A procedure for the CHO/HGPRT mutation assay involving treatment of cells in suspension culture and selection of mutants in soft-agar.
A procedure involving treatment of cells in suspension culture and soft-agar cloning was developed for measuring mutation of Chinese hamster ovary (CHO) cells to 6-thioguanine (6TG) resistance. The use of suspension cultures precluded the need for trypsinization and also permitted a 5-fold increase in cell population for compound exposure and mutant selection as compared to former monolayer techniques. Soft-agar cloning reduced the opportunity for metabolic cooperation and permitted the use of non-dialyzed fetal calf serum which resulted in spontaneous mutant frequencies of 6.6 +/- 3.2 X 10(-6) and cloning efficiencies of 91 +/- 18%. Relative total growth values were calculated based on suspension growth and cloning efficiencies such that an assessment of toxicity could be estimated from treatment through cloning. Dose-dependent mutagenic responses were observed in CHO cells following treatment with ethyl methanesulfonate, methyl methanesulfonate, 4-nitroquinoline-1-oxide, methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Clones of 6TG-resistant cells harvested from soft agar maintained 6TG resistance and methotrexate sensitivity and did not incorporate [3H]hypoxanthine into DNA. These preliminary findings indicate that the use of suspension cultures and soft-agar cloning has improved the efficiency and cost effectiveness of the CHO/HGPRT mutation assay.
D000362:Agar; D000818:Animals; D001790:Blood Physiological Phenomena; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus / Q000235:genetics; D003470:Culture Media; D004351:Drug Resistance; D005260:Female; D005347:Fibroblasts / Q000187:drug effects*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000494:pharmacology; D010053:Ovary; D013866:Thioguanine
D003470:Culture Media; D009153:Mutagens; D000362:Agar; D007041:Hypoxanthine Phosphoribosyltransferase; D013866:Thioguanine