Abstract
Human cytochrome P450 2S1 was recently identified and shown to be inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin and hypoxia. It is highly expressed in epithelial cells of tissues that are exposed to the environment and in many tumors of epithelial origin. The biological function of CYP2S1 has not yet been determined, although its possible role in carcinogen metabolism has been suggested. In this report, we investigated its ability to metabolize carcinogens. To obtain a large quantity of active enzyme for substrate screening, we overexpressed CYP2S1 in Escherichia coli (200 nM culture), using a synthetic gene approach. High-level expression allowed us to achieve purification of CYP2S1 with high specific content and purity (16 nmol/mg). Despite high-level expression, we found that CYP2S1 was not readily reduced by cytochrome P450 reductase, and thus no activity was found using NADPH. However, the oxidative activity of CYP2S1 was supported by cumene hydroperoxide or H2O2, such that CYP2S1 oxidized many important environmental carcinogens, including benzo[a]pyrene, 9,10-dihydro-benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene-7,8-dihydrodiol, aflatoxin B1, naphthalene, and styrene, with high turnover. Most substrates tested were converted to detoxification products, except in the case of benzo[a]pyrene-7,8-dihydrodiol, which was converted into the very potent carcinogenic metabolite 7,8-dihydrodiol-trans-9,10-epoxide at a relatively efficient rate (Km = 12.4 ± 2 μM, turnover = 2.3 min−1). This metabolite formation was also supported both in vitro and in vivo by fatty acid hydroperoxides described in the accompanying report (p. 1044). Together, these data indicate that CYP2S1 contributes to the metabolism of environmental carcinogens via an NADPH independent activity.
- P450, cytochrome P450
- IPTG, isopropyl-β-d-thiogalactopyranoside
- hNPR, human P450 reductase
- BaP, benzo[a]pyrene
- 9,10-H2-BaP, 9,10-dihydro-benzo[a]pyrene
- BaP-7,8-diol, benzo[a]pyrene-trans-7,8-dihydrodiol
- r7,t8,t9,c10-tetrol, benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol
- DMBA, 7,12-dimethylbenz[a]anthracene
- 3,4-diol-DMBA, 7,12-dimethylbenz[a]anthracene-trans-3,4-diol
- 7-OH-DMBA, 7-hydroxy methyl-12-methylbenz(a)anthracene
- 12-OH-DMBA, 12-hydroxymethyl-7- methylbenz[a]anthracene
- HPLC, high-performance liquid chromatography
- KPi, potassium phosphate buffer
- CHP, cumene hydroperoxide
- HR, human reductase
- ΔG, Gibbs free energy of a secondary structure of any given single-stranded DNA or RNA.
Footnotes
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
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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 by a predoctoral fellowship 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.109.057752
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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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