The flavoring and not the nicotine content is a decisive factor for the effects of refill liquids of electronic cigarette on the redox status of endothelial cells

Electronic cigarettes are constantly gaining ground as they are considered less harmful than conventional cigarettes, and there is also the perception that they may serve as a potential smoking cessation tool. Although the acute effects of electronic cigarette use have been extensively studied, the long-term potential adverse effects on human health remain largely unknown. It has been well-established that oxidative stress is involved in the development of various pathological conditions. So far, most studies on e-cigarettes concern the effects on the respiratory system while fewer have focused on the vascular system. In the present study, we attempted to reveal the effects of electronic cigarette refill liquids on the redox state of human endothelial cells (EA.hy926 cell line). For this purpose, the cytotoxic effect of three e-liquids with different flavors (tobacco, vanilla, apple/mint) and nicotine concentrations (0, 6, 12, 18 mg/ml) were initially examined for their impact on cell viability of EA.hy926 cells. Then, five redox biomarkers [reduced form of glutathione (GSH), reactive oxygen species (ROS), total antioxidant capacity (TAC), thiobarbituric acid reactive substances (TBARS) and protein carbonyls (CARBS)] were measured. The results showed a disturbance in the redox balance in favor of free radicals in tobacco flavored e-liquids while vanilla flavored e-liquids exhibited a more complex profile depending on the nicotine content. The most interesting finding of the present study concerns the apple/mint flavored e-liquids that seemed to activate the cellular antioxidant defense and, thus, to protect the cells from the adverse effects of free radicals. Conclusively, it appears that the flavorings and not the nicotine content play a key role in the oxidative stress-induced toxicity of the e-liquids.

Keywords: 8-OH-dG, 8-hydroxy-deoxyguanosine; CARBS, protein carbonyls; CO, carbon monoxide; DCF-DA, 2′,7′-dichlorodihydrofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; DNPH, 2,4-dinitrophenylhydrazine; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DPPHH, 2,2-diphenyl-1-picrylhydrazine; E-cigarettes; E-liquids; ENDS, electronic nicotine delivery systems; EPR, electronic paramagnetic resonance; Endothelial cells; FSC, forward light scattering; GSH; GSH, reduced form of glutathione; HCL, hydrochloric acid; HCN, hydrogen cyanide; MDA, malondialdehyde; Oxidative stress; PBS, phosphate buffered saline; PG, propylene glycol; ROS; ROS, reactive oxygen species; SSC, side light scattering; TAC, total antioxidant capacity; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances; TCA, trichloroacetic acid; Tris-HCl, trishydroxymethylaminomethane hydrochloride; VG, vegetable glycerin.