Optimization of a pre-metabolization procedure using rat liver S9 and cell-extracted S9 in the Ames fluctuation test

Many environmental pollutants pose a toxicological hazard only after metabolic activation. In vitro bioassays using cell lines or bacteria have often no or reduced metabolic activity, which impedes their use in the risk assessment. To improve the predictive capability of in vitro assays, external metabolization systems like the liver S9 fraction are frequently combined with in vitro toxicity assays. While it is typical for S9 fractions that samples and testing systems are combined in the same exposure system, we propose to separate the metabolism step and toxicity measurement. This allows for a modular combination of metabolic activation by enzymes isolated from rat liver (S9) or a biotechnological alternative (ewoS9^R) with in vitro bioassays that lack metabolic capacity. Benzo(a)pyrene and 2-aminoanthracene were used as model compounds to optimize the conditions for the S9 metabolic degradation/activation step. The Ames assay with Salmonella typhimurium strains TA98 and TA100 was applied to validate the set-up of decoupling the S9 activation/metabolism from the bioassay system. S9 protein concentration of 0.25 mg_protein/mL, a supplement of 0.13 mM NADPH and a pre-incubation time of 100 min are recommended for activation of samples prior to dosing them to in vitro bioassays using the regular dosing protocols of the respective bioassay. EwoS9^R performed equally well as Moltox S9, which is a step forward in developing true animal-free in vitro bioassays. After pre-incubation with S9 fraction, chemicals induced bacteria revertants in both the TA98 and the TA100 assay as efficiently as the standard Ames assay. The pre-incubation of chemicals with S9 fraction could serve for a wide range of cellular in vitro assays to efficiently combine activation and toxicity measurement, which may greatly facilitate the application of these assays for chemical hazard assessment and monitoring of environmental samples.