Airway epithelial cell exposure to distinct e-cigarette liquid flavorings reveals toxicity thresholds and activation of CFTR by the chocolate flavoring 2,5-dimethypyrazine

Abstract

Background: The 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.

Methods: We 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.

Results: In 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.

Conclusions: Data 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.

Keywords: 2,5-dimethylpyrazine; Airway epithelium; CFTR; Electronic cigarettes; Odorant receptor; xCELLigence RTCA.

Fig. 1

High-capacity real-time cellular analysis reveals toxicity thresholds of e-liquid flavorings. The toxicity thresholds of individual e-cigarette liquid flavoring constituents on 16HBE14o- cells was determined by cellular impedance changes over 24 h. a-d The highest concentration of each flavoring constituent was representative of the % v/v present in e-cigarette liquid. e-g The highest concentration of each flavoring constituent represented 1/10th the % v/v present in e-cigarette liquid. All flavors except furaneol (f) and vanillin (g) displayed cell index changes consistent with cytotoxicity that was validated with an MTS assay (h). *indicates significant difference from control cells and ^indicates significant difference between concentrations of the same flavoring, P < 0.05 in a one-way ANOVA with a Tukey’s Multiple Comparison Test

Fig. 2

2,5-Dimethylpyrazine reduces the physiological response to cellular signaling critical in airway innate immunity. a 16HBE14o- cells display cytotoxic cellular impedance responses to 2,5-dimethylpyrazine at 0.6 % and higher. b A 24 h exposure to subcytotoxic concentrations of 2,5-dimethylpyrazine causes a concentration-dependent signaling response to forskolin in 16HBE14o- cells. c A 24 h exposure to subcytotoxic concentrations of 2,5-dimethylpyrazine results in a reduction in 16HBE14o- signaling response to ATP. At subcytotoxic concentrations, 2,5-dimethylpyrazine evoked a physiological response at concentrations as low as 0.06 % v/v and dampened the response to cellular signaling key in airway epithelial innate immunity

Fig. 3

2,5-Dimethylpyrazine increases airway epithelial ion conductance. Primary MTE cells were voltage clamped in Ussing chambers and monitored for changes in I_sc and TER following varying doses of 2,5-dimethylpyrazine. a Representative traces of I_sc in response to 2,5-dimethylpyrazine demonstrate a concentration dependent increase in I_sc. b A concentration-dependent curve of the change in I_sc 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. Addition of 2,5-dimethylpyrazine on the apical airway epithelial surface results in an increase in apical ion efflux with a concurrent drop in TER

Fig. 4

2,5-Dimethylpyrazine activates apical ion efflux via CFTR. Primary MTE cells were voltage clamped in Ussing chambers and monitored for changes in I_sc and TER following addition of 0.2 % v/v 2,5-dimethylpyrazine. a Representative traces of I_sc and b TER (left) from MTE cells treated with 2,5-dimethylpyrazine alone (solid line) or 2,5-dimethylpyrazine plus CFTR-172 inh [amiloride (dashed arrow), CFTR-172 inh (dotted arrow), 2,5-dimethylpyrazine (solid arrow)]. Quantification of I_sc and TER (A-B right) of amiloride controls (n = 3) versus cells treated with amiloride and CFTR-172 inh (n = 4) are graphed. The addition of CFTR-172 inh significantly reduced the 2,5-dimethypyrazine-evoked apical ion efflux suggesting Cl⁻ secretion via CFTR. *indicates significant difference P < 0.05 in a one-way ANOVA with a Dunnett’s Multiple Comparison Test

Fig. 5

PKA signaling is upstream of 2,5-dimethylpyrazine-evoked apical ion efflux. Primary MTE cells were voltage clamped in Ussing chambers and monitored for changes in I_sc and TER following addition of 0.2 % v/v 2,5-dimethylpyrazine. a Representative traces of I_sc and b TER (left) from MTE cells pre-treated with H-89 followed by amiloride (dashed arrow) and 2,5-dimethylpyrazine (solid arrow). Quantification of I_sc and TER (A-B right) of amiloride controls (n = 3) versus cells treated with amiloride and H89 (n = 3) are graphed. Significant block of apical ion efflux with H89 suggests 2,5-dimethypyrazine-evoked apical ion efflux is mediated via PKA signaling. *indicates significant difference P < 0.05 in a one-way ANOVA with a Dunnett’s Multiple Comparison Test

Fig. 6

Increased intracellular cAMP dampens 2,5-dimethylpyrazine-evoked apical ion efflux. Primary MTE cells were voltage clamped in Ussing chambers and monitored for changes in I_sc and TER following addition of 0.2 % v/v 2,5-dimethylpyrazine. a Representative traces of I_sc and b TER (left) from MTE cells pre-treated with 8-bromo-cAMP (dotted arrow) followed by amiloride (dashed arrow) and 2,5-dimethylpyrazine (solid arrow). Quantification of I_sc and TER (A-B right) of amiloride controls (n = 3) versus cells treated with 8-bromo-cAMP and amiloride (n = 3) are plotted. The addition of the cAMP analog 8-bromo-cAMP significantly reduced apical ion efflux evoked by 2,5-dimethypyrazine suggesting it activates a receptor that increases cAMP production. *indicates significant difference P < 0.05 in a one-way ANOVA with a Dunnett’s Multiple Comparison Test