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
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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 in tobacco- and menthol-flavored ENDS constituents could elicit a lung-injurious response mediated by nicotinic acetylcholine receptor (α-nAChR or CHRNA) modulation.
Methods
Human 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 (α1, α4, α5, and α7) was quantified by immunoblotting.
Results
Epithelial 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 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 nAChR modulation-mediated inflammation by exposure to these ENDS constituents, even in the absence of nicotine.
