ROS-mediated oxidative stress induced by e-cigarette fluids in Human Gingival Fibroblasts

Introduction: Electronic cigarettes (e-cigarettes) are generally recognized as a safer alternative to combusted tobacco products, but there are conflicting claims about the degree to which these products warrant concern for the health of e-cigarette users. Aim of this work is to investigate the effects of liquids of e-cigarettes on Human Gingival Fibroblasts (HGFs) health, comparing the effects of nicotine containing fluid (nicotine) to the fluid itself (vehicle). Methods: HGFs were treated with different concentrations (0-5mg/mL) of fluids of e-cigarettes for different times (0-72h) and cell proliferation was analyzed by MTT assay. Fluids were administered also after being vaped by cartomizer. Apoptosis occurrence and Bax expression were evaluated by flow cytometry, while mitochondrial membrane potential perturbation and ROS production were analyzed by fluorescence optical microscopy. Results: Both nicotine containing and nicotine free fluids showed a dose and time dependent reduction (up to 20% of the untreated sample) of HGFs proliferation, being the 1mg/ml the most interesting concentration because it induced a proliferation similar to the untreated sample until 24h treatment and a lower proliferation rate afterwards. Bax expression increased in a time dependent manner with a greater extent in the nicotine samples (1.7 fold increase in the 1mg/mL nicotine treated sample) with respect to the vehicle treated samples. Significant cell viability decrease (37% vs 85% of the untreated sample) and apoptosis increase were found after 48h in all the experimental samples with a greater extent in the nicotine sample. ROS production showed a four fold increase in all the samples at 24 h with respect to the control. A perturbation of the mitochondrial membrane potential was observed in the nicotine treated sample after 24 h. Conclusions: Based on our results we can state that the cytotoxicity exerted on HGFs by exposure to e-cigarettes fluids is not entirely ascribable to nicotine. Since the e-cigarettes are presented as a safe alternative to normal ones, expecially for the possibility of “smoking” nicotine free fluids, further studies are necessary to clarify the mechanism involved in the cytotoxicity exerted by such fluids