ISOLATION OF A NOVEL METABOLIZING SYSTEM ENRICHED IN PHASE-II ENZYMESFOR SHORT-TERM GENOTOXICITY BIOASSAYS

Murine S9 liver fractions isolated from mice fed 7.5 g kg(-1) 2(3)-ter t-Butyl-4-hydroxyanisole (BHA) for 3 weeks were tested to determine: ( a) the profile of both phase-I and phase-II xenobiotic metabolizing en zymes; (b) their ability to induce in vitro covalent binding of some p recarcinogens to calf thymus DNA; and (c) their activation in a standa rd genetic toxicology assay. With regard to phase-I pathway, the S9 fr action expressed various cytochrome P-450-(CYP) (classes 1A1, 1A2, 2B1 , 2E1, and 3A)-dependent biotransformation enzymes at levels comparabl e with those present in murine control liver. For post-oxidative enzym es, 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 vitr o DNA binding of the precarcinogenic agents [C-14]-1,4-dichlorobenzene , [C-14]-1,2-dichlorobenzene and [C-14]-1,2-dibromobenzene, mediated b y BHA-induced cytosol and/or microsomal preparation, showed an increas e in specific activity comparable to that observed with phase-I (PB/be ta NF) induced S9. In some instances, covalent binding was even more e levated 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-fo ld increase for [14C]-1,4-dichlorobenzene, while cytosol from PB/beta NF was not. A high mutagenic response on diploid D-7 strain of Sacchar omyces 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 numb er of chemicals are activated by either oxidative or post-oxidative en zymes, the use of metabolizing biosystems, with an enhanced phase-II p athway, together with classical Sg fractions, can improve the sensitiv ity of the assay in detecting unknown genotoxins. (C) 1998 Elsevier Sc ience B.V.