Functional characterization of the novel dioxin-inducible P450, CYP2S1, and its role in metabolism of carcinogens and eicosanoids.

摘要

Dioxins are classified as environmental toxicants and carcinogens. One of the most investigated dioxins is TCDD (2,3,7,8-tetrachlorodibenzo- p-dioxin) which mediates various biological effects via the Aryl Hydrocarbon Receptor (AHR). Our goal is to delineate the TCDD biological effects by studying the function of gene(s) that is regulated by this chemical. Cytochrome P450 2S1 is the new member of dioxin-inducible P450 which was identified in our lab. It is highly expressed in epithelial cells of tissues that are exposed to the environment and in many tumors of epithelial origin. Thus its possible role in carcinogen metabolism was suggested although the biological function of CYP2S1 has not yet been determined. Using a synthetic gene recombinant E.coli expression we obtained large quantity of human CYP2S1 for substrate screening. We found that CYP2S1 is a peculiar P450 which is not readily reduced by cytochrome P450 reductase, thus its activity can't be supported by NADPH/O2. Instead, we found that peroxides, such as cumene hydroperoxide, hydrogen peroxide, and fatty acid hydroperoxides can serve as an oxygen surrogate for CYP2S1-catalyzed oxidation of various substrates including carcinogens like benzo[a]pyrene, benzo[a]pyrene-7,8-diol, DMBA, and aflatoxin B1. Most carcinogens are converted to less reactive metabolites except benzo[a]pyrene-7,8-diol, which was epoxidized to mutagenic epoxide, the 7,8-diol-trans-9,10-epoxide. This was further supported by the fact that benzo[a]pyrene-7,8-diol induced toxicity in mammalian cells overexpressing human CYP2S1. We determined that fatty acid hydroperoxides can serve as physiological cofactors for CYP2S1 due to their low Kms and high turnover numbers.;We found that the eicosanoid peroxides themselves are isomerized and metabolized by CYP2S1 in the absence of co-substrates. CYP2SI can convert pGG2 into MDA, 12-HpHT, 12-HHT, and other suspected products including 15-keto-pGG2 and 12-oxoHT. It also converts pGH2 into MDA, 12-HHT, TXA2 and other products. In addition, CYP2S1 converts fatty acid hydroperoxides, such as 5-HpETE, 12-HpETE, 15-HpETE, and 13-HpODE into keto and epoxy-alcohol derivatives. The metabolism of these eicosanoids is further supported by the observations in mammalian cells overexpressing human and mouse CYP2S1 and in Cyp2s1-null mice. Interestingly, the Cyp2s1-null mice have enlarged seminal vesicles due to hyperproliferation of epithelial cells. Perhaps, CYP2S1 metabolism of the eicosanoids is responsible for this phenotype.