ECL Array and LC-MS Studies of Nitrosamine-Metabolite-Mediated DNA Oxidation

Abstract

Metabolites of nitrosamines in cigarette smoke and E-cigarette (E-cig) vapor cause DNA damage that has been projected to lead to cancers in “genotoxic” pathways. DNA oxidation can also be mediated by reactive oxygen species (ROS) generated from redox cycling reactions involving nitrosamine metabolites, metal ions (Cu2+) and NADPH. DNA adducts of tobacco nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) are well known tobacco-related biomarkers, but only a few reports implicate NNK and NNN in DNA oxidation. We used electrochemiluminescent (ECL) arrays and LC-MS to explore pathways of DNA oxidation driven by metabolites of NNK and NNN facilitated by Cu2+ and NADPH which are present in significant amounts in lung tissue. We used a 3-D printed microfluidic array that first makes reactive metabolites of test compounds using metabolic enzymes (mostly cyt P450s) and DNA in thin films. The assay measurement step detects DNA oxidation selectively by ECL using a new ECL dye, [Os(tpy-benz-COOH)2]2+ that is highly sensitive for primary DNA oxidation product 8-oxodG, which serves as a co-reactant in the ECL generation. Array results reveal that NNK and NNN oxidize DNA at relatively high rates. DNA/metabolic enzyme films on magnetic beads were utilized with nitrosamines, Cu2+, and NADPH for measurement of 8-oxodG in oxidized DNA by LC-MS/MS and specific ROS involved were identified by ROS-specific assays, that showed that nitrosamines metabolism in the presence of Cu2+ and NADPH produces hydroxyl radical (•OH), singlet oxygen (1O2), and hydrogen peroxide (H2O2). Possible reactive metabolites of NNK and NNN involved in ROS generation were identified by LC-MS/MS. Results provide insight into mechanistic pathways of cyt P450-mediated metabolism of NNK and NNN in ROS production and oxidation of DNA.