Inhibition of CYP1A1 enzyme activity in mouse hepatoma cell culture by soybean isoflavones.
The mechanisms by which soybean- and soybean isoflavone-enriched diets inhibit carcinogenesis are not known. We found that the isoflavones genistin and daidzin, and their respective aglucone forms daidzein and genistein, block 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin)-induced CYP1A1 enzyme activity. This inhibition is correlated with the capacity of the isoflavones to prevent CYP1A1-mediated covalent binding of benzo[a]pyrene (BaP) metabolites to DNA. We further evaluated daidzein and genistein, believed to be the active forms of the isoflavones, for the mechanism of the inhibitory process. Although daidzein and genistein appear structurally similar to known aromatic hydrocarbon receptor (AHR) agonists and antagonists, gel mobility shift assays indicated that the isoflavones do not inhibit dioxin-induced activation of the AHR or the accumulation of CYP1A1 mRNA, suggesting that the isoflavones do not act at the transcriptional level. We therefore evaluated the isoflavones for direct effects on the CYP1A1 enzyme. Daidzein and genistein non-competitive with the CYP1A1 substrate BaP for microsomal BaP hydroxylation, with apparent Ki values of 325 microM and 140 microM, respectively. The extent of CYP1A1 inhibition increases with time of preincubation at 37 degrees C, but not at 4 degrees C, in the presence of isoflavone plus NADPH; after 60 min preincubation the inhibition remains non-competitive, with apparent Ki values of 55 microM and 50 microM, respectively. Inhibition is neither prevented nor reversed by the thiol antioxidant dithiothreitol, nor by the iron chelator deferoxamine. Repeated washing of the microsomes does not reverse the inhibition. The dependency on NADPH, temperature and time for inhibition of CYP1A1 suggests that metabolism of either isoflavone or molecular oxygen to reactive species is required. Isoflavone-mediated inhibition of CYP1A1 activity may contribute to the mechanism by which these soybean isoflavones protect against carcinogenesis.
Shertzer, HG; Puga, A; Chang, C; Smith, P; Nebert, DW; Setchell, KD; Dalton, TP
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