Human liver S-9 metabolic activation: proficiency in cytogenetic assays and comparison with phenobarbital/beta-naphthoflavone or aroclor 1254 induced rat S-9.
Induced rat liver S-9 is routinely used for metabolic activation in cytogenetic assays. When a compound gives a positive test result only with rat S-9, the significance for humans should be assessed. To evaluate the use of human S-9, we used sister-chromatid exchanges (SCEs) and chromosome aberrations (Abs) in Chinese hamster ovary cells to test five pro-mutagens, each preferentially activated by a different family of P-450: benzo(a)pyrene (BP), dimethylnitrosamine (DMN), diethylnitrosamine (DEN), aflatoxin B1 (AFB), and 2-acetylaminofluorene (2-AAF). We tested two human S-9 preparations, one from a single liver and a second pooled from two livers known to have good activity for several P-450s. Concentrations and ratios of NADP and isocitrate were adjusted to optimize NADPH generation by the S-9. Abs were scored 20 hr, and SCEs 29-45 hr, after the beginning of a 3 hr treatment. P-450 enzyme activities were generally higher in rat than human S-9. With the single-liver human S-9, increase in SCEs were seen with all chemicals; with both human S-9s, increases in Abs were seen with all chemicals except BP. (The level of P-450 1A1, required for BP activation, is very low in human liver.) Compared with rat S-9, generally higher concentrations of human S-9 and of promutagens were required to see positive results. However, human S-9 effectively activated 2-AAF, whereas neither of the two types of rat S-9 produced Abs with 2-AAF. We also compared rat S-9s induced with Aroclor 1254 or phenobarbital/ beta-naphthoflavone (PB/beta NF). Although there were some differences in P-450 enzyme activities, these did not translate into differences in Abs induction. At low doses of AFB and of BP, PB/beta NF induced S-9 appeared more effective than Aroclor 1254 induced S-9.
Johnson
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TE
Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
Umbenhauer
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DR
Galloway
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SM
D015073:2-Acetylaminofluorene / Q000493:pharmacokinetics / Q000633:toxicity; D016604:Aflatoxin B1 / Q000493:pharmacokinetics / Q000633:toxicity; D000818:Animals; D001140:Aroclors / Q000494:pharmacology*; D001564:Benzo(a)pyrene / Q000493:pharmacokinetics / Q000633:toxicity; D001571:Benzoflavones / Q000494:pharmacology*; D001711:Biotransformation; D016466:CHO Cells; D020111:Chlorodiphenyl (54% Chlorine); D006224:Cricetinae; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism; D004052:Diethylnitrosamine / Q000493:pharmacokinetics / Q000633:toxicity; D004128:Dimethylnitrosamine / Q000493:pharmacokinetics / Q000633:toxicity; D004790:Enzyme Induction; D006801:Humans; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D009152:Mutagenicity Tests; D009153:Mutagens / Q000493:pharmacokinetics / Q000633:toxicity*; D010634:Phenobarbital / Q000494:pharmacology*; D051381:Rats; D019324:beta-Naphthoflavone
D001140:Aroclors; D001571:Benzoflavones; D009153:Mutagens; D020111:Chlorodiphenyl (54% Chlorine); D001564:Benzo(a)pyrene; D004052:Diethylnitrosamine; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D015073:2-Acetylaminofluorene; D016604:Aflatoxin B1; D004128:Dimethylnitrosamine; D010634:Phenobarbital