Polycyclic aromatic hydrocarbons (PAH) are suspect human lung carcinogens and can be metabolically activated to remote quinones, e.g. benzo[a]pyrene-1,6-dione (B[a]P-1,6-dione) and B[a]P-3,6-dione by the action of either P450 monooxygenase or peroxidases and to non-K region o-quinones by aldo-keto reductases (AKRs). B[a]P-7,8-dione also structurally resembles 4-hydroxyequilenin o-quinone. These three classes of quinones can redox cycle, generate reactive oxygen species (ROS) and produce the mutagenic lesion 8-oxo-dGuo, and may contribute to PAH- and estrogen-induced carcinogenesis. We compared the ability of a complete panel of human recombinant AKRs to catalyze reduction of PAH o-quinones in the phenanthrene, chrysene, pyrene and anthracene series. The specific activities for NADPH-dependent quinone reduction were often 100-1,000 times greater than the ability of the same AKR isoform to oxidize the cognate PAH-trans-dihydrodiol. However, the AKR with the highest quinone reductase activity for a particular PAH o-quinone was not always identical to the AKR isoform with the highest dihydrodiol dehydrogenase activity for the respective PAH-trans-dihydrodiol. Discrete AKRs also catalyzed the reduction of B[a]P-1,6-dione, B[a]P-3,6-dione and 4-hydroxyequilenin o-quinone. Concurrent measurements of oxygen consumption, superoxide anion and hydrogen peroxide formation established that ROS were produced as a result of the redox-cycling. When compared with human recombinant NAD(P)H: quinone oxidoreductase (NQO1) and carbonyl reductases (CBR1 and CBR3), NQO1 was a superior catalyst of these reactions followed by AKRs and lastly CBR1 and CBR3. In A549 cells two-electron reduction of PAH o-quinones causes intracellular ROS formation. ROS formation was unaffected by the addition of dicumarol suggesting that NQO1 is not responsible for the two-electron reduction observed and does not offer protection against ROS formation from PAH o-quinones.

This study demonstrates that benzo[g]chrysene-11,12-dihydrodiol (B[g]C-11,12-dihydrodiol) derived from the fjord-region parent hydrocarbon B[g]C is oxidized by rat AKR1C9 with a k cat / K m 100 times greater than that observed with the commonly studied bay-region benzo -11,12dihydrodiol were purified by chiral RP-HPLC. The 11S,12S-stereoisomer was oxidized at the same rate as the racemate. The 11R,12R-stereoisomer did not act as an inhibitor to AKR1C9, indicating that the (-)-R,R-stereoisomer was excluded from the active site. To understand the basis of stereochemical preference, we screened alanine-scanning mutants of active site residues of AKR1C9. These studies revealed that in comparison to the wild type, F129A, W227A, and Y310A enabled the oxidation of both the B[g]C-11S,12S-dihydrodiol and the B[g]C-11R,12R-dihydrodiol. Molecular modeling revealed that unlike B[a]P-7,8-dihydrodiol and B[c]Ph-3,4-dihydrodiol, B[g]C-11,12-dihydrodiol enantiomers are significantly bent out of plane. As a consequence, the (-)-R,R-stereoisomer was prevented from binding to the active site because of unfavorable interactions with F129, W227, or Y310. Additionally, LC/MS validated that the product of the reaction of B[g]C-11,12-dihydrodiol oxidation catalyzed by AKR1C9 was B[g] C-11,12-dione, which was trapped in vitro with the nucleophile 2-mercaptoethanol. The similarity between rates of trans-dihydrodiol oxidation by the rat and human liver specific AKRs (AKR1C9 and AKR1C4) implicate these enzymes in hepatocarcinogenesis in rats observed with the fjordregion PAH.