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/O2. 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 (Km, 3.4 ± 0.8 μM; turnover, 4.51 ± 0.13 min−1), followed by 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (Km, 2.8 ± 0.7 μM; turnover, 3.7 ± 0.1 min−1), 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (Km, 2.7 ± 0.8 μM; turnover, 3.69 ± 0.09 min−1), and 15S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (Km, 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.
- BaP, benzo[a]pyrene
- BaP-7,8-diol, BaP-trans-7,8-dihydrodiol
- BaP-diol-t-epoxide, BaP-r-7,t-8-dihydrodiol-t-9,10-epoxide
- r7,t8,t9,t10-tetrol, BaP-r-7,t-8,t-9,t-10-tetrahydrotetrol
- r7,t8,t9,c10-tetrol, BaP-r-7,t-8,t-9,c-10-tetrahydrotetrol
- P450, cytochrome P450
- HPLC, high-performance liquid chromatography
- 5-HpETE, 5S-Hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid
- 12-HpETE, 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid
- 15-HpETE, 15S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid
- 13-HpODE, 13S-hydroperoxy-9Z,11E-octadecadienoic acid
- BHA, butylated hydroxyanisole
- COX, cyclooxygenase
- MSCV, murine stem cell virus
- EGFP, enhanced green fluorescent protein
- IRES, internal ribosome entry site.
Footnotes
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This research was supported by the National Institutes of Health National Institute of Environmental Health Sciences [Grants R01-ES015384, T32-ES015457] and in part by fellowships from the University of California Toxic Substances Research and Teaching Program.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.057760
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ABBREVIATIONS:
- Received May 13, 2009.
- Accepted August 27, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics
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