American Journal of Respiratory Cell and Molecular Biology

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.

Cigarette smoking is the primary risk factor for developing chronic obstructive pulmonary disease (COPD) (1), an airway disease characterized by irreversible, progressive loss of lung function. It is one of the leading causes of death in the United States (2). We and others have demonstrated that cigarette smoke exposure inhibits the function of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride and bicarbonate ion channel that helps to maintain normal function of secretory epithelia such as the airways (36). This “acquired CFTR dysfunction” has also been observed in patients with COPD, particularly in those who exhibit chronic bronchitis (79), and it may contribute to the pathogenesis of chronic bronchitis by impairing mucus clearance (1013). This is further supported by evidence that CFTR potentiators improve airway surface liquid depth and mucociliary transport in vitro in cigarette smoke–exposed epithelia (1114) and may ameliorate symptoms in patients with chronic bronchitis (15). Decrements in CFTR, as opposed to other anion channels such as calcium- activated chloride channels (CaCCs), have generally been specific (11, 16), although altered potassium channel function (4, 6) has also been suggested.

E-cigarettes (e-cigs), a type of electronic nicotine delivery system (ENDS), are thought to be safer alternatives to traditional cigarettes; as such, they are widely used in place of cigarettes for recreational use or as smoking cessation aids (17, 18). Owing to increasing popularity and the perceived safety profile of e-cigs (1925), there has been significant interest in characterizing their effects on the airways in comparison to the tobacco products they substitute. Although the U.S. Food and Drug Administration recently passed a rule to regulate ENDS as tobacco products (26), there remains limited control over composition of the liquids used in e-cigs and how users modify their personal devices.

In the present study, we sought to determine if e-cigs are as safe as the general populace perceives in regard to the deleterious effects of smoking on the ion transport environment of airway epithelia that are associated with chronic bronchitis. We hypothesized that e-cigs can induce acquired dysfunction of CFTR and potentially other ion transporters because thermal degradation of the hydrocarbons in e-cig liquid may produce reactive aldehydes such as acrolein, a combustion product found in cigarette smoke that we have recently shown to directly modify CFTR protein and reduce CFTR channel activity (14). To assess changes in ion transport, we measured short-circuit current (ISC) in epithelial airway cells after vapor exposure, as well as estimated acrolein output at various exposures using high-resolution quantitative mass spectrometry. Some of these results have been presented as abstracts (27, 28).

Cell Culture

Primary human bronchial epithelial (HBE) cells were isolated from tissue obtained from donors/transplants without cystic fibrosis, COPD, or other diseases known to affect ion transport (Table E1 in the data supplement), in accordance with protocols approved by the University of Alabama at Birmingham Institutional Review Board. Calu-3 (American Type Culture Collection) and passages 1–2 HBE cells were cultured at air–liquid interface until fully differentiated. Tests to rule out mycoplasma contamination are performed quarterly.

Exposures

Red Oak Domestic (ROD; Johnson Creek Vapor Co.) e-cig liquid (lots B2631120, B000878, and C000207) was used for e-cig vapor exposures. ROD is stated to include the following: USP grade vegetable glycerin (VG), deionized water, natural and artificial “tobacco” flavors, USP grade nicotine (1.1%), and USP grade citric acid (as a preservative); 100% VG (Mister-E-Liquid) served as a vehicle control, and propylene glycol (Weeping Willow Oil Co.), another vehicle used in e-liquids, was evaluated as an alternative. Cells were incubated at physiological conditions (37°C, 5% CO2) for up to 1 hour after exposure and before ion transport assessment.

Vaporized Liquid

Before each use, a variable-voltage ENDS device (Vision Spinner II 1600mAh; Visioncig) was fully charged. Cells were placed in a custom-made chamber (Figure 1), which was cleaned with 70% ethyl alcohol between exposures. Using the ENDS device at 4.8 V with a filled 2.1- or 1.5-Ω dual-coil clearomizer (iClear 16; Innokin), each puff was a maximum 8-second vaporization of ROD or VG (with 2.7 L/min vacuum flow to facilitate exposure), unless noted otherwise. The vacuum circuit was clamped between each puff, every minute for 1, 3, 5, or 10 minutes (HBE) or for 15, 30, or 60 minutes (Calu-3).

Aerosolized Liquid

Cells were exposed to continuous nebulization of ROD (15, 30, 60 min) using approximately 1–2 ml of liquid.

Direct Liquid Addition

ROD or VG (1, 3, 10, 30, 100, 300 μl) was added apically to a 5-ml chamber during ion transport studies after CFTR stimulation to determine effects of unvaporized liquid on CFTR activity.

Ion Transport

P2300 Ussing chambers (Physiologic Instruments) were used to measure ion transport as previously described (11). CFTR-dependent ISC was assessed by sequential addition of amiloride (100 μM), a chloride gradient plus amiloride (100 μM), forskolin (20 μM), CFTRInh-172 (10 μM), ATP (10 μM), and bumetanide (10 μM) (Sigma-Aldrich).

Cell Viability

Media collected from cells exposed to each condition were analyzed for lactate dehydrogenase (LDH) activity using a commercially available kit (Sigma-Aldrich).

Statistical Analysis

All quantitative measures of epithelial function (ISC) and acrolein output were analyzed using descriptive statistics. Results between groups were compared using ANOVA or Student’s t test in Prism software (GraphPad Software, Inc.), as appropriate, with P values less than 0.05 considered significant. Tukey’s post hoc test was used for multiple comparisons. Data are presented as mean ± SEM. Further details are noted in the data supplement.

E-Cigarette Vapor Inhibits Chloride Ion Transport in a Dose-Dependent Manner

To estimate the effects of e-cigs on ion transport, we measured ISC in Calu-3 cells, an airway epithelial cell line with high wild-type CFTR but low epithelial sodium channel (ENaC) activity (29), after exposure to VG vapor, ROD vapor, or air (control) for various times (Figure 2A). As expected, there was no significant difference in amiloride-sensitive current (Figure 2B), a measure of ENaC-mediated sodium transport, at any exposure (−0.81 ± 1.02 μA/cm2 with 60-min exposure vs. 1.38 ± 0.77 μA/cm2 air control; P = not significant [NS]); however, these cells are not particularly sensitive to ENaC decrements, given low amiloride-sensitive currents at baseline (29). Vaporized ROD significantly inhibited forskolin-stimulated ion transport, a measure of CFTR-dependent chloride transport, at all exposure durations, whereas there was no statistically significant difference between air control–and VG-exposed cells at exposures shorter than 60 minutes, at which point nonspecific injury ensues (Figure 2C). At 30 minutes, an exposure duration that maximizes CFTR reduction without inducing cellular toxicity (see below), forskolin-stimulated ISC was reduced (ROD, 57.2%; VG, 14.4%; P < 0.0001 compared with air control). This reduction in CFTR activity was confirmed by CFTR-specific inhibition (Figure 2D), which was significant after 30 (59.6% reduction; P < 0.05) and 60 (93.8% decrease; P < 0.001) minutes of exposure. In contrast to cigarette smoke (4, 5, 7, 10), which has a greater effect than CaCCs on CFTR function (30), e-cig vapor also diminished ATP-sensitive current (Figure 2E) that was sensitive to the chloride transport inhibitor bumetanide (Figure 2F). To confirm that the observed decrement in CFTR function was not caused by cytotoxicity after vapor exposure, we assessed baseline transepithelial electrical resistance (TEER) of the cells used to verify monolayer integrity (Figure 2G), as well as LDH activity in the cell media as an indicator of cellular injury (Figure 2H). There was no effect on monolayer integrity or cell viability, except when cells were exposed to 60 minutes of vapor (4.77 ± 0.15 mU/ml air control; 10.62 ± 0.42 mU/ml VG; 9.32 ± 0.86 mU/ml ROD; P = NS). The pH of the media recovered from the basolateral compartment was also unaffected (mean change from air control, 0.026 pH units for VG and 0.011 ROD after 60-min exposure; P = NS) (Figure E1). These findings indicate that vaporizing e-cig liquids can inhibit airway epithelial chloride transport in a dose-dependent fashion, even when neither TEER nor LDH release is affected, whereas further exposure (e.g., 60 min) ultimately affects monolayer integrity (16), diminishing TEER beyond that induced by 100% VG alone.

E-Liquid Vaporization Is Required to Confer Ion Transport Dysfunction

To determine if the observed ion transport defect is due to heating of the liquid to produce vapor, as opposed to toxic effects of the liquid constituents themselves, we assessed the effects of unvaporized e-liquids on ISC in Calu-3 cells. First, we monitored changes in CFTR-mediated ISC as we added increasing volumes of VG or ROD liquid directly to the apical compartment of cells mounted in modified Ussing chambers (chamber volume, 5 ml/compartment). Although there was a volume-dependent reduction in ISC in ROD-exposed cells, there was no significant difference compared with VG in the magnitude of the effect, indicating nonspecific injury from large volumes of VG exposure rather than ROD (Figure 3A). Furthermore, this only occurred at volumes over 10 μl (final concentration over 0.2% vol/vol), probably reflecting nonspecific toxicity of hydrocarbons on cell integrity and likely not achieved with routine vapor inhalation by e-cig users. Next, and to better replicate epithelial exposure rather than direct installation, we exposed Calu-3 cells to nebulized ROD liquid for the same time durations used in vaporized liquid experiments, then measured ISC. There was no meaningful difference in deposition between vaporized and nebulized ROD liquid at exposure durations up to 60 minutes (Figure E2), indicating additions to the airway surface liquid were essentially isovolemic. There was no significant difference in forskolin-stimulated ISC (Figure 3B) or CFTR-specific inhibition of ion transport (Figure 3C) in cells exposed to nebulized ROD liquid, even with prolonged exposure, compared with air control (at 60 min, 17.6% reduction in forskolin response, P = NS; 50.6% decrease in CFTR-specific inhibition, P = 0.06). Similarly, activity of alternative chloride channels was not meaningfully affected (at 60 min, 1.7% reduction in ATP response, P = NS; 16.0% reduction in bumetanide response, P = NS) (Figures 3D and 3E), and there was no indication of monolayer disruption due to these exposures by TEER (Figure 3F). This suggested that heat-induced vaporization of e-liquids is necessary for inhibition of chloride transport in vitro.

E-Cigarette Vapor Reduces Ion Transport in HBE Cells

Next, we tested the effects of e-cig liquid vapor on primary HBE cells, a highly relevant model of the airway epithelium that also expresses ENaC. We exposed cells to ROD or VG vapor for various lengths of time (≤10 min owing to cytotoxicity at longer exposures), then measured ISC to assess changes in sodium and chloride ion transport (Figure 4A). As opposed to Calu-3 monolayers, in primary HBE cells that exhibit sufficient ENaC currents at baseline, we observed a dose-dependent decrement in amiloride-sensitive current (Figure 3B), with a 23.9-fold reduction in ENaC activity at 5 minutes of ROD vapor exposure (−2.1 ± 1.3 compared with −50.5 ± 4.6 μA/cm2 air control; P < 0.0001) that was first apparent at 3 minutes of exposure (P < 0.01). This reduction in ENaC currents was not observed after 3 minutes of VG vapor exposure, indicating relative specificity to ROD-induced injury, although the effects of VG did occur at 5 minutes (−7.2 ± 3.9 μA/cm2; P < 0.0001 vs. air control). At 1 and 3 minutes of ROD exposure, there was no significant effect on CFTR function; however, forskolin-stimulated CFTR currents were significantly reduced after either 5 or 10 minutes of exposure, respectively (by 47.7% and 53.4%; P < 0.05) (Figure 4C); this was confirmed by similar decrements in response to CFTR-specific inhibition (CFTRInh-172) for both durations (Figure 4D). At these longer exposures (5 and 10 min), HBE cells exposed to vaporized 100% VG also exhibited a dose-dependent reduction in CFTR-dependent ion transport compared with air controls; however, forskolin-stimulated changes in ISC were more variable and not statistically significant at either exposure duration (Figure 4C), and the reduction with CFTRInh-172 was similar to ROD at 5 and 10 minutes of exposure (40.9% vs. 47.7% ROD at 5 min; P = NS). Interestingly, HBE cells exposed to VG vapor exhibited an increase in CFTR-dependent ion transport at short exposure durations (1 and 3 min), with a 93.7% increase (P < 0.0001 vs. air control) in forskolin response at 3 minutes, likely reflecting a compensatory response to oxidant stress at subinhibitory exposures or the stimulating effects of low nicotine concentration (31), an effect observed with very low concentrations of cigarette smoke exposure (11).

As observed in Calu-3 cells, responses in ATP- and bumetanide-sensitive currents were also reduced in primary HBE cells after ROD exposure (at 5 min, −10.84 ± 2.98 vs. −18.78 ± 5.11 μA/cm2 air control in response to ATP, P = NS; 2.81 ± 2.91 vs. −5.43 ± 1.22 μA/cm2 air control, P < 0.05) (Figures 4E and 4F), suggesting that CaCCs are negatively affected by e-cig vapor, albeit less prominently than CFTR. VG vapor induced similar trends in HBE cells but required longer durations of exposure than ROD to occur, with reversal of bumetanide-sensitive ISC at 10 minutes (P < 0.0001) compared with air control. Baseline TEER (Figure 4G) and LDH release (4.55 ± 0.65 mU/ml air control vs. 6.00 ± 0.56 mU/ml VG and 5.00 ± 0.15 mU/ml 5-min ROD; P = NS) (Figure 4H) confirmed that ion transport decrements were not due to generalized cytotoxicity.

ROD e-cig vapor produced with shorter, potentially more realistic puff times (2–3 s vs. 8 s) and exposed to HBE cells for the same durations (1–10 min) produced very similar findings (Figures 5A and 5B), including deleterious effects on ENaC activity (Figure 5C), CFTR function (Figures 5A, 5D, and 5E), and purine-activated CaCCs (Figures 5A, 5F, and 5G) compared with air control–exposed monolayers. There were no cytotoxic effects based on baseline TEER (Figure 5H) and no meaningful changes in LDH release (Figure 5I). Interestingly, as opposed to the ion transport inhibition observed with VG, maximum puffs of propylene glycol, an alternate e-cig solvent also used by e-liquid manufacturers, did not exhibit deleterious effects when used alone on measures of ENaC activity (Figure E2A), CFTR activity (Figures E2B and E2C), CaCC activity (Figures E2D and E2E), or TEER (Figure E2F) in HBE cells. Similar to findings with Calu-3 cells, the pH of the basolateral media was not affected by any of the e-cig vapor exposures used (mean change from air control after 10-min exposure, −0.026 ± 0.023 for ROD vapor, −0.004 ± 0.018 for VG vapor, 0.039 ± 0.037 for propylene glycol vapor; P = NS) (data not shown).

Overall, these data indicated that ROD inhibits CFTR and ENaC at clinically relevant exposure durations in primary HBE cells over and above inhibition posed by the transport vehicle VG. As in Calu-3 cells, more prolonged exposures were detrimental to ENaC, CFTR, and CaCCs, even before any evidence of generalized cytotoxicity or decrement in monolayer integrity, reflecting sensitivity of the ion transport apparatus to e-cig vapors.

Vaporizing E-Liquids Produces Acrolein Sufficient to Cause Acquired CFTR Dysfunction

Acrolein is a highly reactive aldehyde that can be produced by combustion and is found in tobacco smoke. We have recently shown that both cigarette smoke and acrolein can directly modify specific amino acid residues on CFTR (14) and inhibit its ion transport function (5), leading to delayed mucus clearance. Although vaporization of e-liquids does not invoke fire, the power applied to produce vapor (11.0 W at 4.8 V with a 2.1-Ω heating coil) can create sufficient energy for chemical reactions to occur (3235), including production of the reactive aldehyde acrolein (34, 35). To test for acrolein production during vaping, we used high-resolution mass spectrometry to quantify acrolein output of various conditions (vaped and nebulized) compared with room air at the relevant time points (15, 30, and 60 min). ROD vapor produced significantly higher amounts of acrolein than control conditions at all time points evaluated (Figure 6A). Consistent with the lack of a deleterious effect of unvaporized ROD liquid except in high concentrations, both 15 and 30 minutes of nebulized ROD produced minimal acrolein (202.5 ± 11.5 and 284.8 ± 35.03 ng/ml, respectively) compared with air control (224.5 ± 15.99; P = NS) (Figure 6B). This suggested that the vaporization process was crucial to acrolein production. To test this, we vaporized e-liquids using two different resistance levels (2.1 and 1.5 Ω), which reduced power applied to the vaporization reaction by approximately 30%. Heating coils with a higher resistance generated 41.7% (P < 0.05) and 42.8% (P < 0.0001) less acrolein with 30 and 60 maximal puffs, respectively, than a device set to a lower resistance (Figure 6A). In addition, the amount of acrolein produced decreases with loss of battery power, which in turn results in a less severe functional decrement in CFTR-mediated ion transport (data not shown). To compare concentrations of acrolein with other relevant exposures, we also quantified acrolein output in ISO (International Organization for Standardization) smoke from standard 3R4F research cigarettes (10 puffs, 35-ml puff volume for one cigarette), as well as 10 minutes of 10 ppm acrolein gas for comparison (Figure 6B). Just 15 puffs of maximal ROD vapor production contained 28,292 ± 404 ng/ml acrolein, approximately twofold over 10 puffs from a single 3R4F research cigarette smoked at ISO (13,443 ± 979 ng/ml). Ten minutes of acrolein gas (10 ppm) produced similar concentrations of acrolein (17,427 ± 436 ng/ml), whereas air control samples contained negligible amounts (225 ± 16 ng/ml). These findings indicated acrolein is produced by e-cig liquid vaporization at amounts sufficient to reduce CFTR activity, which exhibits a half-maximal inhibitory concentration in CFTR channel activity of 3.2 μg/ml (5) and is proportionate to power delivered by the device.

Public perception of e-cig use as a safe alternative to or replacement of traditional cigarette smoking has resulted in increasing popularity of ENDS in recent years, particularly in never-smoker youths. Appropriately, there is significant effort in the scientific community to determine if e-cig use presents any pulmonary hazards. In the present study, we demonstrated that, like tobacco smoke, e-cig vapor results in ion channel dysfunction in airway epithelial cell models; affecting ENaC; CaCCs; and, most importantly owing to its clear association with clinical manifestations and outcomes, acquired CFTR dysfunction (5, 8, 11). We also established that e-cig vaporization produces acrolein—a known respiratory toxicant with broad-reaching effects—via pyrolysis and at amounts similar to those produced by conventional cigarettes (Figure 7). To prevent confounding effects of flavor additives, some of which are known to adversely affect the airways, such as cinnamaldehyde and diacetyl (popcorn) (3642), we used a more traditionally tobacco-flavored e-liquid. This allowed us to determine how vaporization of basic e-liquid ingredients impacts ion transport and acrolein production. These findings indicate a heretofore unknown toxicity of e-cig use, namely acrolein exposure, that is known to be associated with onset and progression of chronic bronchitis and COPD severity (5, 9, 14). It should be noted that as e-liquids continue to evolve, both in their flavorings and their vehicles, additional scrutiny will be needed to assess their effects on airway ion transport function.

ISC data show that e-cig vapor inhibits CFTR function in Calu-3 cells, an airway epithelial cell line with very high wild-type CFTR expression. The significant reduction in CFTR-mediated ion transport observed at 30 minutes and longer suggests that this degree of CFTR expression necessitates longer vapor exposures to see a decrement. Calu-3 cells also demonstrated a lower sensitivity to CFTR-specific inhibition, indicating that alternative chloride channels also play an important role as higher concentrations of vaporized e-liquid are delivered. In addition, the severe reduction in ion transport at 60 minutes of e-cig vapor exposure was not due to detectable cytotoxicity, confirming that e-cig vapor reduces anion conductance, CFTR in particular, as previously observed in smoke exposure models (5, 11, 12, 14). Although we emphasized our analysis on CFTR because of its clear association with clinical abnormalities and aberrant physiology, our data indicate that anion transport by alternative chloride channels was also markedly affected. This is an effect that is unique to e-cigs compared with traditional smoking in our prior studies (5) and that could complicate their deleterious effects on the airway surface further, because transmembrane member (TMEM) channels are believed to partially compensate for the absence of CFTR (4346). Whether this effect is due to the temperature achieved to initiate production of chemically modified products unique to the vaping process, other constituents contained in the liquid/vapor, or dwelling properties of the vapor produced, remains to be studied. The vehicle solvent of the e-liquid could also play a role; VG, as opposed to propylene glycol, may be particularly problematic in this regard.

Interestingly, the decrement in anion transport does not seem to be caused by unvaporized liquid at biologically relevant concentrations. By adding unvaporized e-cig liquid directly to the apical chamber, we were able to assess the effects of the liquid on ISC with increasing volumes. The reduction in ion transport with very high liquid volume addition is likely to be a mechanical effect of the liquids physically blocking the cell surface, because both ROD and VG are highly viscous, and independent of the much more relevant effects of vapor. This is supported by the observation that, although ROD vapor significantly inhibits CFTR activity at various time points, VG vapor does not; yet, both unvaporized ROD and unvaporized VG liquids reduce ISC with increasing volume over time. The lack of significant difference between the effects of unvaporized ROD and VG liquids in the present study, combined with evidence that vaporized ROD has a strong effect and is dependent of the power delivered during the vaporization process, suggests that the observed reduction is related to the vaporization process and accentuated by the contents of the e-liquid. As an alternative method to determine if the observed acquired CFTR dysfunction is due to vaporizing e-cig liquid, we exposed Calu-3 cells to nebulized ROD liquid, then measured ISC after 15-, 30-, or 60-minute exposure. Using this exposure model of aerosolized, unvaporized liquid, we saw no difference in chloride transport between air control and cells exposed to 15–30 minutes of nebulized ROD. Although the reduction observed in cells exposed to 60 minutes of ROD aerosol could be due to the accumulation of viscous liquid on the epithelial surface, this was not related to differences in deposition between vaporized and nebulized liquid onto the airway cells. These data indicate that the severe reduction in chloride channel activity is primarily due to vaporization (i.e., pyrolysis) of e-cig liquid and that it may be challenging to devise a completely safe e-liquid as an alternative.

To extend our findings into a more relevant in vitro model, we repeated the aforementioned ISC experiments in primary non–cystic fibrosis, non-COPD HBE cells after e-cig vapor exposure. These cells exhibited a dose dependency similar to that of Calu-3 cells; however, a significant reduction was observed in response to e-cig vapor at shorter durations of exposure and by e-cig vapor generated by short, 2- to 3-second puff times often implemented by e-cig users, suggesting that primary HBE cells are more sensitive to the deleterious effects, possibly owing to lower wild-type CFTR expression than seen in Calu-3 cells, which highly express the channel. We also observed ENaC inhibition in HBE cells (as opposed to in Calu-3 cells, which lack ENaC expression), an effect that can also be induced by cigarette smoke exposure (4, 11), suggesting that inhaled insults can impact epithelial ion transport in general. Of note, more prolonged exposures (>30 min) beyond which ion transport cannot be accurately assessed were clearly deleterious to the monolayers, disrupting TEER and compatible with injury to the epithelium, representing a potential risk to those with intense or prolonged vaping behaviors.

No changes in pH were observed in any of the basolateral media of cells exposed to vaporized or nebulized liquid when compared with air controls, suggesting that reductions in channel activity may not be due to pH alterations conferred by e-liquid vapor. For example, in the extreme instance of 60-minute vapor exposure, which did cause some damage to the Calu-3 cell monolayers as evidenced by baseline TEER and LDH activity, the mean change in pH from air control was minimal. Because CFTR also transports HCO− in addition to Cl−, it is plausible that the channel dysfunction induced by e-cig vapor exposure could affect pH of the apical surface, although this effect would be expected to be small because CFTR dysfunction is not complete, and changes between cystic fibrosis and non–cystic fibrosis are only approximately 0.1 pH unit (47, 48).

We found that vaporizing e-cig liquid (ROD) can indeed produce significant amounts of acrolein, albeit with exposure times that model repeated or excessive (i.e., cumulative) use. This contradicts claims that e-cigs produce negligible concentrations of acrolein when compared with tobacco cigarettes, particularly with normal vaping topographies that do not result in a “dry puff” (35, 49, 50). This has important implications for e-cig users because these individuals can still be exposed to the same respiratory toxicants found in cigarette smoke that negatively affect airway function. We also determined that acrolein output increases with resistance of the heating coil used and is reduced with loss of battery power; this is especially important when considering the custom modifications users make on personal devices to produce a stronger “hit” or a larger cloud of vapor. We have shown that acrolein plays an important role of inducing acquired CFTR dysfunction (5, 14) by directly modifying specific residues on CFTR protein, thereby inhibiting its functional activity and downstream regulation of mucociliary transport (14). These data cannot definitively rule out the contribution of other constituents, particularly those with the potential for oxidant-induced injury. Cadmium (51, 52) and other heavy metals (52), as well as reactive aldehydes other than acrolein, are likely contributors and could be produced by e-cigs, depending on the nature of the flavorings. Moreover, nicotine, which has been shown to regulate CFTR-dependent ion transport (31) through a complex dose-dependent desensitization of nicotinic receptors and can be found even in e-cig liquids containing minimal ingredients, is a likely contributor, including in the ROD studies presented here, and may be even more problematic if higher concentrations are used. Acrolein is not produced only by lipid peroxidation and heating of carbohydrates found in food and tobacco; it also is produced by thermal degradation of glycerin (32, 35, 53), which is used as a humectant in e-cig liquid. These variations in acrolein output warrant further study as the e-cig industry evolves (53). Indeed, there have been studies performed to assess whether e-cig use results in pulmonary abnormality. Researchers in these studies reported increases in cough, chest tightness, and mucus secretion but no defects in overall respiratory function; however, it is important to note that these studies focused on short-term exposures rather than chronic ones (53). When propylene glycol or VG is heated to a high-enough temperature (>215°C), significant concentrations of other reactive aldehydes, such as formaldehyde and acetaldehyde, are also generated (34). Our data show that vaporized VG (100%) contains high concentrations of acrolein with longer exposures, which is in agreement with another study in which VG heated to over 270°C resulted in acrolein formation (34). This indicates that the commonly used flavor vehicle itself can produce the respiratory irritant in significant amounts, which is particularly notable because the recommended exposure limit for acrolein is 0.1 ppm over 8 hours (54), and suggests that chronic e-cig use, especially with high temperatures, may result in pulmonary detriment.

Given the consistency of our results across model systems, as well as the evolving landscape of e-cig use, both in flavorings and devices implemented, further study is warranted to assess the deleterious effects of e-cigs on airway function. Such work could address recognized limitations of the work presented here, because our findings are limited to cell culture studies and could be expanded to assess e-cig use in vivo. Although we attempted to recapitulate vapor exposures experienced by e-cig users in a realistic fashion and at relevant exposure intensities experienced by e-cig users, inhalation studies in animals or humans would assure the relevance of our findings. The exposure methods performed in the present study were closely aligned with whole cigarette smoke exposures also used by our laboratory (12), and findings regarding acquired ion transport abnormalities have been validated in multiple species (5557) and humans (5, 7, 8), increasing confidence that the findings presented here are meaningful.

The data presented here stress the importance of regulating e-cigs and their customized liquids, which we found are deleterious and may demonstrate a lot-to-lot variability when mixed in-house on an individual basis. The U.S. Food and Drug Administration has recently taken early steps toward this goal by passing a rule to regulate all tobacco products, including ENDS and e-liquids mixed in-house at stores; however, implementation of and compliance with these new regulations is still ongoing (26). Even with such progress, the fact that we have demonstrated that these devices have the capacity to produce the same toxic chemicals which traditional cigarettes do suggests that they have the potential to lead to lung disease with chronic use.

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Correspondence and requests for reprints should be addressed to Steven M. Rowe, M.D., M.S.P.H., Department of Medicine, Gregory J. Fleming Cystic Fibrosis Center, Department of Pediatrics, and Department of Cell Developmental and Integrative Biology, University of Alabama at Birmingham, 1918 University Boulevard, MCLM 702, Birmingham, AL 35294. E-mail: .

* These authors contributed equally to this work.

Present address: Mike O’Callaghan Federal Medical Center, Nellis Air Force Base, Las Vegas Valley, Nevada, and University of Nevada, Las Vegas School of Medicine, Las Vegas, Nevada.

Supported by National Institutes of Health grants R35 HL135816, P30 DK072482, and F31 HL134225.

Author Contributions: V.Y.L., M.D.F., P.L.J., J.E.B., S.V.R., and S.M.R.: conceived of and designed the research; V.Y.L., M.D.F., P.L.J., T.F.B., L.S.W., and M.M.: performed the experiments; V.Y.L., M.D.F., P.L.J., T.F.B., L.S.W., S.J.B., and S.M.R.: analyzed the data; V.Y.L., M.D.F., P.L.J., S.J.B., J.E.B., S.V.R., and S.M.R.: interpreted the results of the experiments; V.Y.L. and M.D.F.: prepared figures; V.Y.L., M.D.F., and S.M.R.: drafted the manuscript; V.Y.L., M.D.F, P.L.J., T.F.B., L.S.W., M.M., S.J.B., J.E.B., S.V.R., and S.M.R.: edited and revised the manuscript; V.Y.L., M.D.F., P.L.J., T.F.B., L.S.W., M.M., S.J.B., J.E.B., S.V.R., and S.M.R.: approved the final version of the manuscript.

This article has a data supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1165/rcmb.2017-0432OC on December 21, 2018

Author disclosures are available with the text of this article at www.atsjournals.org.

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