ABSTRACT: Background While smoking is known to associate with development of multiple diseases, the underlying mechanisms are still poorly understood. Tobacco smoking can modify the chemical integrity of DNA leading to changes in transcriptional activity, partly through an altered epigenetic state. We aimed to investigate the impact of smoking on lung cells collected from bronchoalveolar lavage (BAL). Methods We profiled changes in DNA methylation (5mC) and its oxidized form hydroxymethylation (5hmC) using conventional bisulfite (BS) treatment and oxidative bisulfite treatment with Illumina Infinium MethylationEPIC BeadChip, and examined gene expression by RNA-seq in healthy smokers. Findings We identified 1,667 BS methyl (5mC+5hmC), 1,756 5mC and 67 5hmC differentially methylated positions (DMPs) between smokers and nonsmokers (FDR <0.05, absolute Δβ >0.15). Both 5mC DMPs and to a lesser extent BS methyl (5mC+5hmC) were predominantly hypomethylated. In contrast, almost all 5hmC DMPs were hypermethylated, supporting the hypothesis that smoking-associated oxidative stress can lead to DNA demethylation, via the established sequential oxidation of which 5hmC is the first step. While we confirmed differential methylation of previously reported smoking-associated BS methyl CpGs using former generations of BeadChips in alveolar macrophages, the large majority of identified DMPs, BS methyl (1,639/1,667), 5mC (1,738/1,756), and 5hmC (67/67), have not been previously reported. Most of these novel smoking-associating sites are specific to the EPIC BeadChip and, interestingly, many of them are associated to FANTOM5 enhancers. Transcriptional changes affecting 633 transcripts were consistent with DNA methylation profiles and converged to alteration of genes involved in migration, signaling and inflammatory response of immune cells. Interpretation Collectively, these findings suggest that tobacco smoke exposure epigenetically modifies BAL cells, possibly involving a continuous active demethylation and subsequent increased activity of inflammatory processes in the lungs. -
