Role of human aldo -keto reductases in PAH activation

Polycyclic aromatic hydrocarbons are widespread environmental carcinogens implicated in the causation of human lung cancer. PAH require metabolic activation by epoxide hydrolase (EH) and CYPIA1 to form non-K-region trans-dihydrodiols that can be further activated by CYP1A1 to form diol-epoxides. Previously we have shown that a cytosolic aldo-keto reductase (AYR) from rat liver 3α-HSD/DD (AKR1C9) can divert these traps-dihydrodiols to yield reactive and redox active o-quinones, raising the possibility that this pathway could contribute to PAH carcinogenesis. However, whether this pathway occurs in humans was unknown. In this study we determined whether this pathway can be catalyzed in vitro by human AKR1C isoforms. Furthermore, we investigated whether the isoforms are highly over-expressed in lung cells, and whether the activity is sufficient to generate PAH o-quinones. We now demonstrate that four homogenous recombinant human AKRs (AKR1C1-AKR1C4) oxidized a wide variety of PAH trans-dihydrodiols with regio-selectivity. Of the PAH trans-dihydrodiols tested DMBA-3,4-diol, one of the most potent proximate carcinogens known, was often preferred. Using DMBA-3,4-diol the product of the reaction was shown to be DMBA-3,4-dione which was trapped as a thioether conjugate with 2-mercaptoethanol. DMBA-3,4-dione was shown to form mono- and bis-thioether conjugates via a novel mechanism of sequential 1,6- and 1,4-Michael addition. Using multiple tissue expression array analysis, AKR1C isoforms were shown to be highly expressed in several PAH exposed tissues and in the human lung carcinoma cell line A549. Isoform specific RT-PCR identified AKR1C1-AKR1C3 transcripts in this cell line. Western blot analysis and functional assays showed the presence of active AKR1C isoforms in A549 cells. A549 cell lysates were shown to oxidize DMBA-3,4-diol to DMBA-3,4-dione. The ability to measure DMBA-3,4-dione formation in human cells implicates AKR1C isoforms in the metabolic activation of potent proximate carcinogens in lung cancer. DMBA-3,4-dione is the most electrophilic o-quinone produced by AKRs and its unique ability to form bis-thioether conjugates suggests that bis-conjugates may form with other cellular nucleophiles with toxicological consequences. The constitutively and widely expressed AKR, aldehyde reductase (AKR1A1) was also shown to oxidize PAH trans-dihydrodiols to their corresponding o-quinones. Aldehyde reductase displayed regio- and stereoselectivity in oxidizing PAH trans dihydrodiols of varying ring number and arrangement. Aldehyde reductase preferentially oxidized the metabolically relevant (−)-R,R isomer of BP-7,8-diol to yield BP-7,8-dione implicating the importance of this enzyme in PAH activation in humans. Moreover, AKR1A1 was shown to be co-expressed with EH and CYP1A1. The ability of these human AKR enzymes to divert trans-dihydrodiols to o-quinones suggests that this pathway occurs in site of PAH carcinogenesis in humans