Analysis of CYP1A1/1B1-independent benzo[a]pyrene DNA-adduct formation in human lung cancer cells by LC-MS

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There is substantial evidence to suggest that environmental carcinogens, such as polycyclic aromatic hydrocarbons (PAHs), are able to induce lung cancer through metabolic activation. In order to understand the metabolic pathways of environmental carcinogens, benzo[a]pyrene (B[a]P) was used as a model compound. Activation of B[a]P by CYPs, specifically 1A1/1B1, to 7, 8-dihydroxy-9, 10-epoxy,7, 8, 9, 10-tetrahydrobenzo[a]pyrene (B[a]PDE), is one of the most widely accepted pathways of metabolism. B[a]PDE is able to form stable, covalent adducts with DNA. H358, human bronchoalveolar, cells do not constitutively express CYP1A1/1B1; however, they can be induced with pre-treatment of 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD). An unexpected increase in B[a]PDE-DNA-adduct formation was observed in cells that had not been subjected to TCDD pre-treatment.
 >Parental H358 and TCDD-induced H358 lung cancer cells were treated with increasing concentrations of TCDD before treatment with 2 μM (-)-B[a]P-7, 8-dihydrodiol. Inhibition experiments were performed on H358 cells by treatment with the CYP1 inhibitor, tetramethoxystilbene, and the CYP3A inhibitor, itraconazole. Liver cells lines, HepG2 and Hepa1c1c7 were treated with 2 μM (-)-B[a]P-7, 8-dihydrodiol with and without TCDD pre-treatment for comparison purposes. For all experiments, a stable isotope dilution, liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) assay was used for the analysis of the four stable (±)-anti-B[a]PDE-derived deoxyguanosine (dGuo) DNA-adducts at various time points.
 >In H358 cells we found that increasing the TCDD concentration decreased DNA-adduct formation. These results were the opposite of what is typically observed in liver cell lines. In a separate experiment, H358 parental cells were pretreated with TMS, a known inhibitor of CYP1A1/1B1, before treatment with 2 μM (-)-B[a]P-7,8-dihydrodiol. DNA-adduct levels remained the same for each treatment, suggesting that levels of CYP1A1/1B1 enzymes were insufficient to account for the metabolism of B[a]P-7,8-dihydrodiol to B[a]PDE. Therefore, it is likely that another P450 expressed by the H358 cells is able to metabolize B[a]P and its metabolites to B[a]PDE. In vitro experiments using Supersomes® have revealed that most CYPs are capable of metabolizing (-)-B[a]P-7,8-dihydrodiol to B[a]PDE. Previous studies have shown that that CYP3A5 is present in lung cells. Inhibition experiments using the CYP3A inhibitor, itraconazole, showed a decrease in adduct formation. Therefore, CYP3A5 may be responsible for metabolism of (-)-B[a]P-7,8-dihydrodiol in the H358 lung cells. This work is supported by NIH grants 1R01CA130038, 5P30ES013508, 1F32ES016683, and 5R25CA101871.