Expression and Characterization of Four Recombinant Human Dihydrodiol Dehydrogenase Isoforms: Oxidation of trans-7,8-Dihydroxy-7,8-dihydrobenzo[a]pyrene to the Activated o-Quinone Metabolite Benzo[a]pyrene-7,8-dione,

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Abstract
The bioactivation of polycyclic aromatic hydrocarbons (PAHs) to their ultimate carcinogenic forms proceeds via the formation of proximate carcinogen trans-dihydrodiols. Previous studies demonstrated that rat liver 3α-hydroxysteroid dehydrogenase/dihydrodiol dehydrogenase (3α-HSD/DD), a member of the aldo−keto reductase (AKR) superfamily, oxidizes PAH trans-dihydrodiols to redox-cycling o-quinones. Multiple closely related AKRs exist in human liver; however, it is unclear which, if any, participate in PAH activation by catalyzing the NADP+-dependent oxidation of PAH trans-dihydrodiols. In this study, cDNAs encoding four human DD isoforms were isolated from HepG2 cells using isoform-selective RT-PCR. The recombinant proteins were overexpressed in Escherichia coli, purified to homogeneity, and kinetically characterized. Calculation of KM and kcat values of each isoform for model substrates revealed that they possessed enzymatic activities assigned to native human liver DD1, DD2, DD4, and type 2 3α-HSD (DDX) proteins. The ability of human DDs to oxidize the potent proximate carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-diol) was then examined. A reverse phase HPLC radiochemical assay demonstrated that all four isoforms oxidize (±)-BP-diol in the following rank order: DD2 > DD1 > DD4 > DDX. Each DD consumed the entire racemic BP-diol mixture, indicating that both the minor (+)-S,S- and major (−)-R,R-stereoisomers formed in vivo are substrates. First-order decay plots showed that DD1 and DD2 displayed preferences for one of the stereoisomers, and circular dichroism spectroscopy indicated that this isomer was the (+)-7S,8S-enantiomer. The products of these reactions were trapped as either glycine or thiol ether conjugates of benzo[a]pyrene-7,8-dione (BPQ), indicating that the initial oxidation product was the reactive BPQ. Thus, human liver possesses multiple AKRs which contribute to PAH activation by catalyzing the NADP+-dependent oxidation of PAH trans-dihydrodiols to redox-active o-quinones.

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