Kankanamage Rumasha N T, Ghosh Abhisek Brata, Jiang Di, Gkika Karmel, Keyes Tia, Achola Laura A, Suib Steven, Rusling James F
Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, United States.
School of Chemical Sciences, Dublin City University, Dublin D9, Ireland.
Chem Res Toxicol. 2020 Aug 17;33(8):2072-2086. doi: 10.1021/acs.chemrestox.0c00027. Epub 2020 Aug 4.
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.
吸烟和吸电子烟产生的亚硝胺代谢产物会导致DNA损伤，进而引发基因毒性。虽然亚硝胺4-(甲基亚硝基氨基)-1-(3-吡啶基)-1-丁酮(NNK)和亚硝基去甲烟碱(NNN)的代谢产物形成的DNA加合物是众所周知的与烟草相关的癌症生物标志物，但只有少数研究表明NNN和NNK会导致人体DNA氧化。在从不吸烟的电子烟使用者的尿液中发现了NNK和NNN。本文提出了由NNK和NNN代谢产物在与铜和烟酰胺腺嘌呤二核苷酸磷酸(NADPH)的氧化还原反应中驱动DNA氧化的首个化学途径，该反应会产生活性氧(ROS)。使用一种带有DNA薄膜和代谢酶的微流控阵列来估计DNA氧化的相对速率，该代谢酶在铜和NADPH存在的情况下可产生NNN和NNK的代谢产物。通过电化学发光(ECL)检测使用了一种新型ECL染料[Os(tpy-苯-COOH)]，它对DNA中的主要DNA氧化产物8-氧代-7,8-二氢-2'-脱氧鸟苷(8-氧代脱氧鸟苷，8-oxodG)具有选择性且敏感。磁珠上的酶-DNA薄膜用于产生亚硝胺代谢产物，这些代谢产物与铜和NADPH进入形成ROS的氧化还原循环，液相色谱-质谱联用(LC-MS)用于定量8-氧代脱氧鸟苷并鉴定代谢产物。ROS通过光学传感器进行检测。NNK和NNN的代谢产物+铜+NADPH产生了相对较高的DNA氧化速率。肺部是吸烟和吸电子烟时的暴露途径，人体肺组织含有铜和NADPH，并且在本研究中肺微粒体酶产生的DNA氧化速率最高。此外，电子烟烟雾中的铜含量比香烟烟雾中的高6倍，这可能会加剧DNA氧化。
