Metabolites of Tobacco- and E-Cigarette-Related Nitrosamines Can Drive Cu-Mediated DNA Oxidation.

Nitrosamine metabolites resulting from cigarette smoking and E-cigarette (E-cig) vaping cause DNA damage that can lead to genotoxicity. While DNA adducts of metabolites of nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and -nitrosonornicotine (NNN) are well-known tobacco-related cancer biomarkers, only a few studies implicate NNN and NNK in DNA oxidation in humans. NNK and NNN were found in the urine of E-cigarette users who never smoked cigarettes. This paper proposes the first chemical pathways of DNA oxidation driven by NNK and NNN metabolites in redox reactions with Cu and NADPH leading to reactive oxygen species (ROS). A microfluidic array with thin films of DNA and metabolic enzymes that make metabolites of NNN and NNK in the presence of Cu and NADPH was used to estimate relative rates of DNA oxidation. Detection by electrochemiluminescence (ECL) employed a new ECL dye [Os(tpy-benz-COOH)] that is selective for and sensitive to the primary DNA oxidation product 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in DNA. Enzyme-DNA films on magnetic beads were used to produce nitrosamine metabolites that enter ROS-forming redox cycles with Cu and NADPH, and liquid chromatography-mass spectrometry (LC-MS) was used to quantify 8-oxodG and identify metabolites. ROS were detected by optical sensors. Metabolites of NNK and NNN + Cu + NADPH generated relatively high rates of DNA oxidation. Lung is the exposure route in smoking and vaping, human lung tissue contains Cu and NADPH, and lung microsomal enzymes gave the highest rates of DNA oxidation in this study. Also, E-cigarette vapor contains 6-fold more copper than that in cigarette smoke, which could exacerbate DNA oxidation.