Establishment of a Chinese hamster breeding colony.

A colony of outbred Chinese hamsters, Cricetulus griseus, was established. Attempts to use previously published methods of physically protecting the males were unsatisfactory. Rather, females were selected for nonaggressive behavior. Breeding was among pairs allowed to cohabit for 5 1/2 days. Production was typically in excess of 180 offspring per month, with an average breeder population of 44 females and 30 males. Litters were weaned at 3 weeks of age. At that time, appropriate numbers of offspring of female parents with a history including nonaggressive behavior, fecundity and freedom from seizures were retained for use as future breeders. Females were first bred at 8-12 weeks of age and males at 8-12 months. The remaining animals were maintained in groups of like-sex littermates until used. With these methods, mortality among breeder males due to overly aggressive female behavior was reduced to 3%. Animal husbandry procedures and record keeping requirements were relatively simple, not labor intensive and would be easily adaptable to most facilities.

Evaluation of ochratoxin A for mutagenicity in a battery of bacterial and mammalian cell assays.

Ochratoxin A (OA), a nephrotoxic mycotoxin, was evaluated for genotoxic potential in a battery of in vitro and in vivo assays. OA was not mutagenic to Salmonella typhimurium, either with or without metabolic activation, in the plate incorporation (Ames) test at concentrations of 50-600 micrograms OA/plate or in the gradient plate assay at concentrations of 0.1-1000 micrograms OA/ml. No induction of unscheduled DNA synthesis was evident in primary cultures of rat hepatocytes exposed to concentrations of OA ranging from 0.000025 to 500 micrograms/ml. In the mouse lymphoma forward mutation assay, exposure of L5178Y TK+/- mouse lymphoma cells to OA did not increase the numbers of L5178Y TK-/- mutants. There was no significant difference between the numbers of sister-chromatid exchanges in cells from OA-treated Chinese hamsters and those in cells from the negative-control animals.

Assessment of sister chromatid exchange in spermatogonia and intestinal epithelium in Chinese hamsters.

The induction of sister chromatid exchange (SCE) has been proposed as a predictive test for the identification of mutagens/carcinogens. The in vivo application of this test was investigated by examining the chemical induction of SCE in spermatogonia, intestinal epithelium and bone marrow cells from Chinese hamsters. Sister chromatid differentiation (SCD) was achieved in differentiating spermatogonial cells of male Chinese hamsters by the abdominal subcutaneous (sc) implantation of an agar-coated bromodeoxyuridine (BrdUrd) tablet. A number of genotoxins were administered intraperitoneally (ip) and the induction of SCE in spermatogonia and bone marrow was compared. A significant increase in SCE frequency in spermatogonia occurred following treatment with mitomycin C (MMC), cyclophosphamide (CP), or N,N',N"-triethylenethiophosphoramide (ThioTEPA). Treatment with busulfan, hycanthone (HC), or triethylenemelamine (TEM) failed to induce SCE in vivo in spermatogonia, but these compounds did induce SCE in bone marrow. Differences in cell cycle kinetics were considered to be the major factor involved in the differential induction of SCE in spermatogonia and bone marrow. The induction of SCE in intestinal epithelium was investigated as a system for the identification of genotoxins that may result from the metabolism of xenobiotics by the gastrointestinal flora. Nitro-aromatic compounds were administered orally to Chinese hamsters. Nitro-aromatic compounds were chosen for this study since the mutagenic activity of these compounds is thought to result from their metabolism by bacterial nitroreductase. Metronidazole (MN) and 2-nitro-p-phenylenediamine (2NPPD) induced a dose-related increase in SCE formation in intestinal epithelium but not in bone marrow. Treatment with 3-nitro-o-phenylenediamine (3NOPD) or 4-nitro-o-phenylenediamine (4NOPD) did not induce the formation of SCE in either intestinal epithelium or bone marrow. These findings indicate that studies in axenic animals will be required to elucidate the contribution of the enteric flora to the metabolic activation of some genotoxins.

Chemically-induced sister-chromatid exchange in vivo in bone marrow of Chinese hamsters. An evaluation of 24 compounds.

Chemically-induced sister-chromatid exchange (SCE) was measured in vivo in bone marrow of Chinese hamsters. Chemicals were administered either intraperitoneally or orally and increased SCE frequencies were noted with 6 of 6 direct-acting genotoxins and with 9 of 14 activation-dependent genotoxins. Metronidazole, o-toluidine, 4-nitro-o-phenylenediamine and 2-nitro-p-phenylenediamine, compounds which have shown either mutagenic or carcinogenic activity, did not induce SCE in vivo, 4 non-genotoxins and 4 different control treatments did not induce SCE. The results show that the in vivo SCE method may be useful for the identification of genotoxins and that the outcome of the test is, for certain chemicals, dependent upon the route of exposure.

Chemically-induced unscheduled DNA synthesis in primary rat hepatocyte cultures: a comparison with bacterial mutagenicity using 218 compounds.

The autoradiographic identification of unscheduled DNA synthesis (UDS) in primary cultures of adult rat hepatocytes (HPC) has been proposed as a predictive test for mutagens/carcinogens. To assess the predictive value of this test, results in the hepatocyte UDS assay were compared with data for bacterial mutagenicity using a modified Ames test. Over 200 compounds representing a variety of chemical classes consisting of procarcinogens, ultimate carcinogens, and noncarcinogens were tested in each system. The accurate discrimination of many carcinogens/noncarcinogens was demonstrated by both systems. The induction of UDS in hepatocytes showed an excellent correlation with bacterial mutagenesis in response to polycyclic aromatic hydrocarbons, aromatic amines, biphenyls, nitrosamines, carbamates, azo-compounds, acridines, halogenated compounds, nitrosureas, quinolines, pyridines, purines, pyrimidines, esters and carbamates. Nitrocompounds, although active in bacteria, were poor inducers of UDS. The results support the complementary and confirmatory nature of these tests for genotoxic chemicals and indicate the usefulness of the hepatocyte UDS system as a component in a battery of short-term predictive tests for mutagens/carcinogens.

The induction of unscheduled DNA synthesis by antihistamines in primary hepatocyte cultures.

The autoradiographic identification of chemically-induced, unscheduled DNA synthesis in primary cultures of adult rat hepatocytes is currently being validated as a predictive test for mutagens/carcinogens. Of 8 antihistaminic drugs tested, 2, pyrilamine maleate and tripelennamine HCl, were positive for unscheduled DNA synthesis. Further investigation of the mutagenic/carcinogenic potential of these compounds in alternate test systems is suggested.