Abstract 3236: Lung epithelium DNA methylation: Electronic cigarette users, smokers, and never-smokers
Authors: Song Min-Ae , Smiraglia Dominic J. , Brasky Theodore M. , Weng Daniel Y. , McElroy Joseph P. , Reisinger Sarah A. , Ying Kevin L. , Nickerson Quentin A. , Wewers Mark D. , Shields Peter G. , Freudenheim Jo L. 
Published: August 2018

Abstract
Background: The use of electronic cigarettes (e-cigs) among smokers and never-smokers is increasing, and is considered to be less harmful than cigarettes. However, this has not been studied in target organs for smoking-related diseases, especially the lung. Altered DNA methylation is seen in smokers and contributes to lung carcinogenesis. However, it is not known how e-cigs affect methylation in the lung or whether smoking-induced DNA methylation changes can be reversed in former smokers who switch to e-cigs.
Methods: We conducted a cross-sectional bronchoscopy study of e-cig users (n=12), cigarette smokers (n=10), and never-smokers (n=10) (age 21-30, 84% European-American, 66% male). Bisulfite-converted DNA extracted from lung epithelium collected by bronchial brushing was analyzed for 865,859 CpGs with the llumina Infinium Methylation EPIC Chip in a single batch. Probes were filtered out if on sex chromosomes, cross-reactive, or SNP associated, leaving 735,317 CpGs for analysis. One-way analysis of covariance using M-values was used to identify CpGs differentially methylated for the three groups. False Discovery Rate (FDR) of q&lt;0.1 was considered significant. Ingenuity pathway analysis (IPA) was used for potential biologic implications of identified CpGs.
Results: Smokers averaged 18 cigarettes/day (SD: 4.2) and 6 years of smoking (SD: 4.5). E-cig users vaped an average e-liquid nicotine content of 10 mg/mL (SD: 11) and 8 mL/day (SD: 4), for an average of three years (SD: 1). Mean time since smoking among the e-cig users was 31 months (SD: 15); 3 were never-smokers. We identified 517 differentially methylated CpGs among the three groups (FDR q&lt;0.1). Of them, 128 (25%) were in enhancers, 165 (47%) in promoters, and 162 (31%) in CpG islands/surroundings. For e-cig users, methylation of most differentially methylated CpGs (n=505) was between those for smokers and never-smokers. For smoking- and/or lung cancer-related genes: AHRR, ALDH3A1, ALPK3, CYP1B1, and OXCT1, CpGs were less methylated in smokers than in never-smokers; e-cig users were intermediate. There were 330 unique genes with differentially methylated CpGs; 319 were included in an IPA analysis. The most significantly associated disease was cancer (n=283) and biologic function was drug metabolism (n=10). The top canonical pathways included xenobiotic metabolism signaling (n=13) and wnt/β-catenin signaling (n=9).
Discussion: DNA methylation differed for never-smokers and smokers, with e-cig users' methylation intermediate, consistent with the hypothesis that e-cigs are less harmful than smoking; whether e-cig use is more harmful than never-smoking needs to be studied. Because this study is cross-sectional, association, and not causation, is indicated. Additional observational studies and randomized trials are warranted to understand biologic changes in the lung for smokers and never-smokers using e-cigs.
Citation Format: Min-Ae Song, Dominic J. Smiraglia, Theodore M. Brasky, Daniel Y. Weng, Joseph P. McElroy, Sarah A. Reisinger, Kevin L. Ying, Quentin A. Nickerson, Mark D. Wewers, Peter G. Shields, Jo L. Freudenheim. Lung epithelium DNA methylation: Electronic cigarette users, smokers, and never-smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3236.