Abstract
Rationale: The effects of hookah smoke on respiratory epithelia have not been well characterized. Objectives: To characterize and compare the effects of hookah tobacco smoke and conventional cigarette smoke on the epigenome and transcriptome of human respiratory epithelia. Methods: Normal human small airway epithelial cells and cyclin-dependent kinase 4/human telomerase reverse transcriptase–immortalized human bronchial epithelial cells were cultured for 5 days in normal media in the presence or absence of water pipe condensates or cigarette smoke condensates under relevant exposure conditions. CyQUANT assay (Thermo Fisher Scientific), RNA sequencing, quantitative reverse transcriptase–polymerase chain reaction, and Western blotting techniques were used to examine/compare the effects of hookah and cigarette smoke on cell proliferation, messenger RNA/microRNA expression, and global histone marks. Results: Water pipe condensates– and cigarette smoke condensates–mediated, dose-dependent growth-inhibitory effects in cultured respiratory epithelial cells were measured. Under conditions mediating equipotent mild to moderate growth-inhibitory effects, water pipe condensates as well as cigarette smoke condensates decreased histone H4 lysine 15 acetylation (H4K16ac) and histone H4 lysine 20 trimethylation (H4K20me3) levels in small airway epithelial cells and human bronchial epithelial cells; these histone alterations are putative “hallmarks of cancer.” RNA sequencing analysis demonstrated that cigarette smoke condensates and water pipe condensates mediated dose-dependent alterations in gene expression in small airway epithelial cells and human bronchial epithelial cells. A total of 873 genes were commonly regulated (fold change >2) by water pipe condensates (1.0 mg/ml) and cigarette smoke condensates (0.05 mg/ml) in small airway epithelial cells, whereas a total of 1,577 genes were commonly regulated by water pipe condensates (1.0 mg/ml) and cigarette smoke condensates (0.05 mg/ml) in human bronchial epithelial cells. Ingenuity Pathway Analysis (QIAGEN Bioinformatics) of the 100 genes commonly regulated by water pipe condensates and cigarette smoke condensates in small airway epithelial cells and human bronchial epithelial cells demonstrated that the top three activated upstream regulators are cigarette smoke (z-score = 2.577), benzo[a]pyrene (z-score = 2.905), and nuclear factor (erythroid-derived 2)-like 2 (NFE2L2, Nrf2) (z-score = 2.531). Analysis of downstream effects indicated that growth of epithelial tissue was predicted to be increased (z-score = 2.813; P < 0.001). The top three canonical pathways included xenobiotic metabolism signaling, aryl hydrocarbon receptor signaling, and nicotine degradation III. Conclusions: These preliminary findings strongly suggest that hookah tobacco is not a safe alternative to cigarettes, and warrant further evaluation of the epigenomic effects of hookah smoke in human respiratory epithelia using our established laboratory models and correlative analyses in hookah smokers.