Optimal levels of S9 fraction in the Ames and fluctuation tests: apparent importance of diffusion of metabolites from top agar.

For activation of 2-acetylaminofluorene (AAF) there is an optimal level of rat liver S9 fraction which is considerably lower in the fluctuation test than in the Ames test. The optimal level of S9 is not markedly affected by the dose of AAF used, nor by the ratio of S9 to bacteria, nor by the presence of soft agar. The difference between Ames and fluctuation tests appears to be due to diffusion of some substance or substances from the top agar layer in the Ames test. Diffusion of the co-factors NADP and glucose-6-phosphate is not responsible for the difference in S9 optima, nor is diffusion of soluble S9 constituents although this may considerably affect the performance of the S9 mix. We present evidence that diffusion of non-mutagenic metabolites of AAF from the Ames test top agar may be responsible for the difference in S9 optima. Our results are consistent with a model whereby lipophilic non-mutagenic metabolites accumulate in the microsomes and inhibit further activation. When the metabolites are able to diffuse away, a higher level of S9 will be optimal. The model is consistent with some other phenomena of S9 activation.