Evidence for the aldo-keto reductase pathway of polycyclic aromatic trans -dihydrodiol activation in human lung A549 cells

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Abstract
Polycyclic aromatic hydrocarbons (PAHs) are tobacco carcinogens implicated in the causation of human lung cancer. Metabolic activation is a key prerequisite for PAHs to cause their deleterious effects. Using human lung adenocarcinoma (A549) cells, we provide evidence for the metabolic activation of (±)- trans -7,8dihydroxy-7,8-dihydrobenzo[ a ]pyrene (B[ a ]P-7,8- trans -dihydrodiol) by aldo-keto reductases (AKRs) to yield benzo[ a ]pyrene-7,8-dione (B[ a ]P-7,8-dione), a redox-active o -quinone. We show that B[ a ]P-7,8- trans -dihydrodiol (AKR substrate) and B[ a ]P-7,8-dione (AKR product) lead to the production of intracellular reactive oxygen species (ROS) (measured as an increase in dichlorofluorescin diacetate fluores-cence) and that similar changes were not observed with the regioisomer (±)- trans -4,5-dihydroxy-4,5-dihydrobenzo[ a ]pyrene or the diol-epoxide, (±)- anti -7,8-dihydroxy-9α,10β-epoxy-7,8,9,10-tetrahydro-B[ a ]P. B[ a ]P-7,8- trans -dihydrodiol and B[ a ]P-7,8-dione also caused a decrease in glutathione levels and an increase in NADP + /NADPH ratios, with a concomitant increase in single-strand breaks (as measured by the comet assay) and 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dGuo). The specificity of the comet assay was validated by coupling it to human 8-oxo-guanine glycosylase (hOGG1), which excises 8-oxo-Gua to yield single-strand breaks. The levels of 8-oxo-dGuo observed were confirmed by an immunoaffinity purification stable isotope dilution ([ 15 N 5 ]-8-oxo-dGuo) liquid chromatography-electrospray ionization/multiple reaction monitoring/mass spectrometry (LC-ESI/MRM/MS) assay. B[ a ]P-7,8- trans -dihydrodiol produced DNA strand breaks in the hOGG1-coupled comet assay as well as 8-oxo-dGuo (as measured by LC-ESI/MRM/MS) and was enhanced by a catechol O -methyl transferase (COMT) inhibitor, suggesting that COMT protects against o -quinone-mediated redox cycling. We conclude that activation of PAH- trans -dihydrodiols by AKRs in lung cells leads to ROS-mediated genotoxicity and contributes to lung carcinogenesis.

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