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 (AAr), 6-thioguanine (TGr), ouabain (OUAR), or 5-fluorodeoxyuridine (FUdRr). Mutation data are reported for direct mutagens (EMS, ethyl methanesulfonate; 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 AAr, TGr, OUAR and FUdRr. The induced mutant frequencies as a function of relative cell survival after treatment with EMS, DMN or BP were 2--4 d for AAr, 6--8 d for TGr, 3 d for OUAR, and 1--3 d for FUdRr. 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 less than or equal to 5% wild-type activity; of 30 TGr clones assayed, 83% had less than or equal to 5% wild-type HGPRT activity. Of 42 FUdRr clones assayed, 98% had less than or equal to 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 FUdRr 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.