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Quantitative Liquid Chromatography-Nanoelectrospray Ionization-High-Resolution Tandem Mass Spectrometry Analysis of Acrolein-DNA Adducts and Etheno-DNA Adducts in Oral Cells from Cigarette Smokers and Nonsmokers.

ABSTRACT: Cigarette smoking is an important source of human exposure to toxicants and carcinogens and contributes significantly to cancer morbidity and mortality worldwide. Acrolein, a widespread environmental pollutant, is present in relatively high amounts in cigarette smoke and can react directly with DNA to form DNA adducts, which serve as important biomarkers for the assessment of exposure to acrolein and its potential role in smoking related cancer. Etheno-DNA adducts are promutagenic DNA lesions that can derive from exogenous chemicals as well as endogenous sources, including lipid peroxidation. In this study, we developed a combined method for the quantitation of (6R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8,-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)-one (α-OH-Acr-dGuo), (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8,-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)-one (γ-OH-Acr-dGuo), 1,N⁶-etheno-dAdo (εdAdo), and 3,N⁴-etheno-dCyd (εdCyd) adducts in oral rinse and cytobrush DNA from smokers and nonsmokers by liquid chromatography-nanoelelctrospray ionization-high-resolution tandem mass spectrometry (LC-NSI-HRMS/MS). For oral rinse samples, there was a statistically significant difference between the levels of α-OH-Acr-dGuo, γ-OH-Acr-dGuo, εdAdo, and εdCyd in smokers (12.1 ± 17.9, 163 ± 227, 182 ± 568, and 194 ± 400 adducts/10⁹ nucleotides, respectively) and nonsmokers (1.85 ± 2.08, 5.95 ± 4.23, 7.69 ± 11.7, and 6.07 ± 10.9 adducts/10⁹ nucleotides, respectively). For cytobrush samples, there was a statistically significant difference between the levels of γ-OH-Acr-dGuo and εdAdo in smokers (259 ± 540 and 82.9 ± 271 adducts/10⁹ nucleotides, respectively) and nonsmokers (7.37 ± 5.09 and 16.2 ± 30.2 adducts/10⁹ nucleotides, respectively) but not for α-OH-Acr-dGuo and εdCyd. Our results demonstrate that oral mucosa cells are an excellent source of material for evaluating DNA adducts to be used as biomarkers of tobacco smoke exposure and molecular changes potentially related to cancer.

SUBMITTER: Paiano V

PROVIDER: S-EPMC8185904 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We developed a liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry (LC-NSI-HRMS/MS) method for simultaneous quantitative analysis of 5 oral cell DNA adducts associated with cigarette smoking: (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)-one (γ-OH-Acr-dGuo, 1) from acrolein; (6S,8S and 6R,8R)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxy-6-methylpyrimido[1,2-a]purine-10(3H)-one [(6S,8S)γ-OH-Cro-dGuo, 2; and (6R,8R)γ-OH-Cro-dGuo, 3] from crotonaldehyde; 1,N6-etheno-dAdo (4) from acrylonitrile, vinyl chloride, lipid peroxidation, and inflammation; and 8-oxo-dGuo (5) from oxidative damage. Oral cell DNA was isolated in the presence of glutathione to prevent artifact formation. Clear LC-NSI-HRMS/MS chromatograms were obtained allowing quantitation of each adduct using the appropriately labeled internal standards. The accuracy and precision of the method were validated, and the assay limit of quantitation was 5 fmol/μmol dGuo for adducts 1-4 and 20 fmol/μmol for adduct 5. The assay was applied to 80 buccal cell samples selected from those collected in the Shanghai Cohort Study: 40 from current smokers and 40 from never smokers. Significant differences were found in all adduct levels between smokers and nonsmokers. Levels of 8-oxo-dGuo (5) were at least 3000 times greater than those of the other adducts in both smokers and nonsmokers, and the difference between amounts of this adduct in smokers versus nonsmokers, while significant (P = 0.013), was not as great as the differences of the other DNA adducts between smokers and nonsmokers (P-values all less than 0.001). No significant relationship of adduct levels to risk of lung cancer incidence was found. This study provides a new LC-NSI-HRMS/MS methodology for the quantitation of diverse DNA adducts resulting from exposure to the α,β-unsaturated aldehydes acrolein and crotonaldehyde, inflammation, and oxidative damage which are all associated with carcinogenesis. We anticipate application of this assay in ongoing studies of the molecular epidemiology of cancers of the lung and oral cavity related to cigarette smoking.

Project description:BackgroundNicotine-containing electronic cigarette (e-cig) use has become widespread. However, understanding the biological impact of e-cigs compared with smoking on the lung is needed. There are major gaps in knowledge for chronic effects and for an etiology to recent acute lung toxicity leading to death among vapers.MethodsWe conducted bronchoscopies in a cross-sectional study of 73 subjects (42 never-smokers, 15 e-cig users, and 16 smokers). Using bronchoalveolar lavage and brushings, we examined lung inflammation by cell counts, cytokines, genome-wide gene expression, and DNA methylation.ResultsThere were statistically significant differences among never-smokers, e-cig users, and smokers for inflammatory cell counts and cytokines (FDR q < 0.1). The e-cig users had values intermediate between smokers and never-smokers, with levels for most of the biomarkers more similar to never-smokers. For differential gene expression and DNA methylation, e-cig users also more like never-smokers; many of these genes corresponded to smoking-related pathways, including those for xenobiotic metabolism, aryl hydrocarbon receptor signaling, and oxidative stress. Differentially methylated genes were correlated with changes in gene expression, providing evidence for biological effects of the methylation associations.ConclusionsThese data indicate that e-cigs are associated with less toxicity than cigarettes for smoking-related pathways. What is unknown may be unique effects for e-cigs not measured herein, and a comparison of smokers completely switching to e-cigs compared with former smokers. Clinical trials for smokers switching to e-cigs who undergo serial bronchoscopy and larger cross-sectional studies of former smokers with and without e-cig use, and for e-cigs who relapse back to smoking, are needed.ImpactThese data can be used for product regulation and for informing tobacco users considering or using e-cigs. What is unknown may be unique effects for e-cigs not measured herein, and clinical trials with serial bronchoscopy underway can demonstrate a direct relationship for changes in lung biomarkers.

Project description:IntroductionThe severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus responsible for the COVID-19 pandemic, gains entry into the host cell when its Spike protein is cleaved by host proteases TMPRSS2 and furin, thereby markedly increasing viral affinity for its receptor, angiotensin-converting enzyme-2 (ACE2). In rodent and diseased human lungs, tobacco cigarette (TCIG) smoke increases ACE2, but the effect of electronic cigarette vaping (ECIG) is unknown. It is unknown whether nicotine (in both TCIGs and ECIGs) or non-nicotine constituents unique to TCIG smoke increase expression of key proteins in COVID-19 pathogenesis.MethodsImmune (CD45+) cells collected before the pandemic in otherwise healthy young people, including TCIG smokers (n = 9), ECIG vapers (n = 12), or nonsmokers (NS) (n = 12), were studied. Using flow cytometry, expression of key proteins in COVID-19 pathogenesis were compared among these groups.ResultsTCIG smokers and ECIG vapers had similar smoking or vaping burdens as indicated by similar plasma cotinine levels. TCIG smokers compared with NS had a significantly increased percentage of cells that were positive for ACE2 (10-fold, p < .001), TMPRSS2 (5-fold, p < .001), and ADAM17 (2.5-fold, p < .001). Additionally, the mean fluorescence intensity (MFI) consistently showed greater mean ACE2 (2.2-fold, p < .001), TMPRSS2 (1.5-fold, p < .001), furin (1.1-fold, p < .05), and ADAM17 (2-fold, p < .001) in TCIG smokers compared with NS. In ECIG vapers, furin MFI was increased (1.15-fold, p < .05) and TMPRSS2 MFI tended to be increased (1.1-fold, p = .077) compared with NS.ConclusionsThe finding that key instigators of COVID-19 infection are lower in ECIG vapers compared with TCIG smokers is intriguing and warrants additional investigation to determine if switching to ECIGs is an effective harm reduction strategy. However, the trend toward increased proteases in ECIG vapers remains concerning.Implications(1) This is the first human study to report a marked increase in proteins critical for COVID-19 infection, including ACE2, TMPRSS2, and ADAM17, in immune cells from healthy tobacco cigarette smokers without lung disease compared with e-cigarette vapers and nonsmokers. (2) These findings warrant additional investigation to determine whether switching to electronic cigarettes may be an effective harm reduction strategy in smokers addicted to nicotine who are unable or unwilling to quit. (3) The increase in proteases in electronic cigarette vapers remains concerning.

Dataset Information

Quantitative Liquid Chromatography-Nanoelectrospray Ionization-High-Resolution Tandem Mass Spectrometry Analysis of Acrolein-DNA Adducts and Etheno-DNA Adducts in Oral Cells from Cigarette Smokers and Nonsmokers.

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