Inhibition of CYP1A1 enzyme activity in mouse hepatoma cell culture by soybean isoflavones

The mechanisms by which soybean- and soybean isoflavone-enriched diets inhi bit carcinogenesis are not known. We found that the isoflavones genistin an d daidzin, and their respective aglucone forms daidzein and genistein, bloc k 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin)-induced CYP1A1 enzyme activity. This inhibition is correlated with the capacity of the isoflavone s to prevent CYP1A1-mediated covalent binding of benzo[a]pyrene (BaP) metab olites to DNA. We further evaluated daidzein and genistein, believed to be the active forms of the isoflavones, for the mechanism of the inhibitory pr ocess. 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 t he isoflavones do not act at the transcriptional level. We therefore evalua ted the isoflavones for direct effects on the CYP1A1 enzyme. Daidzein and g enistein are non-competitive with the CYP1A1 substrate BaP for microsomal B aP hydroxylation, with apparent K-1 values of 325 mu M and 140 mu M, respec tively. The extent of CYP1A1 inhibition increases with time of preincubatio n at 37 degrees C, but not at 4 degrees C, in the presence of isoflavone pi ns NADPH; after 60 min preincubation the inhibition remains non-competitive , with apparent K-1 values of 55 mu M and 50 mu M, 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 t ime for inhibition of CYP1A1 suggests that metabolism of either isoflavone or molecular oxygen to reactive species is required. Isoflavone-mediated in hibition of CYP1A1 activity may contribute to the mechanism by which these soybean isoflavones protect against carcinogenesis. (C) 1999 Elsevier Scien ce Ireland Ltd. All rights reserved.