Role of aldo -keto reductase enzymes in polycyclic aromatic hydrocarbon activation

Polycyclic aromatic hydrocarbons (PAH) are environmental pollutants and suspected human carcinogens. Cigarette smoke, considered the most important risk factor for lung cancer, contains a variety of PAH. PAH require metabolic activation to exert their deleterious effects. Aldo-keto reductases (AKR) oxidize proximate carcinogen PAH trans-dihydrodiols to catechols, which auto-oxidize forming reactive oxygen species (ROS) and o-quinones. Fjord-region PAH are more mutagenic and carcinogenic versus unsubstituted PAH. In this study, rat AKR1C9 oxidized fjord -region benzo[g]chrysene-11,12-dihydrodiol with a kcat/Km 100-times greater than unsubstituted bay-region benzo[a]pyrene-7,8-dihydrodiol. During oxidation, only one stereoisomer was used. Alanine-scanning mutants of active site residues revealed that several active site residues enabled oxidation of both stereoisomers of benzo[g]chrysene-11,12-dihydrodiol. This suggests that AKRs may contribute to the carcinogenicity of fjord-region PAH. One deleterious consequence of o-quinone formation is ROS generation during futile redox cycling. Consequently, ROS inflict oxidative damage to DNA. However, the identity of quinone reductases, which enhance ROS formation by reducing PAH quinones to catechols in cells, is unknown. In this study, human recombinant AKR1A1, 1B1, 1B10, 1C1-4, 7A2, 7A3, NAD(P)H quinone oxidoreductase 1 (NQ01), and carbonyl reductase (CBR) isoforms were tested as quinone reductases. Structural series of PAH o-quinones, benzo[a]pyrene diones, and estrogen replacement therapy metabolite 4-hydroxyequilenin-o-quinone were tested as substrates with each enzyme. NQO1 and the AKRs reduced all types of quinones. The specific activities with AKRs exceeded those observed for trans-dihydrodiol oxidation. CBRs reduced few PAH quinones, implying CBRs are not the major cellular quinone reductases. NQO1 had higher specific activities than AKRs. However, the Km for NADPH with NQO1 is higher than the AKRs, which could limit quinone reduction by NQO1 in cells. Contributions of AKRs and NQO1 to ROS formation in human lung adenocarcinoma A549 cells treated with PAH o-quinones with AKR1C or NQO1 inhibitors was tested. No change in ROS formation occurred with these inhibitors, suggesting that NQO1 does not reduce quinones in A549 cells. However, AKRs not blocked by the AKR1C inhibitor may play roles in quinone reduction of PAH quinones in A549 cells. This could lead to increased oxidative stress following o-quinone formation and ultimately contribute to chemical and hormonal carcinogenesis