Study population

Self-reported never smokers (“nonsmokers”, n = 7), and self-reported light-use waterpipe-only smokers (“waterpipe smokers”, n = 7) were recruited from the general population in the New York metropolitan area (Table 1). One of the waterpipe smokers had a history of conventional cigarette smoking (less than 5 pack/years) which occurred >20 years prior to enrollment in this study, whereas all other waterpipe smokers and nonsmokers had no history of cigarette smoking. The term “light-use” was used to define waterpipe smokers who reported smoking less than or equal to 5 sessions per week. All subjects were evaluated at the Weill Cornell NIH Clinical and Translational Science Center and Department of Genetic Medicine Clinical Research Facility, using Weill Cornell Institutional Review Board-approved clinical protocols and written informed consent obtained. The criteria for “healthy” was based on medical history, physical exam, complete blood count, coagulation studies, liver function tests, urine studies, chest X-ray, EKG and pulmonary function tests as previously described [17, 24]. All subjects were negative for HIV1 and had normal α1-antitrypsin levels (for full inclusion/exclusion criteria, see S1 Methods). Urine nicotine and cotinine levels were determined using liquid chromatography-tandem mass spectrometry (ARUP laboratories, Salt Lake City, UT) [26].

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Table 1. Demographics of the study population and biological samples¹.

https://doi.org/10.1371/journal.pone.0171112.t001

DNA methylation analysis

To study the effect of waterpipe smoking on the DNA methylation profile of the SAE, the microarray-based high resolution HpaII tiny fragment Enriched by Ligation-mediated PCR (HELP) assay was performed using a 720K Roche-NimbleGen custom array (capturing 117,521 HpaII fragments) on SAE DNA from a total of 14 samples (n = 7 nonsmokers and n = 7 waterpipe smokers) [27]. Quality control of the arrays included assessment of MspI and HpaII intensity distribution and spatial uniformity of the Cy3 and Cy5 signals [28]. For all queried HpaII fragments, intensities were processed to determine the Q centered (Qcent) ratio, and the log₂ multi-sample, quantile normalized unmethylated / methylated (HpaII/MspI) ratio. The resulting Qcent parameter was exported to Excel with fragment annotation details for statistical analysis. To aid in categorization of the methylation states of specific fragments, the HpaII/MspI ratio was inverted to MspI/HpaII, therefore defining hypomethylated (less methylated) loci with a negative log₂ ratio value and hypermethylated (increased methylation) loci a positive log₂ ratio value. The phenotypes were evaluated in Partek Genomics Suite Software version 6.6 (Partek Inc., St. Louis, MO) for sources of variation. A 4-way ANOVA was performed to assess waterpipe smoking on methylation of the 117,521 HpaII fragments, with Qcent ratios corrected by age, gender, ethnicity and region of SAE (left vs right lower lobe). Fold-change was determined as: [least square mean waterpipe smokers /least square mean nonsmokers]. Probe fragments with a waterpipe smoking vs nonsmoking p value <0.05 calculated by a Student’s t-test and a fold-change >±1.5 were designated as the threshold (no significant probe fragments were identified with a Benjamini Hochberg corrected p value <0.05). The closest gene was determined for these fragments and annotation files were used to map individual HpaII fragments relative to transcription start site of the closest gene. The raw data are publically available at the Gene Expression Omnibus (GEO) site (http://www.ncbi.nlm.nih.gov/geo/), accession number GSE92662.

Gene expression analysis

To study the effect of waterpipe smoking on the gene expression profile of the SAE assessed for methylation analysis, total RNA from the SAE of the same nonsmokers (n = 7) and waterpipe smokers (n = 7) was processed to generate cDNA and perform genome-wide gene expression analysis using the HG-U133 Plus 2.0 array (Affymetrix, Santa Clara, CA) according to Affymetrix protocols. Overall microarray quality was verified by the criteria: (1) 3'/5' ratio for GAPDH ≤3; and (2) scaling factor ≤10.0 [29]. CEL files were processed by Partek for quality control, identification of outliers, and determination of expression level for all probesets, using Robust Multi-chip Average (RMA) method with Partek default parameters. The phenotypes were evaluated in Partek for sources of variation. A 4-way ANOVA was performed to assess waterpipe smoking on gene expression corrected by age, gender, ethnicity and region of SAE (left vs right lower lobe). A p value <0.05 calculated by a Student’s t-test with no fold-change cutoff were designated as the threshold. Validation of the HG-U133 Plus 2.0 array gene expression data by RNA sequencing in a subset of the same samples was carried out as detailed in S1 Methods. The raw data are publically available at the Gene Expression Omnibus (GEO) site (http://www.ncbi.nlm.nih.gov/geo/), accession number GSE92662.

Correlation of gene expression with methylation

The correlation between waterpipe smoking-dependent DNA methylation and gene expression of the SAE was assessed using a starburst plot comparing the p values for waterpipe smoking-dependent methylation to the p value for smoking-dependent gene expression. This analysis was performed for 673 unique genes from the 727 HpaII probe fragments differentially methylated in waterpipe smokers compared to nonsmokers (p<0.05, fold-change > ±1.5) that also had corresponding HG-U133 Plus 2.0 gene expression data.

Statistics

Comparison of demographic parameters among groups was performed by two-tailed Student’s t-test. A 4-way ANOVA was performed on the DNA methylation HELP assay data to examine the influence of covariates on waterpipe smoking response. For the HG-U133 Plus 2.0 gene expression data, a 4-way ANOVA was also performed to examine the influence of covariates on smoking response.

Waterpipe smoking associated methylation changes of the small airway epithelium

DNA methylation, the attachment of methyl groups to cytosine bases followed by guanine (CpG sites), is a heritable and reversible gene regulatory modification that plays a critical role in regulating cell type and tissue-specific gene expression [37, 38]. DNA methylation is highly modified in response to cigarette smoke and altered in multiple lung diseases including asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), suggesting that DNA methylation may play an important role in the pathogenesis of these diseases [24, 25, 39–51]. Comparing the SAE DNA methylation of waterpipe smokers to nonsmokers, our results demonstrate that light-use waterpipe smoking is associated with genome-wide DNA methylation changes affecting hundreds of genes. Interestingly, we observed predominant hypermethylation of the affected genes in the SAE of waterpipe smokers relative to the SAE of nonsmokers which contrasts with our previous findings comparing the SAE of cigarette smokers to nonsmokers where predominant hypomethylation was observed [24]. These differences in DNA methylation patterns between waterpipe smoking and cigarette smoking may result from the different chemical composition of waterpipe and cigarette smoke. For example, compared to one cigarette, one waterpipe session exposes the smoker to 2 to 4 times the amount of nicotine, 7 to 11 times the amount of carbon monoxide, 100 times more tar, 17 times the amount of formaldehyde, 2 to 5 times the amount of high molecular weight carcinogenic polyaromatic hydrocarbons and 3 times the amount of phenol [7, 9, 14, 15]. In addition, high levels of benzene, volatile aldehydes and other toxins originating from flavoring have been detected in waterpipe smoke [10–13, 16]. These chemical differences between waterpipe and cigarette smoke likely have differential effects on cellular processes that regulate DNA methylation resulting in differences in global methylation levels and patterns. Despite these global differences, comparison of the effects of waterpipe smoking and cigarette smoking on DNA methylation of the SAE demonstrated an overlap of differentially methylated genes in the SAE of both waterpipe smokers and cigarette smokers compared to nonsmokers suggesting a common effect on modifying the SAE epigenome.

Alterations in molecular pathways

Characterization of the molecular pathways associated with our waterpipe smoking-dependent differential methylated gene set showed significant enrichment of pathways previously associated with cigarette smoking and COPD in the SAE, including aryl hydrocarbon receptor signaling, xenobiotic metabolism signaling and G-protein coupled receptor signaling, further supporting the concept that waterpipe smoking has harmful effects on lung biology [24, 25]. There was also enrichment of pathways previously not associated with cigarette smoking and lung disease including cholecystokinin/gastrin-mediated signaling. The gastrointestinal peptides cholecystokinin (CCK) and gastrin are a structurally diverse group of secreted molecular messengers that regulate multiple normal and abnormal biological processes including development, inflammation, tissue regeneration, and neoplastic transformation [52]. Both CCK and gastrin exert their effects by binding to specific G-protein coupled receptors on the surface of a target cell and upon binding, trigger production of secondary messengers and subsequent Ca2+ release for activation of multiple kinase signal transduction pathways that relay the mitogenic signal to the nucleus [52, 53]. Important mediators that play a central role in relaying these activation signals related to cholecystokinin/gastrin-mediated signaling pathway include PLCB2 (phospholipase C, beta 2), PLCB3 (phospholipase C, beta 3), ITPR3 (inositol 1,4,5-trisphosphate receptor, type 3), RHOA (Ras homolog family member A), RHOF (Ras homolog family member F), SRC (src proto-oncogene), SOS2 (son of sevenless homolog 2), MAPK14 (mitogen-activated protein kinase 14) and the transcription factors JUN (jun proto-oncogene) and FOS (FBJ murine osteosarcoma viral oncogene homolog) [52], all of which display abnormal DNA methylation at the gene level in the SAE of waterpipe smokers vs nonsmokers. The role of cholecystokinin/gastrin-mediated signaling in human lung biology is unknown, however a recent study using a bleomycin-induced mouse model of pulmonary fibrosis demonstrated enrichment of this pathway in the target genes for altered miRNA expression in the fibrotic lung suggesting this pathway may play a role in the disease process [54]. Interestingly, some of the intracellular mediators for cholecystokinin/gastrin-mediated signaling also play a role in other signaling pathways including nicotine signaling [55]. Furthermore, multiple genes (e.g., FOS, JUN, PLCB2, PLCB3, SOS2 and SRC) displaying abnormal DNA methylation at the gene level in the SAE of waterpipe smokers vs nonsmokers were present in multiple pathways suggesting that waterpipe smoking may impact a wide variety of biological processes by disrupting a small number of key genes.

In addition to assessing the genome-wide DNA methylation changes associated with waterpipe smoking, we investigated whether these methylation changes were associated with alterations in the SAE transcriptome of the associated gene by correlating gene expression patterns from patient matched samples. Using this approach we identified 11.3% of the differentially methylated genes displayed a significant change in gene expression with pathway analysis of this gene set showing enrichment for the eIF2 signaling and regulation of eIF4 and p70S6K signaling pathways which play an important role in regulating mRNA translation and subsequent protein synthesis during homeostasis and in response to environmental stimuli [33–36]. Interestingly, four genes (EIF2S1, RPS5 and RPS15 and SOS2) displaying both DNA methylation and gene expression changes in the SAE of waterpipe smokers were present in both pathways suggesting possible functional redundancy in the biological roles of these pathways. Due to the critical role of proteins in regulating a large variety of biological processes, protein synthesis, folding and subsequent degradation (i.e. protein homeostasis or “proteostasis”) are fundamental to maintain optimal cellular function and tissue homeostasis during normal conditions and in response to environmental stress [56, 57]. This is of particular importance to the lung, which due to its anatomical structure is in direct contact with the outside world and continuously challenged by inhaled insults including cigarette smoke. One pathway critical for maintaining cellular homeostasis is termed the “integrated stress response” which is composed of four homologous stress-sensing kinases which are activated in response to cellular stress including protein folding efficiency in the endoplasmic reticulum (ER) and multiple environmental insults including viral infection and cigarette smoke [58–60]. In the presence of specific stress and/or insult, the integrated stress response is activated resulting in phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) and subsequent inhibition of protein synthesis in the cell [33]. This global shutdown of protein synthesis serves a number of protective roles including relieving ER stress by reducing the rate of proteins entering the ER and allowing the unfolded protein response (UPR) to resolve the accumulation of misfolded proteins that compromise ER function [61, 62]. Phosphorylation of eIF2α promotes translation of a subset of mRNAs that help the cell adapt to these stresses including the transcription factor ATF4 (activating transcription factor 4) [33, 60–62]. A recent study in COPD identified a 98 gene airway gene expression signature which included many genes that were targets of the ATF4 transcription factor suggesting chronic activation of the integrated stress response abnormal proteostasis in COPD [63]. In addition to COPD, disruption of protein homeostasis has been associated with the development and progression of additional chronic lung diseases and genetic disorders including IPF, asthma, cystic fibrosis and α1-antitrypsin deficiency [59, 64–66]. The finding that light-use waterpipe smoking is associated with both methylation and transcriptomic changes in a number of genes linked to pathways that regulate protein translation and synthesis suggests waterpipe smoking impacts protein homeostasis of the SAE which may play a role in the pathogenesis of waterpipe-dependent lung disease.

Impact and limitations of the study

We acknowledge the limitations of the study including the small sample population and lack of validation of the DNA methylation data by an independent methodology. Although the samples were all >98.6% pure epithelium, due to the small percentage (0.8–1.4%) of inflammatory cells in the SAE brushings we cannot rule out the possibility that some of the epigenetic and transcriptional changes we observe in response to waterpipe smoking are originating from non-epithelial cell populations. However, based on our knowledge, this is the first report regarding the impact of waterpipe smoking on epigenetics of the SAE, a cell population critical to the initiation and pathology of cigarette smoking induced lung disease. Due to the increasing use of waterpipe smoking among young adults and limited studies showing its biological and molecular impact on the lung, the results of this study will help in the design of larger epidemiologic and biologic studies on the harmful effects of waterpipe smoking.

In summary, the data demonstrates that light-use waterpipe smoking in young adults is associated with a broad range of genome-wide DNA methylation-related changes, with many of these methylation-related changes associated with changes in the SAE transcriptome. Relative to traditional cigarette smoking, waterpipe smoking impacts the SAE in both similar and unique ways at the DNA methylation level adding to the accumulating evidence that waterpipe smoking is harmful and detrimental to lung health.