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Mutagenicity testing in mammalian cells. II. Validation of multiple drug-resistance markers having practical
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.
Insights
Chinese hamster ovary cells were used to identify drug-resistant mutants, revealing locus-specific sensitivities to mutagens. The thymidine kinase (tk) locus showed the greatest response, suggesting its utility for mutagen screening.
Area of Science:
- * Genetics and molecular toxicology
- * Mammalian cell mutation assays
Background:
- * Chinese hamster ovary (CHO) cells are widely used for genotoxicity testing.
- * Developing sensitive assays for mutagen detection is crucial for risk assessment.
Purpose of the Study:
- * To characterize mutation frequencies and patterns in CHO cells using various drug-resistance markers.
- * To evaluate the utility of different loci for detecting mutagenic effects.
Main Methods:
- * Utilized CHO cell lines heterozygous for adenine phosphoribosyltransferase (aprt) and thymidine kinase (tk) loci.
- * Selected spontaneous and induced mutants resistant to 8-azaadenine (AAr), 6-thioguanine (TGr), ouabain (OUAR), and 5-fluorodeoxyuridine (FUdRr).
- * Treated cells with direct mutagens (EMS, MNNG, NQO) and promutagens (DMN, BP) activated by rat-liver homogenates.
Main Results:
- * Established optimal plating densities for all drug-resistance markers.
- * Determined induced mutant frequencies as a function of relative cell survival for different mutagens and loci.
- * Observed locus-specific differences in sensitivity, with the tk locus showing the largest mutational response.
- * Characterized biochemical properties of resistant mutants, with high percentages exhibiting deficient enzyme activity.
Conclusions:
- * The thymidine kinase (tk) locus is highly responsive to mutagenic treatment.
- * 5-fluorodeoxyuridine resistance (FUdRr) exhibits rapid expression kinetics and may detect a broad spectrum of genetic lesions.
- * The FUdRr marker is a promising candidate for a sensitive screening assay for potential mutagens.