Salivary biomarkers of inflammation, angiogenesis, and aging in hookah smokers, cigarette smokers, and non-smokers

Hookah smoking is an increasingly prevalent form of tobacco consumption, particularly among young adults. Although conventional cigarette smoking is well established as a modulator of inflammatory and biological processes in the oral environment, salivary inflammatory and protective responses associated with hookah smoking remain less well characterized. This study investigated whether hookah smoking is associated with a distinct salivary profile of inflammatory cytokines (IL-1β, IL-2, IL-6, and TNF-α), vascular endothelial growth factor (VEGF), and Klotho compared with conventional cigarette smoking and non-smoking. Methods: Unstimulated whole saliva was collected from 87 individuals classified into three groups: hookah smokers ( n = 32), conventional cigarette smokers ( n = 26), and non-smokers ( n = 29; control group). Samples were collected between 10:00 and 12:00 h, and salivary concentrations of IL-1β, IL-2, IL-6, TNF-α, VEGF, and Klotho were quantified using enzyme-linked immunosorbent assays (ELISA). Unadjusted between-group comparisons were performed using non-parametric tests, and associations with clinical and behavioral variables were assessed using Spearman’s rank correlations with correction for multiple testing. Multivariable regression analyses were performed to account for potential confounding by age, sex, alcohol consumption, and, in analyses restricted to smokers, duration of tobacco use. Receiver operating characteristic (ROC) curve analyses were performed as exploratory assessments of biomarker discrimination. Results: Hookah smokers exhibited higher salivary concentrations of IL-2, IL-6, IL-1β, and TNF-α and lower concentrations of VEGF and Klotho compared with non-smokers. After adjustment for age, sex, and alcohol consumption, these differences remained significant (all p < 0.001). Conventional cigarette smokers also exhibited higher IL-2, IL-1β, and TNF-α and lower VEGF and Klotho concentrations than controls, whereas the adjusted difference in IL-6 was not significant. Direct adjusted comparisons between smoking modalities demonstrated higher IL-2, IL-6, IL-1β, and TNF-α and lower VEGF concentrations in hookah smokers than in cigarette smokers, whereas Klotho did not differ between the smoking groups. After additional adjustment for duration of tobacco use among smokers, differences in IL-2, IL-1β, TNF-α, and VEGF remained significant. No biomarker–clinical variable correlation remained significant after correction for multiple testing. Conclusion: Both hookah and conventional cigarette smoking were associated with altered salivary inflammatory and regulatory biomarker profiles compared with non-smoking. Importantly, hookah smokers exhibited a distinct profile compared with conventional cigarette smokers, particularly characterized by higher IL-2, IL-1β, and TNF-α and lower VEGF concentrations after adjustment for relevant potential confounders, including duration of tobacco use. These findings suggest that different forms of tobacco exposure may be associated with distinct biological responses in the oral environment.