electronic cigarettes (e-cigarettes) heat and aerosolize a liquid, commonly containing nicotine and flavorings, mixed with an excipient such as propylene glycol and/or glycerin, producing an aerosol that is inhaled. E-cigarette use is increasing, particularly among young people (5, 25), but the potential health effects of e-cigarettes remain uncertain. Due to this uncertainty, the Forum of International Respiratory Societies concluded that “the health and safety claims regarding electronic nicotine delivery devices should be subject to evidentiary review” and that they “should be restricted or banned until more information about their safety is available” (45).

There have been no long-term exposure studies in humans (41) and very few exposure studies in animals (11, 15, 26, 27, 48). Results of animal studies consistently show adverse respiratory effects of exposure to e-cigarettes although, in some cases, methodological flaws, particularly in the delivery of the liquid, hamper the findings. For example, in one study (11) mice were exposed to nebulized e-cigarette liquid, a process that will alter the physicochemical properties of the aerosol (9). Another study exposed mice to e-cigarette liquid via intratracheal instillation twice weekly for 10 wk (27). The authors report an exacerbation of allergy-induced inflammation and airway hyperresponsiveness in e-cigarette liquid-exposed mice, compared with mice treated with ovalbumin only, but these results should be viewed with caution due to the unrealistic e-cigarette exposure method and their use of the discredited enhanced pause technique for inferring airway hyperresponsiveness (28). In a more recent study, mice were exposed to aerosol from an “NJOY menthol bold” e-cigarette for 3 h/day for 2 wk before infection with Streptococcus pneumonia or influenza (48). E-cigarette aerosol alone induced a significant increase in oxidative stress, macrophage-mediated inflammation, impaired bacterial clearance, increased lung viral titers, and enhanced virus-induced morbidity and mortality. In a similar study (15), mice were exposed to e-cigarette aerosol for 1 h/day for 4 wk. This led to minor changes in some inflammatory markers (KC and IL-1ra) within the airways [although no changes in bronchoalveolar lavage (BAL) inflammatory cells] and elevation of Pentraxin 3 in serum. Lerner et al. (26) exposed mice to e-cigarette aerosol containing nicotine for 5 h/day for 3 consecutive days. This brief exposure regime resulted in reduced lung glutathione levels and increases in certain proinflammatory cytokines [IL-6, monocyte chemotactic protein-1 (MCP-1), IL-1α, and IL-13] in BAL fluid. Again, there was no increase in cellular inflammation in BAL.

The aim of this study was to investigate the respiratory health effects of e-cigarettes using a mouse model and an exposure regime that more closely reflects real-life aerosol exposures. Our exposures started when mice were 4 wk of age, representing adolescence in humans, and we attempted to replicate typical e-cigarette use in humans (~120 puffs/day, average duration of use of ~12 wk (8). We hypothesized that e-cigarette aerosol exposure would significantly impact pulmonary responses but that these responses would be less severe than those seen in cigarette-exposed mice.

E-cigarettes were vaped using our custom-designed aerosolizer. For collection of particles and aerosol, we set this device to produce 35 ml of aerosol once per minute for 30 min. Aerosol was diluted by 2 l/min of medical air. To match this output, mainstream cigarette smoke was generated by two Winfield Red cigarettes smoked using the inExpose machine (SCIREQ). Cigarettes were puffed using the International Organization for Standardization standard of 35-ml puff, once per minute, and medical air flowed through the system at 2 l/min. Appropriate filters and collection tubes were attached to precalibrated Airchek XR5000 pumps (SKC, Eighty Four, PA). Filters and tubes were stored appropriately and transported to the ChemCentre (Bentley, Australia) for analyses as described in Table 1 .

E-cigarette liquids were analyzed by Western Australian Organic and Isotope Geochemistry, Curtin University (Bentley, Australia). Between 1 and 5 mg of e-cigarette liquid were dissolved in pyridine to give a concentration of 2.5 mg/ml. Seventy microliters of solution were transferred to a 300-μl microinsert inside a gas chromatography vial. One-hundred microliters of N , O -bis(trimethylsilyl)trifluoroacetamide were added to each vial. The vials were tightly capped, homogenized with a vortex mixer, and placed in an oven at 70°C for 1 h. The derivatized samples were diluted with pyridine for GC-MS analysis. GC-MS analysis was performed using a HP 6890 GC coupled to a 5973 MSD. The column was a ZB-5MSi, 30-m length, 0.25-mm inner diameter, and 0.25-μm phase thickness. One microliter of sample was injected using a cool on-column injection system. The GC oven was held at 50°C for 1 min, increased at 6°C/min to 320°C, and held for 24 min.

After lung function measurement, the tracheal cannula was instilled with 5% formaldehyde at 10 cmH 2 0 for six mice per treatment. Fixed lungs were embedded in paraffin wax, and the left lobe was sectioned for histology and assessment of mean linear intercept (chord) length (L m ). Five-micrometer-thick lung sections were cut at proximal, middle, and distal parts of the lung. Sections were stained with Masson’s trichrome, Alcian blue-periodic acid-Schiff or hematoxylin and eosin for assessment of airway smooth muscle mass/epithelial thickness/collagen, mucous/mucous-producing cells, and inflammation, respectively. Airway smooth muscle mass and epithelial thickness were assessed using stereological software (newCAST; Visiopharm, Hørsholm, Denmark). The cross-sectional area of epithelium and airway smooth muscle and the internal perimeter of the basement membrane (P bm ) were measured. The square root of all areas was normalized to P bm to correct for differences in airway size ( 16 ). The percentage of collagen was assessed in whole lung cross sections using ImageJ software (National Institutes of Health) and a modification of a previously published technique ( 19 ). The proportion of mucus-producing cells in the epithelium was calculated as previously described ( 43 ). Mean linear intercept length was calculated to assess alveolar surface area loss and enlargement of distal air spaces. Briefly, a lung section was randomly selected from each mouse. The section was masked and 20 random fields of view were generated using stereological software (newCAST, Visiopharm, Hørsholm, Denmark) and a motorized microscopic stage. Within each field of view we measured all straight line segments that spanned the air space between two sequential intersections of the alveolar surface on three random test lines ( 20 ). This resulted in at least 250 L m measurements per individual.

For measurement of thoracic gas volume (TGV) and lung mechanics, mice were anesthetized, tracheostomized, and attached to a mechanical ventilator (HSE Harvard Minivent; Hugo Sachs Harvard Elektronik, March-Hugstetten, Germany). They were ventilated at a rate of 400 breaths/min with a tidal volume of 8 ml/kg and 2 cmH 2 O of positive-end expiratory pressure. This ventilation regime was sufficient to allow measurement of TGV and lung mechanics without paralysis. We used plethysmography to measure TGV as described previously ( 22 ). At end expiration, the trachea was occluded and the intercostal muscles electrically stimulated (six 2- to 3-ms, 20-V pulses) to induce inspiratory efforts. During stimulation, tracheal pressure and plethysmograph box pressure were measured. TGV was then calculated using Boyle’s law, after we had corrected for the thermal properties and impedance of the plethysmograph ( 17 ). We then measured respiratory system impedance (Z rs ) using a wave-tube system adapted for use in small animals ( 39 , 47 ) and a modification of the forced oscillation technique ( 47 ) as previously described ( 24 ). The constant phase model was fit to Z rs to generate the parameters of airway resistance (R aw ), tissue damping (G), and tissue elastance (H). Z rs was measured at functional respiratory capacity and also during a slow inflation-deflation maneuver from 0 to 20 cmH 2 O transrespiratory pressure, allowing us to construct absolute pressure-volume curves and assess the volume dependence of lung mechanics. After measurement of TGV and lung mechanics, mice were transferred to a small animal ventilator (flexiVent; SCIREQ) for assessment of responsiveness to methacholine (MCh; acetyl β-methacholine chloride; Sigma-Aldrich, MO) as previously described ( 23 ).

Four different varieties of e-cigarette “juice” (Mt. Baker Vapor, Lynden, WA) were used. All were “American Tobacco” flavor, and they varied based on nicotine content (0 or 12 mg/ml) and main excipient [propylene glycol (PG) or glycerin (VG)]. Thus we had 0-PG, 12-PG, 0-VG, and 12-VG. Gas chromatography-mass spectrometry (GC-MS) analyses of the e-cigarette aerosols showed that PG and VG aerosols were quite distinct, with both PG-based aerosols containing 100% propylene glycol. VG-based aerosols were primarily glycerin, with small amounts of propylene glycol; 0-VG contained 95.30% glycerin and 4.70% propylene glycol, while 12-VG contained 97.53% glycerin and 2.47% propylene glycol. We custom designed a computer-controlled device to accurately and repeatedly aerosolize e-cigarette juice based on an Innokin iTaste MVP2.0 aerosolizer (Innokin Technology, Shenzhen, China). It consisted of a computer-controlled syringe pump and bidirectional solenoids connected to the aerosolizer ( Fig. 1 ). It was set to produce 35 ml of aerosol per minute for each exposure to match tobacco smoke exposures. Throughout exposure, medical air flowed through the system at 2 l/min.

Four-week-old female BALB/c mice were purchased from the Animal Resources Centre (Murdoch, WA, Australia) and housed in individually ventilated cages (Sealsafe; Tecniplast, Buguggiate, Italy) on nonallergic, dust-free bedding (Shepards Specialty Papers, Chicago, IL). Mice were supplied with an allergen-free diet (Specialty Feeds, Glen Forrest, WA, Australia) and water ad libitum. Between the ages of 4 and 12 wk, mice were exposed to medical air (control), tobacco smoke, or one of four e-cigarette aerosols. All mice were whole body exposed in 27-liter exposure chambers. While whole body exposure does not preclude off-target effects from oral uptake of excipient/nicotine through grooming, such effects are likely to be negligible compared with the magnitude of direct respiratory effects measured for our outcomes. We used four chambers for this study: one for “Air,” one for “Smoke,” one for e-cigarettes containing nicotine, and one for e-cigarettes without nicotine. The chambers were thoroughly cleaned between each exposure. Animals were exposed for 1 h/day, 5 days/week from week 4 to 10 of life. From week 11 to 12 of life (as mice became accustomed to cigarette smoke and e-cigarette aerosols), exposures were increased to 1 h, twice daily, 5 days/week. Twelve mice were exposed to each exposure regime.

We also measured the dose per puff generated by the devices for each case, followed by mass median aerodynamic diameter (MMAD), and average chamber concentration throughout each experiment ( Table 10 ). Figure 9 shows typical particle number-based aerosol size spectra for each aerosol. On average the VG-based e-liquids have an MMAD ~25% larger than cigarette smoke, whereas the PG-based e-liquids have an MMAD ~25% smaller. Despite the greater number concentration of the PG liquids, they have a ~20% lower mass dose than the VG-based liquids.

Similarly, a range of volatile organic compounds (VOCs) were measured in Smoke (19 out of 30), and in all cases, Smoke contained substantially more of these compounds than any of the e-cigarette aerosols. Only hexane and 2-ethoxy ethanol were detected in all aerosols, and these were only in trace amounts compared with Smoke. Via semiquantitation relative to toluene, we were able to determine the total concentration of VOCs in each sample ( Table 9 ). This analysis showed that Smoke contained approximately four times as much VOCs as e-cigarette aerosols (which were all similar).

Comprehensive aerosol and smoke chemical analyses were performed ( Table 1 ), and the results are summarized in Table 9 . Briefly, nicotine was detected in Smoke (6,700 μg/m 3 ), 12-PG (99 μg/m 3 ), and 12-VG (76 μg/m 3 ) aerosol but not in 0-PG or 0-VG aerosol. Particulates were also highest in Smoke (81 mg/m 3 ), followed by VG-based aerosol (0-VG = 13 mg/m 3 ; 12-VG = 19 mg/m 3 ). PG-based aerosol contained substantially less particulates than VG-based aerosol (0-PG = 0.9 mg/m 3 ; 12-PG = 2.3 mg/m 3 ). The majority of metals we tested for were not detected in any of the samples; however, we did detect titanium, chromium and barium in all samples (including Air). A range of carbonyl compounds were detected in the samples ( Table 9 ), with Smoke containing the greatest amount of most carbonyls. In comparison with other e-cigarette aerosol, 0-PG aerosol contained the greatest concentrations of acetaldehyde, acetone, acrolein, formaldehyde, propionaldehyde, tolualdehyde, and valeraldehyde. There were few other clear trends in e-cigarette aerosols with respect to carbonyl levels.

For analyses of liquids, there was no difference in retention time or mass spectra between the VG and PG samples. The peaks are therefore labeled “glycerin” for all samples ( Table 8 ). VG/PG made up the vast majority of all 4 four-cigarette liquids. Hexose was found in PG-based liquids but not VG-based liquids. Surprisingly, nicotine was not detectable in 12-VG liquid, despite it being measured in 12-VG aerosol. Only two other identifiable compounds were found in all four liquids, 2-chlorophenol and octadecanoic acid. Trace amounts of 2-amino-octanoic acid; 1,2,3-butantriol; triacetin; hexadecanoic acid; and propionic acid were found in some e-cigarette liquids, accounting for ~1% by volume. Two compounds, also present in trace amounts, were unidentifiable by GC-MS.

Fig. 7. E-cigarette aerosol does not increase lung cellular inflammation, alter lung collagen or muscle, or alter lung mucous or mucous-producing cells. Female mice were exposed to tobacco smoke (Smoke), e-cigarette aerosol (0-PG, 12-PG, 0-VG, and 12-VG), or control (Air) for 8 wk. Twenty-four hours after the final exposure lungs were inflation fixed with formaldehyde before being sectioned and stained with hematoxylin and eosin ( left ), Masson’s trichrome ( middle ), or Alcian blue-periodic acid-Schiff ( right ). Original magnification: ×40; bar length = 65 μm. These images are representative of all mice. Mice exposed to tobacco smoke (Smoke) showed significantly more cellular inflammation than any e-cigarette aerosol exposure group.

Histological assessment revealed few differences among groups ( Fig. 7 and Table 7 ). There was visible cellular inflammation in Smoke mice (which was reflected in BAL cellular inflammation) but no significant differences in epithelium thickness ( P = 0.466, airway smooth muscle area ( P = 0.133), or percentage collagen ( P = 0.296). Luminal area was significantly higher for 12-PG and 0-VG mice compared with Smoke ( Table 7 ; P < 0.050 in both cases). Mice exposed to e-cigarette aerosols without nicotine (0-PG and 0-VG) had significantly fewer mucous-positive cells in their epithelium compared with Air controls ( P < 0.049 in both cases). 0-PG mice also had significantly less mucous-positive cells compared with Smoke mice ( P = 0.028). There were significant differences among groups with respect to L m ( Table 7 ). Mean linear intercept length was significantly greater in Smoke mice compared with all other treatments ( P < 0.050 in all cases), except for 0-VG ( P = 0.247).

Smoke mice had significantly higher levels of KC in their BAL supernatant compared with all other treatments ( P < 0.027 in all cases; Table 6 ). Smoke mice also had more IL-12p40 compared with 0-PG and 12-VG mice ( P < 0.040 in both cases) and more IL-1α than 12-VG mice ( P = 0.05). E-cigarette aerosol exposure did not alter the level of any mediator in comparison with Air exposure. For a small number of analytes, there was no difference among the treatments (IL12-p70, MIP-1α, MIP-1β, and TNF-α; P > 0.125 in all cases; Table 6 ). For the majority of analytes assessed, a significant proportion of individual measurements (or all measurements) were below the lower limit of quantification making statistical comparisons among treatments impossible without extrapolation (IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17, eotaxin, GM-CSF, G-CSF, RANTES, MCP-1, and IFNγ).

0-VG-exposed mice showed similar increases in responsiveness to MCh to Smoke mice, while PG mice were similar to Air mice regardless of nicotine presence or absence ( Table 5 and Fig. 6 ). Compared with Air mice, 0-VG mice had greater R aw ( P = 0.005; Fig. 6 A ), G ( P < 0.001; Fig. 6 B ) and H ( P < 0.001; Fig. 6 C ) at the highest dose of MCh ( Table 5 ). Similarly, Smoke mice had greater R aw ( P = 0.042) and G ( P = 0.002) at the highest dose of MCh compared with Air mice. At the highest dose of MCh, both 0-VG and Smoke mice also had higher R aw than 0-PG and 12-PG mice ( P < 0.042 in all cases). Regardless of nicotine content, PG-exposed mice were not significantly more responsive than Air mice in terms of R aw ( P < 0.826 in both cases), G ( P < 0.414 in both cases; Fig. 6 B ), or H ( P < 0.166 in both cases; Fig. 6 C ). Smoke and 12-VG mice were significantly more responsive than Air mice in G ( P < 0.049 in both cases).

There were also significant impacts of e-cigarette aerosol exposure on the volume dependence of G and H ( Table 4 and Fig. 5, B and C ) where we measured an upward/leftward shift in the curve for mice exposed to e-cigarette aerosol, compared with those exposed to Air or Smoke. G at P rs = 20 cmH 2 O was significantly higher in 0-VG (16,663 ± 1,260 hPa/l), 0-PG (15,795 ± 928 hPa/l), and 12-PG (15,235 ± 713 hPa/l) mice compared with Air (14,678 ± 1,289 hPa/l) and Smoke (14,378 ± 828 hPa/l; P < 0.029 in all cases). H at P rs = 20 cmH 2 O was significantly higher in all e-cigarette aerosol-exposed groups (ranged between 122,776 ± 12,165 and 127,076 ± 8,784 hPa/l) compared with Air (105,864 ± 6,970 hPa/l) or Smoke (102,500 ± 9,084 hPa/l; P < 0.003 in all cases).

E-cigarette aerosol exposure significantly impacted the volume dependence of lung mechanics ( Table 4 and Figs. 4 and 5 ). When inflated to a respiratory system pressure (P rs ) of 20 cmH 2 O, the lung volume of mice exposed to 0-VG (0.70 ± 0.06 ml), 0-PG (0.71 ± 0.05 ml), or 12-PG (0.73 ± 0.10 ml) e-cigarette aerosol was significantly less than that of Air mice (0.81 ± 0.07 ml; P < 0.049 in all cases; Table 4 and Fig. 4 ). The lung volume of mice exposed to 0-VG or 0-PG e-cigarette aerosol was also significantly less than that of Smoke mice (0.807 ± 0.05 ml; P < 0.022 in both cases). There was no difference in TGV at P rs = 20 cmH 2 O for Smoke and Air mice ( P = 0.874).

There was no effect of any exposure on lung volume ( P = 0.500). All groups had an average lung volume of between 0.20 ± 0.06 and 0.23 ± 0.06 ml ( Table 3 ). E-cigarette aerosol exposure significantly impaired lung function at functional residual capacity (FRC; Table 3 ). Airway resistance at FRC was significantly higher in mice exposed to e-cigarette aerosol without nicotine compared with Air-exposed mice ( P < 0.004 in both cases). Mice exposed to e-cigarette aerosol containing nicotine did not have significantly higher R aw at FRC compared with Air mice ( P > 0.360 in both cases). Smoke exposure did not increase R aw at FRC compared with Air ( P = 0.325). Tissue damping at FRC was significantly higher in mice exposed to any e-cigarette aerosol compared with those exposed to Air ( P < 0.002 in all cases; Table 3 ) and was higher for 0-PG, 0-VG, and 12-VG mice compared with those exposed to Smoke ( P < 0.003 in all cases). Similarly, tissue elastance at FRC was significantly higher in mice exposed to any e-cigarette aerosol compared with those exposed to Air ( P < 0.037 in all cases; Table 3 ). Mice exposed to 0-VG or 0-PG had significantly higher H at FRC compared with mice exposed to Smoke ( P < 0.018 in both cases). There was no difference in H at FRC among e-cigarette aerosol-exposed groups ( P < 0.225 in all cases). Smoke exposure did not increase G ( P = 0.444) or H ( P = 0.949) at FRC compared with Air. There was no effect of any exposure on hysteresivity ( P = 0.657; Table 3 ).

Despite a random allocation into treatments, at study commencement there were significant differences in weights among treatments ( Table 2 ). In general, mice exposed to tobacco smoke or e-cigarette aerosol containing nicotine increased in weight at a slower rate compared with mice exposed to e-cigarette aerosol without nicotine or air control ( Table 2 and Fig. 2 ), and after 8 wk of exposure, mice in the Air group (20.72 ± 1.13 g) were significantly heavier than mice in all other treatments ( P < 0.001), which were not different from each other ( P > 0.374 in all cases). Linear regression analyses showed that Air mice gained weight at a faster rate than Smoke ( P = 0.009)- and 12-VG-exposed mice ( P = 0.021). 0-VG mice gained weight faster than Smoke-exposed mice ( P = 0.004), 12-PG-exposed mice ( P = 0.018), and 12-VG-exposed mice ( P = 0.001). There was no difference in rate of weight gain among Air-, 0-PG-, and 0-VG-exposed mice ( P > 0.065 in all cases).

DISCUSSION

Our results show that exposure to e-cigarette aerosol can negatively impact lung function and increase responsiveness to MCh in vivo. These physiological effects occur in the absence of significant pulmonary inflammation. We found that exposure to e-cigarette aerosols based on VG-induced functional impairments akin to tobacco smoke exposure. Exposure to PG-based aerosols caused less severe impairments. There was little difference in functional outcomes caused by the presence or absence of nicotine.

As e-cigarette use is increasing rapidly in adolescents, we commenced exposures of mice at 4 wk of age, representing a time that lung development is nearing completion (40), but somatic growth is still occurring. It is roughly equivalent to human adolescence. E-cigarette aerosol exposure and Smoke exposure appeared to result in growth impairment, with these mice being 9–10% lighter than Air mice after 8 wk of exposure. Weight gain was also apparently slower in mice exposed to tobacco smoke or e-cigarette aerosol containing nicotine compared with those exposed to e-cigarette aerosol without nicotine, or air. These findings should be tempered by the knowledge that there were significant differences in starting weights among groups and that there were significant differences in weight gain among individuals. Regardless, our findings were not surprising as human smokers are known to weigh less than age- and sex-matched nonsmokers (1), and nicotine is a known appetite suppressant (34).

Impairments in lung function measured in e-cigarette aerosol-exposed mice occurred in the absence of pulmonary inflammation and in the absence of gross morphological differences in lung structure. With respect to pulmonary inflammation, this finding is fundamentally consistent with other animal e-cigarette exposure models despite methodological differences. Previous published studies that have exposed mice to e-cigarette aerosol via inhalation (15, 26, 48) used a shorter exposure duration than our study and, therefore, may not be a meaningful analogue for long-term use in humans. In previous studies, lung function was not measured. Sussan et al. (48) found that e-cigarette aerosol exposure resulted in 58% more BAL macrophages and significantly decreased BAL IL-6. There was no change in neutrophilia, MCP-1, or MIP-2, which is surprising based on the increased macrophage inflammation. E-cigarette exposure did not increase BAL inflammatory cells in other studies that employed inhalation exposure (15, 26); however, minor changes in the levels of some proinflammatory cytokines were measured. In our study, e-cigarette aerosol exposure did not increase BAL inflammation. In fact, mice exposed to 12-PG aerosol had significantly fewer macrophages than Air controls. The reasons for this reduction are difficult to elucidate, especially as there was no decrease in macrophages for the 0-PG group. It is possible that the interplay between nicotine presence and absence [nicotine is known to have some anti-inflammatory properties (2)] and the physicochemical properties of the aerosol play a part. With respect to effects of cigarette smoke or e-cigarette aerosol exposure on lung structure, we found that Smoke mice had a significant increase in L m compared with all other treatments (except 0-VG). An increase in L m in Smoke mice is consistent with enlargements of distal air spaces in as seen in cigarette-smoke-induced lung disease (20). The fact L m was not significantly different between Smoke and 0-VG mice may provide some mechanistic evidence for the functional differences we observed. That said, we did not see increased L m in our other functionally impaired treatment (12-VG), so other factors must also be taken into account. In our model, where exposures started relatively early in life, there is also the possibility that instead of resulting in alveolar destruction Smoke (and 0-VG) exposure resulted in impaired alveolar development. We did not measure any differences in key parameters of lung structure including airway smooth muscle mass or lung collagen content; however, the percentage of positively stained mucous-producing cells in the epithelium was significantly lower in 0-VG and 0-PG mice compared with Air controls. Our data show no clear relationship between exposure type (i.e., excipient/nicotine presence or absence) and mucous-positive cells, again suggesting that e-cigarette aerosol exposure for 8 wk has limited or no effect on lung structure.

Mice exposed to e-cigarette aerosol or tobacco smoke did not have smaller lungs than Air controls (Table 2). It is known that in utero cigarette smoke exposure impairs lung growth in mice (22). In humans, cigarette smoking during adolescence is known to reduce forced vital capacity (49). Our finding supports the notion that beginning exposure at a time when lung development is effectively complete potentially has less severe impacts than if exposures were commenced earlier.

E-cigarette aerosol exposure significantly impacted lung function at FRC and the volume dependency of parenchymal lung mechanics. There are no previous studies that report the effects of prolonged e-cigarette aerosol exposure on these parameters; however, some studies in humans report that acute e-cigarette use results in small increases in airway resistance and/or reductions in conductance (38, 50). We also identified a consistent trend of greater R aw , G, and H in mice exposed to e-cigarette aerosol without nicotine, compared with those exposed to aerosol containing nicotine. Measurements of R aw , G, and H at FRC were not significantly different among Smoke, 12-PG, and 12-VG mice; however, all of these parameters were higher in the 0-PG- and 0-VG-exposed mice. This was an unexpected finding, as nicotine inhalation in humans is known to induce bronchoconstriction, most likely through a vagally mediated mechanism (14). Furthermore, cigarette smoke inhalation is known to induce bronchoconstriction, which is thought to be due both the nicotine and particulate matter. Thus we expected that R aw at FRC would be higher in the 12-PG and 12-VG groups compared with the 0-PG and 0-VG groups. At high transrespiratory pressures, G and H were significantly higher in e-cigarette aerosol-exposed mice than either Air or Smoke mice. The shift in the curves shown in Fig. 5 shows that e-cigarette aerosol exposure increases the resistance of the peripheral airways and reduces lung compliance. As we have previously reported (22), this type of change could be due to an increase in parallel airway heterogeneity (30, 31) or it could reflect more closed peripheral lung units (29). Both of these are unlikely in our model as hysteresivity is not significantly different among treatments at FRC, at P rs = 20 cmH 2 O, or after MCh challenge (Tables 3–5). Additionally, we can see no clear evidence of airway/lung unit collapse in our lung sections (Fig. 7). We originally thought that lung units may have become closed/blocked via a build-up of e-cigarette excipient in the lung periphery based on a recent clinical case report whereby long-term e-cigarette use was associated with exogenous lipoid pneumonia (32). This condition can develop after inhalation of an oil, such as VG. The authors surmise that the source of exogenous lipoid pneumonia was recurrent inhalation of VG-based e-cigarette aerosol as symptoms resolved after e-cigarette use ceased. This clinical case report also provides support for why we measured more severe impacts of VG-based e-cigarette aerosol. In saying that, however, as we found no difference in BAL VG levels among treatments as measured by ELISA (data not shown), accumulation of VG is also insufficient to explain functional differences in our mouse model. While there is little information in the literature on the effects of inhaling heated and aerosolized VG, even short-term inhalation exposure to aerosolized VG can result in squamous metaplasia of the epithelium lining the epiglottis (44), which is consistent with repeated inhalation of an irritant.

E-cigarette aerosol exposure increased responsiveness to MCh and VG-based aerosol caused greater impairments than PG-based aerosol (Fig. 6). The largest effects were seen in R aw . These findings show that e-cigarette aerosol increased airway narrowing in an otherwise healthy individual and that the type of e-cigarette excipient is of critical importance in determining the severity of response. It is perhaps important to note here that our studies were performed using the BALB/c strain of mouse, which is known to exhibit heightened responses to MCh (compared with other strains of mouse), when exposed to a variety of environmental stimuli (4, 53). This fact should be taken into account when considering the generalizability of our results. Regardless, we expected that inhalation of PG-based aerosol would also result in airway hyperresponsiveness, however this was not the case. PG has been certified as “generally recognized as safe” by the U.S. Food and Drug Administration (21); however, this classification does not typically include inhalation exposure. PG is known to cause eye, throat, and airway irritation after short-term exposure (35, 50, 52), while long-term exposure can increase the risk of asthma in children (7). A possible reason for PG-based e-cigarette aerosol not inducing airway or parenchymal responsiveness to the same degree as VG-based aerosol may be due to the physicochemical composition of the aerosols. Specifically, PG-based aerosols contained ~10 times less particulate matter compared with VG-based aerosols. Particulate matter exposure is well known to contribute to asthma development and exacerbation (12). Similar mechanisms may be at play in determining airway and parenchymal responsiveness. One study has shown an increase in respiratory system resistance in mice after exposure to nebulized e-cigarette liquid containing nicotine, although there was no effect of inhaling a nicotine-free liquid (11). This publication is not a full scientific paper, so it is difficult to draw any firm conclusions from the data. Similarly, another study reported an exacerbation of allergy-induced airway hyperresponsiveness in e-cigarette liquid-exposed mice (27). However, in this study, mice received e-cigarette liquid via intratracheal instillation, and responsiveness was inferred from the enhanced pause parameter.

Physicochemical analyses of the e-cigarette liquids and aerosols were conducted to determine if any of the constituents were associated with the functional impairments measured. Particulate matter is a pollutant with demonstrated impacts on lung development and lung function (42). The PG-based aerosols contained considerably less particulate matter compared with VG-based aerosols. However, as Smoke mice did not display the most severe impairments but had the highest particulate matter mass this may only partially explain the more severe response seen in VG-exposed mice compared with PG-exposed mice. Associations between inhaled particles and health are largely due to the size of particles and less to the mass of particles (37, 46). We found that VG-based aerosols contained the largest particles and PG-based aerosols contained the smallest particles (smoke particles were in between), so it is unlikely that, in this study, particulate matter mass or size accounted for the differences in lung function impairments observed among the groups.

We measured a number of other toxicants in the e-cigarette aerosols, including some metals, carbonyl compounds, and VOCs. These have been reported in e-cigarette aerosols previously (6, 13). Some of these have been associated with poor lung function (3, 33, 51), but their effects are not well established (36). In this study, there were no consistent differences in toxicant concentrations among the different aerosols and tobacco smoke, and, therefore, it is not possible to determine if any of these contributed to the observed impairments.

Our study is the first to show that exposure to e-cigarette aerosol during adolescence is not harmless and results in significant impairments in lung function in mice. Importantly, our data suggest that the e-cigarette excipient used is critical in determining the severity of the response. The physicochemical composition of an e-cigarette aerosol may be a key driver behind functional impairments. Further study exploring these differences and the mechanisms involved in determining e-cigarette-induced pulmonary effects are warranted.