Fatty acid hydroperoxides support cytochrome P450 2S1-mediated bioactivation of benzo[a]pyrene-7,8-dihydrodiol

Abstract

In the accompanying report (p. 1031), we showed that a novel dioxin-inducible cytochrome P450, CYP2S1, efficiently metabolizes benzo[a]pyrene-trans-7,8-dihydrodiol (BaP-7,8-diol) into the highly mutagenic and carcinogenic benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (BaP-diol-t-epoxide), using cumene hydroperoxide in lieu of NADPH/O 2. Lipid hydroperoxide-supported P450 oxidation has been reported in several cases. However, it has not yet been described for the bioactivation of BaP-7,8-diol. In this report, we demonstrate that CYP2S1 can use various fatty acid hydroperoxides to support epoxidation of BaP-7,8-diol at a much higher rate than with cumene hydroperoxide. Kinetic analyses with several fatty acid hydroperoxides revealed that 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE) was the most potent oxidant tested (K m, 3.4 ± 0.8 μM; turnover, 4.51 ± 0.13 min -1), followed by 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (K m, 2.8 ± 0.7 μM; turnover, 3.7 ± 0.1 min -1), 5S-hydroperoxy-6E,8Z, 11Z,14Zeicosatetraenoic acid (K m, 2.7 ± 0.8 μM; turnover, 3.69 ± 0.09 min -1), and 15S-hydroperoxy-5Z,8Z,10E,14Z- eicosatetraenoic acid (K m, 11.6 ± 0.3 μM; turnover, 0.578 ± 0.030 min -1). The antioxidant butylated hydroxyanisole inhibited CYP2S1-catalyzed epoxidation by 100%, suggesting that epoxidation proceeds by a free radical mechanism. Other cytochromes P450, including CYP1A1, CYP1B1, CYP1A2, and CYP3A4, were also able to epoxidize BaP-7,8-diol using various fatty acid hydroperoxides, although at slower rates than CYP2S1. The cytotoxicity of BaP-7,8-diol significantly increased in mammalian cells overexpressing CYP2S1, and BaP-diol-t-epoxide formation in these cells also increased in the presence of 13-HpODE. Together, these results suggest that fatty acid hydroperoxides can serve as physiological cofactors in supporting in vivo CYP2S1-catalyzed oxidation of BaP-7,8-diol, and that fatty acid hydroperoxides and CYP2S1 may play important roles in benzo[a]pyrene-induced carcinogenesis. Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics.