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

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.

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.

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.

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. Competing Interest Statement The authors have declared no competing interest. Footnotes Figures adjusted to fit in the 8.5x11 page size List of Abbreviations - COPD - Chronic obstructive pulmonary disease - ELISA - Enzyme-linked immunosorbent assay - PG/VG - Propylene glycol/Vegetable glycerol - AO/PI - Acridine orange/Propidium iodide - IL-6 - Interleukin-6 - IL-8 - Interleukin-8 - ENDS - Electronic nicotine delivery systems - PATH - Population Assessment of Tobacco and Health - ARDS - Acute respiratory distress syndrome - TNF - Tumor necrosis factor - BALF - Bronchoalveolar lavage fluid - ALI - Acute lung injury - ALI - Air-liquid interphase - NF-κB - Nuclear factor kappa B - FBS - Fetal bovine serum - HEPES - 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid - DMEM - Dulbecco’s Modified Eagle Medium - TEER - Transepithelial electrical resistance - EDTA - Ethylenediaminetetraacetic acid - HBEC - Primary human bronchial epithelial cells - CHRNA - Nicotinic Acetylcholine Receptors