A microtiter plate assay for the selection of 6-thioguanine-resistant mutants in Chinese hamster V79 cells in the presence of phorbol-12-myristate-13-acetate.
6-Thioguanine-resistant mutants can be efficiently recovered from Chinese hamster V79 cells incubated at high cell densities in microtiter plates (10(3)-10(4) cells/0.2 ml growth medium/0.4 cm2) when selected with 30 microM 6-thioguanine and 0.1 microgram/ml phorbol-12-myristate-13-acetate, an inhibitor of metabolic cooperation among V79 cells. Mutant frequencies in the microtiter plates were calculated from a direct count of mutant colonies. After treatment of the V79 cells with the carcinogen benzo[a]pyrene in a fibroblast-mediated assay, the mutation frequencies determined with the microtiter assay system were quantitatively similar to those obtained with a conventional procedure in which selection with 6-thioguanine was performed in petri dishes. The mutagenic activities of 3 polycyclic aromatic hydrocarbons (activated in the cell-mediated assay) were assessed with the microtiter plate selection procedure. The active carcinogen benzo[a]pyrene at 1 microgram/ml yielded about 100 mutants per 10(5) colony-forming cells. The same dose of a less active carcinogen, cyclopenta[c,d]pyrene, yielded about 20 mutants per 10(5) colony-forming cells, and benz[a]anthracene, not an active carcinogen, was inactive as a mutagen at all doses tested. Because of the small requirements for growth medium and tissue culture vessels compared with other assays, this microtiter plate assay can serve as an inexpensive system for detecting the mutagenic activity of environmental chemicals in mammalian cells.
D000818:Animals; D001551:Benz(a)Anthracenes / Q000494:pharmacology; D001564:Benzo(a)pyrene; D001580:Benzopyrenes / Q000494:pharmacology; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance; D005347:Fibroblasts; D009152:Mutagenicity Tests / Q000379:methods*; D011721:Pyrenes / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate; D013866:Thioguanine / Q000494:pharmacology*
D001551:Benz(a)Anthracenes; D001580:Benzopyrenes; D011721:Pyrenes; C016328:cyclopenta(c,d)pyrene; D001564:Benzo(a)pyrene; C030935:benz(a)anthracene; D013866:Thioguanine; D013755:Tetradecanoylphorbol Acetate