Vaporized E-Cigarette Liquids Induce Ion Transport Dysfunction in Airway Epithelia

Abstract

Cigarette smoking is associated with chronic obstructive pulmonary disease and chronic bronchitis. Acquired ion transport abnormalities, including cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction, caused by cigarette smoking have been proposed as potential mechanisms for mucus obstruction in chronic bronchitis. Although e-cigarette use is popular and perceived to be safe, whether it harms the airways via mechanisms altering ion transport remains unclear. In the present study, we sought to determine if e-cigarette vapor, like cigarette smoke, has the potential to induce acquired CFTR dysfunction, and to what degree. Electrophysiological methods demonstrated reduced chloride transport caused by vaporized e-cigarette liquid or vegetable glycerin at various exposures (30 min, 57.2% and 14.4% respectively, vs. control; P < 0.0001), but not by unvaporized liquid (60 min, 17.6% vs. untreated), indicating that thermal degradation of these products is required to induce the observed defects. We also observed reduced ATP-dependent responses (-10.8 ± 3.0 vs. -18.8 ± 5.1 μA/cm² control) and epithelial sodium channel activity (95.8% reduction) in primary human bronchial epithelial cells after 5 minutes, suggesting that exposures dramatically inhibit epithelial ion transport beyond CFTR, even without diminished transepithelial resistance or cytotoxicity. Vaporizing e-cigarette liquid produced reactive aldehydes, including acrolein (shown to induce acquired CFTR dysfunction), as quantified by mass spectrometry, demonstrating that respiratory toxicants in cigarette smoke can also be found in e-cigarette vapor (30 min air, 224.5 ± 15.99; unvaporized liquid, 284.8 ± 35.03; vapor, 54,468 ± 3,908 ng/ml; P < 0.0001). E-cigarettes can induce ion channel dysfunction in airway epithelial cells, partly through acrolein production. These findings indicate a heretofore unknown toxicity of e-cigarette use known to be associated with chronic bronchitis onset and progression, as well as with chronic obstructive pulmonary disease severity.

Keywords: acquired cystic fibrosis transmembrane conductance regulator dysfunction; chronic obstructive pulmonary disease; e-cigarette; ion transport.

Figure 1.

Exposure chamber. (A) Example of exposure setup for e-cigarette vapor exposures. (B and C) Custom-designed chamber used to expose cells to e-cigarette vapor or nebulized liquid, shown open (B) and closed (C).

Figure 2.

Vaporized e-cigarette liquid induces a dose-dependent reduction in chloride transport in Calu-3 cells. (A–E) Calu-3 cells expressing high amounts of wild-type cystic fibrosis transmembrane conductance regulator (CFTR) and low epithelial sodium channel (ENaC) were exposed to various durations of air, vaporized vegetable glycerin (VG), or vaporized Red Oak Domestic (ROD) e-cigarette liquid, followed by assessment of short-circuit current (I_SC). (A) Representative tracings for 30-min exposures. (B) Changes in ENaC, (C and D) CFTR-dependent ion transport, and (E and F) alternative chloride channels were quantified. (G and H) To confirm that severe reductions in ion channel activity were not caused by cytotoxicity, baseline transepithelial resistance (TEER; G) and lactate dehydrogenase (LDH; H) activity were measured. n = 4–28 monolayers per condition. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (compared with air control), and ^≠P < 0.05 (VG vs. ROD), as assessed by two-way ANOVA (B–G) or one-way ANOVA (H).

Figure 3.

Unvaporized e-cigarette liquids do not impact chloride ion transport. (A) Increasing volumes (and thus concentrations) of unvaporized VG or ROD e-cigarette liquid were acutely added to the apical compartment of Calu-3 cells to assess real-time changes in chloride transport after CFTR stimulation, compared with untreated control. (B–E) Calu-3 cells were exposed to ROD e-cigarette liquid nebulized for 15, 30, and 60 minutes, followed by quantification of changes in I_SC induced by forskolin (B), CFTR inhibitor 172 (C), ATP (D), and bumetanide (E). n = at least 5 (acute liquid addition) or 10 (nebulized ROD) monolayers per condition. (F) Baseline TEER was used to confirm cell monolayer integrity following each exposure. *P < 0.05, ***P < 0.001, ****P < 0.0001, ^#P = 0.06, and ⁺P = 0.09 compared with control as analyzed by two-way ANOVA.

Figure 4.

E-cigarette vapor reduces ion transport in primary non–cystic fibrosis (CF) human bronchial epithelial cells in a dose-dependent fashion. Non-CF, non–chronic obstructive pulmonary disease human bronchial epithelial cells were exposed to 1, 3, 5, and 10 minutes of ROD (1.1% nicotine) or 100% VG e-cigarette vapor, then assessed with Ussing chamber electrophysiology. (A) Representative I_SC tracings for ROD-exposed cells. (B–F) Changes in ion transport upon addition of amiloride (B), forskolin (C), CFTR-specific inhibitor 172 (D), ATP (E), and bumetanide (F) were quantified. (G and H) Baseline TEER (G) and LDH (H) activity were assessed to confirm that reductions in ion transport were not caused by exposure-induced cytotoxicity. n = 4–12 monolayers per condition (generated from at least three donors). *P < 0.05, **P < 0.01, ****P < 0.0001 (compared with air control), ^#P < 0.0001 versus VG, ^aP < 0.05, and ^bP < 0.01 versus ROD as assessed by two-way ANOVA. ^≠P < 0.0001 versus ROD.

Figure 5.

E-cigarette vapor produced by shorter puffs induced a dose-dependent reduction in human bronchial epithelial cell ion transport. Non-CF, non–chronic obstructive pulmonary disease human bronchial epithelial cells were exposed to short, 2- to 3-second puffs every minute for 1, 3, 5, and 10 minutes of ROD (1.1% nicotine) vapor, then assessed with Ussing chamber electrophysiology. (A) Representative I_SC tracings comparing air control monolayers with those exposed to 10 puffs of ROD vapor. (B–G) Changes in ion transport at baseline (B) and upon addition of amiloride (C), forskolin (D), CFTR-specific inhibitor 172 (E), ATP (F), and bumetanide (G) were quantified. (H and I) Baseline TEER (H) and LDH (I) activity were assessed to confirm that reductions in ion transport were not caused by exposure-induced cytotoxicity. n = 3–5 monolayers per condition (generated from at least three donors). *P < 0.05, **P < 0.01, and ****P < 0.0001 (compared with air control) as assessed by two-way ANOVA; ^#P = 0.0585 as analyzed by one-way ANOVA.

Figure 6.

Vaporized e-cigarette liquid produces significant amounts of acrolein. (A) Quantitative mass spectrometry was used to measure acrolein output of ROD vapor for various durations using two heating coil resistances compared with air control. (B) Comparison of acrolein production from 15, 30, and 60 puffs of ROD vapor, aerosolized ROD, acrolein (Acr, 10 ppm, 10 min), one 3R4F cigarette (10 puffs over 10 min), and air control. n = at least 4 wells per condition. *P < 0.05 and ****P < 0.0001 as analyzed by two-way ANOVA; ^aP < 0.0001 versus ROD vapor at all-time points, ^bP < 0.01 versus acrolein, ^cP < 0.0001 versus acrolein, ^dP < 0.01 versus 3R4F, and ^eP < 0.0001 versus 3R4F as assessed by one-way ANOVA.

Figure 7.

Schematic of proposed mechanism for e-cigarette (E-cig) vapor–induced ion transport dysfunction. E-cig vapor inhibits epithelial chloride transport mediated by CFTR and calcium- activated chloride channels (CaCCs). It can also produce acrolein, which has been shown to induce CFTR dysfunction.