Isolation of a novel metabolizing system enriched in phase-II enzymes for short-term genotoxicity bioassays.
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 [14C]-1,4-dichlorobenzene, [14C]-1,2-dichlorobenzene and [14C]-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/beta 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 [14C]-1,4-dichlorobenzene, while cytosol from PB/beta NF was not. A high mutagenic response on diploid D7 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.
Paolini
M
M
Department of Pharmacology, University of Bologna, Italy. paolini@biocfarm.unibo.it
Pozzetti
L
L
Silingardi
P
P
Della Croce
C
C
Bronzetti
G
G
Cantelli-Forti
G
G
D000818:Animals; D000975:Antioxidants; D001711:Biotransformation; D001969:Bromobenzenes / Q000378:metabolism / Q000633:toxicity; D002083:Butylated Hydroxyanisole; D002273:Carcinogens / Q000378:metabolism* / Q000633:toxicity; D002722:Chlorobenzenes / Q000378:metabolism / Q000633:toxicity; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis; D018736:DNA Adducts / Q000096:biosynthesis*; D004790:Enzyme Induction; D005260:Female; D008099:Liver / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D051379:Mice; D009152:Mutagenicity Tests / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics; D013347:Subcellular Fractions / Q000201:enzymology*
D000975:Antioxidants; D001969:Bromobenzenes; D002273:Carcinogens; D002722:Chlorobenzenes; D018736:DNA Adducts; D002083:Butylated Hydroxyanisole; C004726:2-dichlorobenzene; D003577:Cytochrome P-450 Enzyme System; C066656:1,4-dibromobenzene; C018511:4-dichlorobenzene