Metabolism of Carcinogens and Drugs by Human P450s

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SUMMARY Cytochrome P450 (P450) enzymes are involved in the oxidations of ~95% of chemicals, including many drugs, steroids, and chemical carcinogens. Variations in P450s are issues in drug-drug interactions, endocrine diseases, cancer, and other medical problems. One Aim is the identification of functions of orphan P450s. We identified skin mitochondrial P450 27C1 as a retinoid 3,4-desaturase, but the function of 3,4-dehydroretinoids is unknown. Studies are focused on human keratinocytes and include immunohistochemical localization, subcellular location, absolute quantitation of P450 27C1 and redox partners in skin using proteomics, roles of retinoid binding proteins, analysis of effects of relevant retinoids on keratinocytes, examination of inducibility by retinoids and light, promoter analysis, and analysis of genes regulated by P450 27C1 desaturation, in order to define the function of this enzyme. We also plan to use LC-MS metabolomic screens to identify endogenous substrates for human P450s 2S1, 4X1, and 20A1. We will also screen Cyp2s1–/– vs. wild-type mice and a zebrafish P450 20A1 knockout model. A mechanism of human P450 51A1 (lanosterol 14a-demethylase) is proposed involving Compound I (FeO3+), which will be addressed using an isotopic labeling approach with 2H- labeled 32-formyl lanosterol and analysis of the side product formic acid. Detailed kinetic approaches will be applied to the question of the complex 3-step oxidation of deoxycorticosterone to the important steroid aldosterone by P450 11B2, a drug target in hypertension. These kinetic approaches to analysis of processivity will be applied to several other human P450s. Collectively the proposed experiments will provide useful information about the functions and mechanisms of several human P450s and have continued relevance in the fields of endocrinology and drug metabolism.