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Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants implicated in the causation of human lung cancer. One pathway of metabolic activation that yields reactive oxygen species (ROS) involves the oxidation of PAH trans-dihydrodiols by aldo-keto reductases (AKRs) to yield reactive and redox-active o-quinones, which can cause oxidative DNA damage (e.g. 8-oxo-dGuo). Evidence for this pathway in lung cells has been hampered by the difficulty in detecting 8-oxo-dGuo levels reliably in intact cells. We used the single cell gel electrophoresis (Comet) assay to detect DNA strand breaks in human lung adenocarcinoma (A549) cells (which express AKR1A1 and AKR1C isoforms) following treatment with BP-7,8-diol (AKR substrate), and BP-7,8-dione (AKR-product) and (+)-anti-BPDE. In the presence of DNA synthesis inhibitors (cytosine arabinoside and hydroxyurea) all three PAH metabolites produced significant DNA strand breaks in a concentration-dependent manner. Inclusion of ROS scavengers attenuated or suppressed BP-7,8-diol and BP-7,8-dione-mediated DNA strand breaks suggesting that the DNA strand scission was AKR and ROS dependent, respectively. (+)-Anti-BPDE-mediated DNA strand breaks, however, were not affected by ROS scavenger treatment. The specificity of the Comet assay was improved by coupling the assay to 8-oxoguanine-glycosylase (hOGG1), which by catalyzing the excision of 8-oxo-dGuo yields strand-breaks that are dependent upon this lesion. This coupled-assay was validated by treating A549 cells with either the powerful oxidant (potassium bromate) or the methylating agent (MMS), as positive and negative controls, respectively and then measuring DNA-strand breaks or the amount of 8-oxo-dGuo formed. 8-Oxo-dGuo levels were measured by the use of stable isotope ([15N5]-8-oxo-dGuo) dilution LC-electrospray ionization (ESI)MS/MS, coupled with immunoaffinity purification and the employment of precautions to prevent adventitious oxidation of dGuo. There was excellent agreement between strand-breaks observed with the Comet Assay and 8-oxo-dGuo detected by immunoaffinity stable isotope dilution LC-MS/MS providing confidence in the detection method. hOGG1 treatment produced more DNA single strand breaks in BP-7,8-dione-exposed A549 cells than without hOGG1 treatment, this correlated with the amount of 8-oxo-dGuo detected by LC-MS, and the changes were dependent upon BP-7,8-dione concentration. Our results suggest that ROS formation by AKR-generated PAH o-quinones contributes to PAH-mediated lung carcinogenesis. (Supported by P01-CA092357 and R01-CA39504)

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA