3H male mice that had been treated with dieldrin (CAS: 60-57-1) as well as in animals treated with N-diethylnitrosamine (CAS: 55-18-5) and in untreated control mice. Adenomas were seen in some animals at the second laparotomy when there had been no tumor at the first laparotomy. In a few mice there was histologic progression from adenoma to carcinoma. A change in predominant cell type in adenomas from clear to basophilic or eosinophilic was also observed in some cases. Additional hepatocellular carcinomas were observed in some animals necropsied at 2 years of age. These observations suggest that spontaneous hepatic tumors and tumors in mice treated with either complete carcinogens or nongenotoxic compounds have a strong tendency to progress. Tumor regression in mice appears to be unusual. No consistent relationship of histologic grade of hepatocarcinoma to the type of chemical employed was observed.

Dieldrin-induced mallory bodies in hepatic tumors of mice of different strains.

Mallory bodies (MBs) were induced in hepatic tumors by administration for up to 85 weeks of a diet containing 10 ppm dieldrin to 50 C3H/He and 62 C57BL/6J x C3H/He B6C3F1 male mice. MBs were seen in 15 of 28 (54%) mice which developed benign hepatic tumors and 33 of 45 (73%) mice with hepatocellular carcinoma, but in only 3 of 39 (8%) mice without hepatic tumors. In mice with tumors, the MBs were predominantly confined to tumor tissue and persisted in a carcinoma transplanted into a nude mouse. MBs were not observed, however, in hepatic tumors of 67 C57BL/6J, 49 C3H/He, or 81 B6C3F1 mice given 12 micrograms diethylnitrosamine i.p. on Days 0, 3, 9, and 15. Thirty-one of 195 control mice of all three strains had hepatic tumors. Only one of the controls had a tumor with an MB, and no MBs were seen in nontumor-bearing livers of controls animals. These observations, coupled with the results of a previous study in which MBs were observed in hepatocytes of dieldrin-treated C57BL/6J mice, indicate that mice treated with dieldrin are a reliable animal model for the study of MBs.

Enzyme histochemical characteristics of spontaneous and induced hepatocellular neoplasms in mice.

Hepatocellular neoplasms are known to differ in enzyme activity from the surrounding non-neoplastic liver. We have compared histochemically the enzyme activity of spontaneous hepatocellular tumors in mice with tumors induced by diethylnitrosamine and dieldrin. Some neoplasms had increased activity, others had decreased enzyme activity, yet other had the same activity as the surrounding liver. Alkaline phosphatase, glucose-6-phosphatase, succinic dehydrogenase and adenosine triphosphatase, as well as glycogen levels were studied. Carcinomas differed from adenomas in having elevated enzyme activity significantly more often than adenomas. However, the carcinomas showed elevated glycogen levels less frequently than adenomas. Histochemically, pulmonary metastases resembled the primary hepatocellular carcinomas from which they were derived. Tumors of dieldrin animals were notable in having increased activity of all the enzymes which we studied more frequently than tumors of diethylnitrosamine animals or of controls. Differences in enzyme activity between the three mouse strains were slight.

Mallory body formation in hepatic nodules of mice ingesting dieldrin.

Mallory bodies (MBs) were observed in hepatic nodules induced by long-term administration of a diet containing 10 p.p.m. of dieldrin to C57BL/6 mice. MBs were first detected after 46 weeks and were seen in 26 of 41 mice which developed hepatic nodules. The MBs were limited to the nodules in 25 mice. Twenty-one of the nodules were carcinomas and 20 of those contained MBs. Our observations suggest that MBs may be a marker for neoplastic transformation and may prove useful in experimental studies of carcinogenesis.

Atrioventricular bundle degeneration associated with sudden death in the dog.

Twelve dogs that were necropsied after sudden unexpected death, sudden episodes of viciousness, or seizure disorder were found to have degeneration of the atrioventricular bundle of the heart. In addition, hypoxic-type degeneration was found in the hippocampus and the dorsal one-half of the midportion of the cerebral cortex of the dogs.

Leiomyosarcoma of the urinary bladder in a cat.

An 81/2-year-old female domestic short-haired cat was admitted with signs of chronic cystitis. Radiography revealed marked and irregular thickening of the bladder wall. Biopsy revealed the thickening to be a malignant tumor of muscle origin and treatment with proteolytic enzymes was instituted. After one month, the cat was euthanatized because of uremia associated with progressive tumor growth. At necropsy the bladder was grossly thickened. The histopathologic diagnosis was leiomyosarcoma.