title: 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.
mesh_terms: D015112:4-Nitroquinoline-1-oxide / Q000494:pharmacology; D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D000228:Adenine Phosphoribosyltransferase / Q000235:genetics*; D000818:Animals; D001580:Benzopyrenes / Q000494:pharmacology; D001711:Biotransformation; D006224:Cricetinae; D003412:Cricetulus; D004128:Dimethylnitrosamine / Q000494:pharmacology; D004351:Drug Resistance; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D005467:Floxuridine / Q000378:metabolism; D005819:Genetic Markers; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008862:Microsomes, Liver / Q000378:metabolism; D009152:Mutagenicity Tests / Q000379:methods*; D010042:Ouabain / Q000378:metabolism; D010430:Pentosyltransferases / Q000235:genetics*; D051381:Rats; D013866:Thioguanine / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics*
chemical_list: D001580:Benzopyrenes; D005819:Genetic Markers; D005467:Floxuridine; D008769:Methylnitronitrosoguanidine; D015112:4-Nitroquinoline-1-oxide; D010042:Ouabain; D005020:Ethyl Methanesulfonate; D010430:Pentosyltransferases; D000228:Adenine Phosphoribosyltransferase; D013937:Thymidine Kinase; D013866:Thioguanine; D000225:Adenine; D004128:Dimethylnitrosamine; C006654:8-azaadenine