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Isolation of a novel metabolizing system enriched in phase-II enzymes for short-term genotoxicity bioassays

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, ISSN: 1383-5718, Vol: 413, Issue: 3, Page: 205-217
1998
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Murine S9 liver fractions isolated from mice fed 7.5 g kg −1 2(3)- tert -Butyl-4-hydroxyanisole (BHA) for 3 weeks were tested to determine: (a) the profile of both phase-I and phase-II xenobiotic metabolizing enzymes; (b) their ability to induce in vitro covalent binding of some precarcinogens to calf thymus DNA; and (c) their activation in a standard genetic toxicology assay. With regard to phase-I pathway, the S9 fraction expressed various cytochrome P -450-(CYP) (classes 1A1, 1A2, 2B1, 2E1, and 3A)-dependent biotransformation enzymes at levels comparable with those present in murine control liver. For post-oxidative enzymes, the S9 expressed high levels of glutathione S -transferases (up to 12-fold increase), glutathione S -epoxide-transferase (up to 2.6-fold), UDP-glucuronosyl transferase (up to 5.3-fold) and epoxide hydrolase (up to 2.6-fold) activities, as compared to untreated mice. The in vitro DNA binding of the precarcinogenic agents [ 14 C]-1,4-dichlorobenzene, [ 14 C]-1,2-dichlorobenzene and [ 14 C]-1,4-dibromobenzene, mediated by BHA-induced cytosol and/or microsomal preparation, showed an increase in specific activity comparable to that observed with phase-I (PB/ β NF) induced S9. In some instances, covalent binding was even more elevated using the BHA-induced systems as compared with traditional S9 fractions. For example, cytosol derived from BHA-administered mice was able to induce a significant binding to calf thymus DNA up to 26.2-fold increase for [ 14 C]-1,4-dichlorobenzene, while cytosol from PB/ β NF was not. A high mutagenic response on diploid D 7 strain of Saccharomyces cerevisiae as exemplified by a marked induction of mitotic gene conversion and point (reverse) mutation confirmed that BHA-derived S9 fractions activate precarcinogens to final genotoxins. Because a number of chemicals are activated by either oxidative or post-oxidative enzymes, the use of metabolizing biosystems, with an enhanced phase-II pathway, together with classical S9 fractions, can improve the sensitivity of the assay in detecting unknown genotoxins.

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