Development of an optimal S9 activation mixture for the L5178Y TK+/- mouse lymphoma mutation assay

In previously described activation systems [Clive D, Spector JFS (1975): Mutat Res 31:17-29] for the mouse lymphoma mutation assay the cofactor isocitrate is rapidly exhausted and the resultant loss of NADPH can halt metabolic processes. Presented here are data obtained with a non-toxic balance of NADP (1.4 mg/ml), isocitrate (6.0 mg/ml), and S9 (less than or equal to 4%) in Fischer's medium which produces a more stable supply of the required cofactors. By spectrophotometric analysis, the molar concentration of NADPH remains at greater than or equal to 50% or more of the maximum over the usual 4-hr treatment period. Accompanying this increase in NADPH duration was increased toxicity and mutant frequency at most doses among cells treated with the reference mutagens 3-methylcholanthrene (MCA), 2-acetylaminofluorene (AAF), benzo(a)pyrene (BAP), 9,10-dimethyl-1,2-benzanthracene (DMBA), or cyclophosphamide (CPA), but not with dimethylnitrosamine (DMN)-possibly a reflection of the single enzyme mediated step in the metabolism of this chemical. These observations also suggest that results attributed to varying the amounts of S9 in an activation mixture may be due to suboptimal cofactor levels and further emphasize the need to maintain sufficient NADPH exposure to evaluate the effects of metabolic enzyme levels or compare the relative activities of analogous chemicals.