Research Article: Waterpipe smoking induces epigenetic changes in the small airway epithelium

Date Published: March 8, 2017

Publisher: Public Library of Science

Author(s): Matthew S. Walters, Jacqueline Salit, Jin Hyun Ju, Michelle R. Staudt, Robert J. Kaner, Allison M. Rogalski, Teniola B. Sodeinde, Riyaad Rahim, Yael Strulovici-Barel, Jason G. Mezey, Ahmad M. Almulla, Hisham Sattar, Mai Mahmoud, Ronald G. Crystal, Yuanpu Peter Di.


Waterpipe (also called hookah, shisha, or narghile) smoking is a common form of tobacco use in the Middle East. Its use is becoming more prevalent in Western societies, especially among young adults as an alternative form of tobacco use to traditional cigarettes. While the risk to cigarette smoking is well documented, the risk to waterpipe smoking is not well defined with limited information on its health impact at the epidemiologic, clinical and biologic levels with respect to lung disease. Based on the knowledge that airway epithelial cell DNA methylation is modified in response to cigarette smoke and in cigarette smoking-related lung diseases, we assessed the impact of light-use waterpipe smoking on DNA methylation of the small airway epithelium (SAE) and whether changes in methylation were linked to the transcriptional output of the cells. Small airway epithelium was obtained from 7 nonsmokers and 7 light-use (2.6 ± 1.7 sessions/wk) waterpipe-only smokers. Genome-wide comparison of SAE DNA methylation of waterpipe smokers to nonsmokers identified 727 probesets differentially methylated (fold-change >1.5, p<0.05) representing 673 unique genes. Dominant pathways associated with these epigenetic changes include those linked to G-protein coupled receptor signaling, aryl hydrocarbon receptor signaling and xenobiotic metabolism signaling, all of which have been associated with cigarette smoking and lung disease. Of the genes differentially methylated, 11.3% exhibited a corresponding significant (p<0.05) change in gene expression with enrichment in pathways related to regulation of mRNA translation and protein synthesis (eIF2 signaling and regulation of eIF4 and p70S6K signaling). Overall, these data demonstrate that light-use waterpipe smoking is associated with epigenetic changes and related transcriptional modifications in the SAE, the cell population demonstrating the earliest pathologic abnormalities associated with chronic cigarette smoking.

Partial Text

Waterpipe smoking (also called hookah, shisha, or narghile) is a tobacco use method traditionally associated with the Middle East [1–6]. However, its use is becoming more prevalent in the US and Western societies especially among young adults [4–6]. In contrast to cigarettes, waterpipe smoking involves placing the tobacco in a bowl surrounded by burning charcoal. When the smoker inhales, air is pulled through the charcoal and into the bowl holding the tobacco which results in the smoke being bubbled through water, carried through a hose, and subsequently inhaled [7]. The process of passing the smoke through water leads to a common belief amongst many waterpipe smokers that water filters out “toxins” from the smoke and, therefore, waterpipe is a safer smoking alternative to cigarettes [8]. However, the resulting smoke still includes many volatilized and pyrolyzed tobacco products together with carbon monoxide and charcoal components with the potential to induce toxic effects on the lung [7, 9–16].

To study the effect of waterpipe smoking on the DNA methylation profile of the small airway epithelium (SAE), DNA from the SAE of 7 nonsmokers and 7 waterpipe smokers was assessed by the HELP assay (Table 1). Principal component analysis using all methylation probesets as an input dataset demonstrated clear separation of the samples by waterpipe smoking phenotype when corrected by the covariates age, gender, ethnicity and region of SAE (left vs right lower lobe, Fig 1A). To identify differentially methylated probesets between waterpipe smokers and nonsmokers we followed the same approach of Pascual et al. [30] and our previous study of cigarette smoking induced DNA methylation of the SAE [24] and considered p<0.05 and a fold-change > ±1.5 as the threshold for analysis. Using this approach, a total of 727 differentially methylated probesets between waterpipe smokers and nonsmokers were identified representing 673 unique genes (see S1 Data File) with approximately 69% of these differentially methylated probesets located within 2 kb of the transcription start site of a gene. Of the 727 significant probesets, 64.6% (470/727) were hypermethylated and 35.4% hypomethylated (257/727; Fig 1B). Unsupervised hierarchical cluster analysis using the 727 waterpipe smoking-dysregulated probesets revealed complete separation of waterpipe smoker and nonsmoker subjects (Fig 1C).

The use of waterpipe to smoke tobacco is increasing worldwide, second only to cigarette smoking [1–6]. Epidemiologic studies in the US, Europe and other countries suggest the increase in prevalence of waterpipe smoking is mainly among young adults and teens, with 10 to 48% of adolescent and young adults admitting to smoking waterpipe, with 10 to 35% being current waterpipe smokers [4–6]. We have previously demonstrated that light-use waterpipe smoking by young adults is associated with a number of abnormal parameters related to lung health including, increased cough and sputum, a reduction in diffusion capacity, increases in blood carboxyhemoglobin, increased levels of pulmonary capillary-derived endothelial microparticles and global changes in the transcriptomes of alveolar macrophages and the small airway epithelium (SAE), two cell populations critical to maintain normal lung health [17]. In the present study, to understand the role of the epigenome in regulating the transcriptomic changes induced by waterpipe smoking in the SAE, we have built on these findings and assessed the effects of waterpipe smoking on the DNA methylation of the SAE, the initial site of pathologic changes in the lung of cigarette smokers [18–23].




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