Isolation of S9 fractions from mouse and rat with increased enzyme activities after repeated administration of cytochrome P-450 and P-448 inducers.

Abstract

Cytochrome P-450 (cyt P-450), NADPH cytochrome P-450 reductase and various microsomal monooxygenase activities [e.g. aminopyrine N-demethylase, p-nitroanisole O-demethylase, dinemorphan N-demethylase, ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase (ERD)], were determined in hepatic post-mitochondrial supernatant from mice and rats. Experiments were performed on male and female animals treated with a combination of sodium phenobarbital and beta-naphthoflavone according to the standard protocol schedule for short-term genotoxicity testing. A second inductive treatment after 2, 3, 4 or 5 weeks was provided. The increase in cyt P-450 and in all enzymatic activities measured was enhanced in both species by a second induction treatment, particularly when given after 4 weeks. ERD activity was the only monooxygenase activity which was sex-dependent, being more active in female than in male animals. To extend the biochemical data, experiments were performed with the proposed S9 fractions on styrene, which previously has proved difficult to detect in short-term in vitro mutagenicity tests. Using the new induction conditions positive results were obtained with the D7 strain of Saccharomyces cerevisiae. It was concluded that a simple pre-induction of the animals 3-4 weeks before the main induction treatment leads to a more active S9 fraction for in vitro genotoxicity studies.