COUMARIN CHEMOPROTECTION AGAINST AFLATOXIN BETA(1)-INDUCED GENE MUTATION IN A MAMMALIAN-CELL SYSTEM - A SPECIES-DIFFERENCE IN MUTAGEN ACTIVATION AND PROTECTION WITH CHICK-EMBRYO AND RAT-LIVER S9

Coumarin (1,2-benzopyrone), a natural food constituent, prevents polyc yclic aromatic hydrocarbon-induced neoplasms in rats and mice, but has not been studied with other chemical carcinogens. We examined coumari n chemoprotection against aflatoxin B-1 using the 6-thioguonine resist ance mutation assay in two different Chinese hamster ovary cell lines (K1BH4 and AS52) with liver S9 from rats and 19-day-old chick embryos for aflatoxin B-1 bioactivation. Laboratory rodents metabolize coumari n through 3-hydroxylation, whereas 7-hydroxylation predominates in chi ck embryos and humans. Chick embryo liver S9 was approximately 25-fold more effective in activating aflatoxin B-1 to the mutagenic and cytot oxic metobolite(s) than rat liver S9. Coumarin added at 50 and 500 mu M with chick embryo liver S9 reduced the mutant frequency of 1 mu M af latoxin B-1 by 40 and 85%, respectively. Coumarin up to 500 mu M had n o effect on aflatoxin B-1 mutagenicity with rat liver S9. When liver S 9 from chick embryos pretreated with coumarin was used for aflatoxin B -1 bioactivation, mutant frequency and cytotoxicity were decreased com pared to liver S9 from vehicle treated controls. Liver S9 from coumari n-treated rats did not significantly affect mutant frequency or cytoto xicity. HPLC analysis of chick embryo liver S9 incubated with 1 mu M a flatoxin B-1 showed a dose-dependent decrease by coumarin of aflatoxin B-1 activation to the 8,9-epoxide ranging from 70% of controls at 5 m u M coumarin to 4% of controls at 500 mu M coumarin. In contrast, coum arin produced a dose-dependent increase in 20 mu M aflatoxin B-1 activ ation by rat liver S9, reaching twice the control levels at 500 mu M c oumarin. These findings, using a mammalian cell system as a mutagenic endpoint, demonstrate marked species differences in chemoprotection by coumarin. Environ. Mol. Mutagen. 32:64-74, 1998 (C) 1998 Wiley-Liss, Inc.