Mutagenicity testing in mammalian cells: II. Validation of multiple drug-resistance markers having practical application for screening potential mutagens

Abstract

Chinese hamster ovary (CHO) cell lines heterozygous at both the adenine phosphoribosyltransferase ( aprt) and thymidine kinase ( tk) loci were used for single-step selection of spontaneous and induced mutants resistant to 8-azaadenine (AA r), 6-thioguanine (TG r), ouabain (OUA r), or 5-fluorodeoxyuridine (FUdR r). Mutation data are reported for direct mutagens (EMS, ethyl methane-sulfonate; MNNG, N-methyl- N′-nitro- N-nitrosoguanidine; NQO, 4-nitroquinoline 1-oxide) and promutagens (DMN, dimethylnitrosamine; BP, benzo[ a]-pyrene) activated by rat-liver homogenates. Optimal plating densities were established for AA r, TG r, OUA r and FUdR r. The induced mutant frequencies as a function of relative cell survival after treatment with EMS, DMN or BP were 2–4 d for AA r, 6–8 d for TG r, 1–3 d for FUdR r. The induced mutant frequencies as a function of relative cell survival after treatment with EMS, DMN or BP showed locus-specific differences in sensitivity. Of 61 clonal isolates resistant to AA and assayed for APRT activity, 87% had ⩽ 5% wild-type activity; of 30 TG r clones assayed, 83% had ⩽ 5% wild-type HGPRT activity. Of 42 FUdR r clones assayed, 98% had ⩽ 1% wild-type TK activity. 50 clones selected in medium containing FUdR displayed cross-resistance to 5-bromodeoxyuridine (BUdR) and trifluorothymidine (TFT) and all were sensitive to HAT (hypoxanthine-amethopterin-thymidine) medium. The tk locus showed the largest mutational response as a function of cell survival after mutagen treatment. The rapid expression kinetics for FUdR r and the possibility that the locus detects a broader spectrum of genetic lesions than the other drug-resistance markers are discussed in terms of a sensitive screening assay for detecting potential mutagens.