Studies of an S9-based metabolic activation system used in the mouse lymphoma L5178Y cell mutation assay

The purpose of this work was to define, in this laboratory, the conditions of most general utility for the metabolic activation of chemicals to mutagens in the mouse lymphoma L5178Y cell tk+tk- ----tk-tk- assay. The approach used was to optimize the generation of non-toxic concentrations of NADPH from NADP glucose-6-phosphate (G6P) and G6P dehydrogenase, then use that system to examine the effects of increasing concentrations of Aroclor-1254-induced Fischer 344 male rat liver post-mitochondrial supernatant fluid (S9) upon the mutagenicity of chemicals. The promutagens were 2-acetylaminofluorene, cyclophosphamide, dimethylnitrosamine, methanol, 3-methylcholanthrene and procarbazine. It was determined that: (i) cell population growth was reduced at NADPH concentrations of greater than or equal to 1 mM; (ii) to ensure a maximum conversion of NADP to NADPH the G6P/NADP ratio should be two or greater; (iii) excess G6P (2.5 mM) is not harmful to the cells; (iv) toxicity due to S9 was observed at 12.5 mg whole liver equivalents (WLE)/ml (i.e. 50 microliters of a 25% liver S9 preparation per ml); (v) concentrations of S9 between 2.5 and 7.5 mg WLE/ml appeared to be adequate for the activation of the six promutagens to demonstrably mutagenic products.