Abstract

BackgroundThe potential for adverse respiratory effects following exposure to electronic (e-) cigarette liquid (e-liquid) flavorings remains largely unexplored. Given the multitude of flavor permutations on the market, identification of those flavor constituents that negatively impact the respiratory tract is a daunting task. In this study we examined the impact of common e-liquid flavoring chemicals on the airway epithelium, the cellular monolayer that provides the first line of defense against inhaled particulates, pathogens, and toxicants.MethodsWe used the xCELLigence real-time cell analyzer (RTCA) as a primary high-capacity screening tool to assess cytotoxicity thresholds and physiological effects of common e-liquid flavoring chemicals on immortalized human bronchial epithelial cells (16HBE14o-). The RTCA was used secondarily to assess the capability of 16HBE14o- cells to respond to cellular signaling agonists following a 24 h exposure to select flavoring chemicals. Finally, we conducted biophysical measurements of well-differentiated primary mouse tracheal epithelial (MTE) cells with an Ussing chamber to measure the effects of e-cigarette flavoring constituents on barrier function and ion conductance.ResultsIn our high-capacity screens five of the seven flavoring chemicals displayed changes in cellular impedance consistent with cell death at concentrations found in e-liquid. Vanillin and the chocolate flavoring 2,5-dimethylpyrazine caused alterations in cellular physiology indicative of a cellular signaling event. At subcytotoxic levels, 24 h exposure to 2,5-dimethylpyrazine compromised the ability of airway epithelial cells to respond to signaling agonists important in salt and water balance at the airway surface. Biophysical measurements of 2,5-dimethylpyrazine on primary MTE cells revealed alterations in ion conductance consistent with an efflux at the apical airway surface that was accompanied by a transient loss in transepithelial resistance. Mechanistic studies confirmed that the increases in ion conductance evoked by 2,5-dimethylpyrazine were largely attributed to a protein kinase A-dependent (PKA) activation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel.ConclusionsData from our high-capacity screening assays demonstrates that individual e-cigarette liquid flavoring chemicals vary in their cytotoxicity profiles and that some constituents evoke a cellular physiological response on their own independent of cell death. The activation of CFTR by 2,5-dimethylpyrazine may have detrimental consequences for airway surface liquid homeostasis in individuals that use e-cigarettes habitually.

Highlights

  • The potential for adverse respiratory effects following exposure to electronic (e-) cigarette liquid (e-liquid) flavorings remains largely unexplored

  • Minimum Essential Medium with Earle’s salts (MEM), Fetal Bovine Serum (FBS), 2,5-dimethylpyrazine, amiloride, damascenone, forskolin, linalool, α-ionone, ethyl maltol, furaneol and vanillin were from Sigma-Aldrich

  • Primary mouse tracheal epithelial (MTE) cells were voltage clamped in Ussing chambers and monitored for changes in in short-circuit current (Isc) and transepithelial resistance (TER) following varying doses of 2,5-dimethylpyrazine. a Representative traces of Isc in response to 2,5-dimethylpyrazine demonstrate a concentration dependent increase in Isc. b A concentration-dependent curve of the change in Isc following 2,5-dimethylpyrazine. c Representative traces of TER in response to varying concentrations of 2,5-dimethylpyrazine demonstrate a concentration dependent drop in TER

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Summary

Introduction

The potential for adverse respiratory effects following exposure to electronic (e-) cigarette liquid (e-liquid) flavorings remains largely unexplored. Electronic (e-) cigarettes are the most common type of electronic nicotine delivery systems (ENDS) that simulate smoking independent of the combustion of tobacco. The global market for ENDS has rapidly expanded and it is predicted that within the decade, sales of ENDS will surpass that of conventional combustible tobacco cigarettes [1]. Despite the rapid increase in popularity of ENDS the potential for harmful respiratory effects following use of these products remains largely unexplored. In their simplest form e-cigarettes contain a fluid-filled (e-liquid) cartridge with a battery-powered atomizer. An early study of e-cigarettes suggested that the levels of several harmful constituents are lower in e-cigarette aerosols when compared with cigarette smoke [7], some constituents unique to ecigarette aerosols, namely flavorings, have been shown to be cytotoxic in cell models [8]

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