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

BACKGROUND: Menthol 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 HYPOTHESIS: We hypothesized that exposure to common constituents of tobacco- and menthol-flavored ENDS could elicit a lung-injurious response mediated by modulation of nicotinic acetylcholine receptors (α-nAChRs or CHRNA). METHODS: Human bronchial epithelial cells, BEAS-2B, were treated with commonly used menthol and tobacco constituents on transwell inserts. Transepithelial barrier resistance (TEER) and millivolts (mV) across epithelial cells were measured over 24 h. To assess the elicited inflammatory response, cytokines IL-8 and IL-6 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 h. Alpha nicotinic receptor protein abundance (α1, α4, α5, and α7) was quantified by immunoblotting. RESULTS: Epithelial integrity decreased over time, with significant decreases in TEER and voltage by ENDS constituents. A significant increase in IL-6 in conditioned media was observed in PG/VG, carvone, and WS-23 treated cells. Carvone-treated cells also elicited significantly elevated IL-8 in conditioned media. Further, increased α1, α4, α5, and α7 nAChR were seen in cells treated with PG/VG, acetoin, carvone, and WS-23. CONCLUSION: These 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 α-nAChRs modulation-mediated inflammation by exposure to these ENDS constituents, even in the absence of nicotine.