Comparative toxicity of menthol- and tobacco-flavored electronic cigarette constituents causing inflammation, epithelial barrier dysfunction, and nicotinic acetylcholine receptor modulation in the absence of nicotine.

BackgroundMenthol and tobacco-flavored nicotine delivery systems (ENDS) are widely used as safer alternatives to combustible cigarettes. These flavored products include constituents such as propylene glycol/vegetable glycerin (PG/VG), benzoic acid, acetoin, L-menthone, 98% menthone, 2-isopropyl-N,2,3-trimethylbutanamide (WS-23), vanillin, and carvone. However, little is known about the potential adverse effects of the constituents in these flavored products. Rationale and hypothesisWe hypothesized that exposure to common constituents in tobacco- and menthol-flavored ENDS constituents could elicit a lung-injurious response mediated by nicotinic acetylcholine receptor (-nAChR or CHRNA) modulation. MethodsHuman bronchial epithelial cells, BEAS-2B, cells were treated with commonly used menthol and tobacco constituents on trans well inserts. Transepithelial barrier resistance (TEER) and millivolts (mV) across epithelial cells were measured over a 24-hour time. To assess the elicited inflammatory response, cytokines IL8 and IL6 were quantified in the conditioned media. Cytotoxicity caused by these constituents was evaluated by acridine orange/propidium iodide (AO/PI) staining of the cells after 24 hrs. alpha nicotinic receptor protein abundance (1, 4, 5, and 7) was quantified by immunoblotting. ResultsEpithelial integrity was decreased over time with a significant decrease in TEER and voltage by ENDS constituents. A significant increase in IL6 in conditioned media was observed in PG/VG, carvone, and WS-23 treated cells. Carvone-treated cells also elicited significantly elevated IL8 in conditioned media. Further, increased 1, 4, 5, and 7 nAChR were seen in cells treated with PG/VG, Acetoin, Carvone, and WS-23. ConclusionThese findings suggested that common constituents in menthol- and tobacco-flavored ENDS induce lung inflammation, epithelial barrier dysfunction, and lung injury. Further, our data implicate potential lung disease pathogenesis via nAChR modulation-mediated inflammation by exposure to these ENDS constituents, even in the absence of nicotine.