Skip to main content
NCBI home page
American Journal of Physiology - Lung Cellular and Molecular Physiology logoLink to American Journal of Physiology - Lung Cellular and Molecular Physiology
. 2021 Apr 7;320(6):L1064–L1073. doi: 10.1152/ajplung.00525.2020

Differential responses to e-cig generated aerosols from humectants and different forms of nicotine in epithelial cells from nonsmokers and smokers

Yael-Natalie H Escobar 1, Cameron B Morrison 2, Yuzhi Chen 3, Elise Hickman 1, Charlotte A Love 1, Meghan E Rebuli 1,5, Jason D Surratt 3,4, Camille Ehre 2,5, Ilona Jaspers 1,5,✉
PMCID: PMC8285624  PMID: 33825493

Abstract

In the United States, millions of adults use electronic cigarettes (e-cigs), and a majority of these users are former or current cigarette smokers. It is unclear, whether prior smoking status affects biological responses induced by e-cigs. In this study, differentiated human nasal epithelial cells (hNECs) from nonsmokers and smokers at air-liquid interface were acutely exposed to the e-cig generated aerosols of humectants, propylene glycol (PG), and glycerol (GLY). Mucin levels were examined in the apical washes, and cytokine levels were assessed in the basolateral supernatants 24 h postexposure. The aerosol from the GLY exposure increased mucin 5, subtype AC (MUC5AC) levels in the apical wash of hNECs from nonsmokers, but not smokers. However, the aerosol from GLY induced pro-inflammatory responses in hNECs from smokers. We also exposed hNECs from nonsmokers and smokers to e-cig generated aerosol from PG:GLY with freebase nicotine or nicotine salt. The PG:GLY with freebase nicotine exposure increased MUC5AC and mucin 5, subtype B (MUC5B) levels in hNECs from nonsmokers, but the nicotine salt exposure did not. The PG:GLY with nicotine salt exposure increased pro-inflammatory cytokines in hNECs from smokers, which was not seen with the freebase nicotine exposure. Taken together, these data indicate that the e-cig generated aerosols from the humectants, mostly GLY, and the type of nicotine used cause differential effects in airway epithelial cells from nonsmokers and smokers. As e-cig use is increasing, it is important to understand that the biological effects of e-cig use are likely dependent on prior cigarette smoke exposure.

Keywords: e-cigs, GLY, nicotine, PG, smokers

INTRODUCTION

The increasing popularity of electronic cigarettes (e-cigs) has led to a growing field of research regarding e-cigs and their health effects. This research is made difficult by the diversity of e-cig devices (1) and variety of e-liquid compositions (2). E-liquids are primarily made of flavorings, nicotine, and the humectants, propylene glycol (PG) and glycerol (GLY). The diversity of flavorings in e-liquids has been well documented (3), and a great deal of research has been placed on understanding potential toxicities of the flavoring compounds (4–13). However, although flavorings can vary between e-cig brands, nicotine and the humectants can be found in nearly all e-cigs. Nicotine is not a new chemical exposure, but it has experienced a resurgence of focus in light of the high-level popularity of JUUL, a pod-based e-cig which uses nicotine salt (14). Nicotine salts are generated by the addition of an organic acid to freebase nicotine, where the acid protonates nicotine and lowers the pH of the e-liquid, making it less harsh on the user than an e-liquid with an equivalent concentration of freebase nicotine without an acid present (15, 16). The popularity of JUUL devices, which are able to deliver nicotine at higher doses with minimal irritation, has prompted many other e-cig companies to sell e-liquids or pod-based devices with nicotine salt at even higher nicotine concentrations and cheaper prices than JUUL (17). The humectants, PG and GLY, are also not new chemicals and do not cause many adverse effects in inhalational exposure models when nebulized (18–20). However, when PG and GLY are heated, they are known to undergo thermal degradation and form aldehydes (21) and free radicals (22). We and others have shown that in vitro exposures of airway epithelial cells to e-cig generated aerosols from PG and GLY increase in pro-inflammatory (23, 24) and cellular stress responses (24).

Despite the potential harmful effects of e-cigs, there are millions of people in the United States who currently use them. The percentage of adults and youth who use e-cigs has increased in recent years, with 19.6% of high schoolers in 2020 using e-cigs (25, 26). Among adults, it is estimated that 3.2% use e-cigs, but the prevalence of e-cig use is higher at 7.6% in younger adults (18–24 yr old) (25). The most popular reason for e-cig use is as an attempt to reduce or quit conventional cigarette smoking altogether (10, 27, 28). There is little research comparing the effects of e-cig use in smokers versus nonsmokers or determining whether prior smoking history modifies responses induced by e-cigs. It is known, however, that smoking can induce epigenetic modifications and genetic effects, which can cause lasting changes in cell function even after smoking cessation (29, 30). We and others have previously demonstrated that airway epithelial cells from smokers retain certain “smoking features” after culturing and differentiation, which results in modified cell function (29, 31). Hence, it is reasonable to speculate that prior smoking history affects e-cig-induced responses at the level of the epithelium.

Furthermore, smoking is associated with increased lung inflammation and chronic bronchitis symptoms, such as increased sputum production and airflow obstruction (32, 33). Smokers have been shown to have elevated neutrophils and macrophages in their bronchoalveolar lavage fluid as well as increased levels of common pro-inflammatory cytokines: interleukin (IL) 6 (IL-6), IL-8, and IL-1β (34). Smokers tend to have higher phlegm production than nonsmokers along with higher mucin concentrations, which is now a marker of chronic bronchitis (35). Specifically, current and former smokers have been shown to have enhanced concentrations of mucin 5, subtype AC (MUC5AC) (35), one of the two main polymeric mucin macromolecules in mucus along with mucin 5, subtype B (MUC5B) (36).

The purpose of this study was to compare mucin and immune mediator responses to e-cig generated aerosols from PG and GLY with and without freebase nicotine or nicotine salt in airway cells from nonsmokers and smokers. By using differentiated human nasal epithelial cells (hNECs) from smokers and nonsmokers, we show that e-cig generated aerosol from GLY increases MUC5AC levels in nonsmoker cells but not in smoker cells. We also discovered that the different forms of nicotine, freebase nicotine and nicotine salt, induce vastly different mucin and immune mediator responses from exposed hNECs. This is of particular importance because even though most e-cig users are former or current conventional cigarette smokers, there is an increase in e-cig use among never smokers (37), particularly in young adults (38).

METHODS

Human Nasal Epithelial Cell Procurement and Culturing

Superficial nasal scrape biopsies were obtained from healthy human volunteers (nonsmokers n = 8 and smokers n = 6 or 7) (Tables 1 and 2) as previously described (39). The volunteers provided written informed consent for the acquisition of nasal scrape biopsies and were compensated. Nonsmokers were defined as having smoked less than 100 cigarettes in their lifetime, and smokers were defined as smoking a minimum of five cigarettes per day. This protocol was approved by the Institutional Review Board for Biomedical Research of the University of North Carolina at Chapel Hill School of Medicine. Nasal cells were similarly cultured as previously described by Muller et al. (39), but with an updated media to improve cell proliferation. Briefly, nasal cells were collected and expanded to passage 2 in PneumaCult-Ex Plus Medium (Stemcell 05040) supplemented with hydrocortisone (0.48 µg/mL), penicillin (100 U/mL), streptomycin (100 µg/mL), and amphotericin B (0.25 µg/mL). The cells were then plated on 0.4-μm transwell plates and cultured in the same media until confluency. Once confluent, cells were taken to air-liquid interface (ALI) with PneumaCult ALI Medium (Stemcell 05001) with 1% penicillin and streptomycin. Cell media was changed, and apical surfaces were washed 3× a week with HBSS++ (Gibco, Thermo Fisher Scientific). Complete differentiation was achieved 4 wk post-ALI, and cells were exposed 5–7 wk post-ALI.

Table 1.

Subject demographics

Nonsmokers Smokers
n 8 6
Age 31.25 ± 1.99 38 ± 3.55
Sex, female/male 5/3 3/3
Ethnicity, White/African American/Asian American 5/2/1 3/3/0
Open in a new tab

Values are means ± SE.

Table 2.

Subject demographics

Nonsmokers Smokers
n 8 7
Age 31.25 ± 1.99 37.14 ± 3.13
Sex, female/male 5/3 4/3
Ethnicity, White/African American/Asian American 5/2/1 4/3/0
Open in a new tab

Values are means ± SE.

E-Cig Device and E-Liquids

A third-generation e-cig, DNA 200 Lava Box (Volcano eCigs, Honolulu, HI), with a SMOK TFV4 mini tank using subohm TF-S6 sextuple Kanthal coils with a 0.4 Ω resistance (SMOK, Shenzhen IVPS Technology Co. Limited, Shenzhen, China) was used for these exposures. The manufacturers recommend the device be used between 30 and 100 W. Based on the previous studies, we used the device at 85 W, achieving significant deposition of particles onto the transwell plate (∼1.81 ± 0.0289 mg/cm2). Pure PG (Thermo Fisher Scientific, USP grade), pure GLY (Sigma-Aldrich, USP grade, St. Louis, MO), and a 55:45 volume-to-volume (vol/vol) mixture of PG:GLY without nicotine, with 12 mg/mL of freebase nicotine (Sigma-Aldrich 820877) or with 12 mg/mL of a nicotine salt from a commercial vendor (Liquid Nicotine Wholesalers, Phoenix, AZ), were vaporized using separate tanks for each exposure type to avoid cross contamination. The pH values of the different e-liquid compositions, PG:GLY without nicotine, PG:GLY with 1.2% freebase nicotine, and PG:GLY with 1.2% nicotine salt, were 7.09, 8.25, and 5.88, respectively. The pH was measured with an accumet AB15. The deposition of PG:GLY with freebase nicotine or nicotine salt particles onto the transwell plate was determined to be 1.43 ± 0.23 mg/cm2 and 1.36 ± 0.08 mg/cm2, respectively (Supplemental Fig. S1; all Supplemental Material is available at https://doi.org/10.6084/m9.figshare.13118345.v3).

Exposure Parameters

Before each exposure, all e-cig tanks were filled completely with their respective humectant. Our exposure parameters consisted of a 4-s puff every 30 s with a flow rate of 2.5 L/m for a puff volume of 166 mL. Each exposure lasted 10 min for a total of 20 puffs per exposure. During the exposure, the levels of humectants within the e-cig tank were monitored and maintained at a minimum of 2/3 of capacity to avoid dry puffing conditions.

In Vitro Exposures

Cells were exposed in an exposure system previously described by our group (24). In brief, immediately before exposing fully differentiated hNECs, the basolateral media was exchanged for fresh media and the apical surface of hNECs was washed with 100 µL of HBSS++. In the first set of experiments, hNECs from nonsmokers and smokers, together, were exposed to air and e-cig generated aerosol from PG, GLY, or PG:GLY mixture at a 55:45 (vol/vol) ratio without nicotine. In the second set of experiments, hNECs from nonsmokers and smokers were exposed to air or e-cig generated aerosol from PG:GLY at a 55:45 (vol/vol) ratio with 12 mg/mL of freebase nicotine or with 12 mg/mL of nicotine salt. Immediately after exposure, the basolateral media was changed to fresh media. Apical wash and basolateral supernatant were then collected 24 h postexposure.

Mucin Analysis

Mucin Western blotting analysis was performed as previously described (40). Briefly, cell cultures were washed with 110 µL of HBSS++ 24 h postexposure. The order in which samples were run on the gel was scrambled, and the technician blinded to what the samples were in order to avoid any bias. Samples were run on an 0.8% agarose gel (80 V, 60 min) and vacuum transferred (Boekel Appligene, Boekel Scientific, Feasterville, PA) to a 0.45-µm nitrocellulose membrane (Amersham, GE Healthcare, Chicago, IL). After transfer, membranes were blocked for 1 h at RT (Blotto, Thermo Fisher, Waltham, MA) and probed with 45M1 mouse anti-MUC5AC (Invitrogen MA5-12178, Invitrogen, Carlsbad, CA) and H-300 rabbit anti-MUC5B (Santa Cruz SC-20119, Santa Cruz Biotechnology, Dallas, TX) overnight at 4°C. Membranes were rinsed in PBS and then probed for secondary detection with 1 µg/mL donkey anti-mouse (LI-COR 926–68022) and donkey anti-rabbit (LI-COR 926–32213, LI-COR, Lincoln, NE) for 1 h at RT. Relative mucin abundance was quantified using LI-COR Odyssey software and was still blinded during quantification. Some of the representative immunoblot images were cut and rearranged to better mimic the order in which the data are presented in the graphs.

Cytokine Analysis

Cytokines in the basolateral supernatant of exposed cells were analyzed with a V-PLEX Human Cytokine 30-Plex Kit (Meso Scale Discovery, K15054D-1) per manufacturer instructions. For the comparison of the effects of e-cig generated aerosol from PG and GLY to air, samples were analyzed at a 1:4 dilution. Of the 29 unique cytokines measured, there was adequate signal detected in 26 of the measured analytes. Adequate signal for an analyte was defined as 25% or more of the samples having signal above the blank. IL-16, IL-17, and tumor necrosis factor β (TNF-β) were removed from the analysis as they did not meet signal detection criteria. For the comparison of e-cig generated aerosol from PG:GLY with freebase nicotine or nicotine salt to air, samples were analyzed with same multiplex ELISA kit as above and were analyzed at the manufacturer recommended minimum dilution. Of the 29 unique cytokines, adequate signal was detected in 26 of the measured cytokines. IL-17, TNF-β, and monocyte chemotactic protein-4 (MCP-4) were removed from the analysis as they did not meet the signal detection criteria described here.

Statistical Analysis

Data are presented as means ± SE. The mucin data were analyzed with a matched two-way ANOVA with a post hoc Fisher’s LSD test. The cytokine data were analyzed using a matched two-way ANOVA followed by two-stage step-up method of Benjamini, Krieger, and Yekutieli with a false discovery rate of 0.05 to correct for multiple comparisons. Significance was determined by P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001 as compared to respective air controls. P ≤ 0.05 significant difference between air control samples of nonsmokers versus smokers in analytes was measured in the cytokine analysis. Analysis was performed using GraphPad Prism v. 8.0.0 for Windows, GraphPad Software, San Diego, CA, https://www.graphpad.com. Heatmaps were generated using the “pheatmap” package with log-transformed average data and unbiased clustering by Euclidean distance in R to visualize differences in cytokines between groups (41, 42).

RESULTS

hNECs from nonsmokers and smokers (Table 1) were exposed to air and e-cig generated aerosols from PG, GLY, and a 55:45 (vol/vol) PG:GLY mixture with the e-cig device set at 85 W using the exposure system previously characterized. The hNECs were exposed for 10 min, and mucin levels in the apical wash of exposed hNECs were measured 24 h postexposure with agarose mucin gel electrophoresis. The e-cig generated aerosols from GLY and PG:GLY significantly increased MUC5AC levels in the apical wash of exposed hNECs from nonsmokers as compared to their respective air control, which was not observed in hNECs from smokers (Fig. 1, A and B). MUC5B levels from nonsmoker hNECs exposed to e-cig generated aerosol from GLY and PG:GLY did increase, albeit not statistically significant (Fig. 1, C and D). There were no significant differences between baseline air exposed hNECs from nonsmoker and smokers.

Figure 1.

Figure 1.

Open in a new tab

MUC5AC and MUC5B levels in the apical wash of exposed hNECs. Representative immunoblots of MUC5AC (A) and MUC5B (C) from nonsmoker and smoker hNEC apical washes 24 h postexposure to air, e-cig generated aerosol from PG, GLY, and PG:GLY. Relative densitometry of MUC5AC (B) and MUC5B (D). Means ± SE. Nonsmokers n = 8, smokers n = 6. *P ≤ 0.05, **P ≤ 0.01. Some of the representative immunoblot images were cut and rearranged in order to better mimic the order in which the data are presented in the graphs. GLY, glycerol; hNECs, human nasal epithelial cells; MUC5AC, mucin 5, subtype AC; MUC5B, mucin 5, subtype B; PG, propylene glycol.

The basolateral supernatants of hNECs were analyzed with a multiplex ELISA cytokine panel for 29 unique analytes, with adequate signal being measured in 26 of the 29 analytes. Overall, the e-cig generated aerosol from humectants dysregulated 11 of the cytokines measured and this was largely driven by the e-cig generated aerosol from GLY (Fig. 2 and Supplemental Table S1). The e-cig generated aerosol from GLY increased cytokine levels of IL-10, IL-15, and IL-1β in hNECs from both nonsmokers and smokers. However, the e-cig generated aerosol from GLY only increased IL-2, IL-6, IL-8, interferon gamma-induced protein 10 (IP-10), and vascular endothelial growth factor (VEGF) in hNECs from smokers, whereas e-cig generated aerosol from GLY increased interferon gamma (IFN-γ) and IL-4 protein levels in hNECs from nonsmokers. Monocyte chemotactic protein (MCP-1) and macrophage inflammatory protein-1β (MIP-1β) levels were decreased in hNECs from nonsmokers exposed to e-cig generated aerosol from PG and PG:GLY, respectively (Supplemental Table S1). There were also differences in the baseline levels of air-exposed cells between hNECs from nonsmokers and smokers, with hNECs from smokers having significantly lower levels of IP-10 and MCP-4 as compared to hNECs from nonsmokers (Supplemental Table S1).

Figure 2.

Figure 2.

Open in a new tab

Dysregulated cytokine levels largely driven by e-cig generated aerosol from GLY. Unbiased clustering of log-transformed averages of detectable cytokines from the basolateral supernatant of hNECs from nonsmokers and smokers 24 h postexposure to air, e-cig generated aerosol from PG, GLY, or PG:GLY. Nonsmokers n = 8, smokers n = 6. Air, air-only exposure; GLY, glycerol; hNECs, human nasal epithelial cells; NS, nonsmoker; PG, propylene glycol.

As most e-liquids contain some type of nicotine and typically use a combination of PG:GLY, we conducted a follow-up experiment exposing hNECs from nonsmokers and smokers to air or the e-cig generated aerosol from humectants PG:GLY at a 55:45 ratio with freebase nicotine or nicotine salt at a concentration of 12 mg/mL (Table 2). In this study, we used a concentration of freebase nicotine and nicotine salt of 12 mg/mL, more common for third-generation box mod e-cig device, rather than 50 or 60 mg/mL typically seen for disposable devices or pod-based devices like JUUL. Initially, nicotine salts were used in pod/cartridge-based e-cig devices that do not have as high of power output as a box mod. However, the popularity of nicotine salt has spread (17) and can be found in several refill e-liquids (15). The acid present in the nicotine salt was characterized via RPLC/ESI-HR-QTOFMS with standards of previously identified acids in nicotine salts: lactic, benzoic, tartaric, malic, salicylic, and levulinic acids. We identified the acid present in the nicotine salt that we used as lactic acid (Supplemental Fig. S2).

The PG:GLY with freebase nicotine increased apical MUC5AC and MUC5B levels as compared to the air control in hNECs from nonsmokers (Fig. 3, A–C). However, when hNECs from nonsmokers were exposed to PG:GLY with the nicotine salt, MUC5AC and MUC5B secretion was not increased (Fig. 3, D–F). In contrast, e-cig generated aerosol from PG:GLY with either type of nicotine (freebase or salt) did not change MUC5AC or MUC5B in hNECs from smokers (Fig. 3).

Figure 3.

Figure 3.

Open in a new tab

MUC5AC and MUC5B levels in the apical wash of hNECs exposed to different forms of nicotine. Representative immunoblots of MUC5AC and MUC5B from apical washes of hNECs from nonsmokers and smokers exposed to air and PG:GLY with freebase (A) or nicotine salt (D). Relative densitometry of MUC5AC (B, E) and MUC5B (C, F) levels from the apical wash of hNECs from nonsmokers and smokers exposed to air, PG:GLY with freebase nicotine (A–C), or PG:GLY with nicotine salt (D–F) 24 h postexposure. Means ± SE. Nonsmokers n = 8, smokers n = 7. *P ≤ 0.05. Some of the representative immunoblot images were cut and rearranged in order to better mimic the order in which the data are presented in the graphs. hNECs, human nasal epithelial cells; MUC5AC, mucin 5, subtype AC; MUC5B, mucin 5, subtype B.

We also measured secreted cytokines in the basolateral supernatant of exposed hNECs from nonsmokers and smokers 24 h postexposure to air or e-cig generated aerosol from PG:GLY with nicotine (freebase or salt). The freebase nicotine and the nicotine salt exposures had vastly different effects on the secreted cytokines of exposed hNECs (Fig. 4). The freebase nicotine exposure caused increased levels of IL-12p70, IL-7, IL-8, and VEGF and decreased levels of GM-CSF, IP-10, MCP-1, and TARC in hNECs from nonsmokers. In hNECs from smokers, the freebase nicotine exposure only caused a decrease in TARC levels. The nicotine salt exposure caused increased levels of IL-12p70, IL-1b, IL-2, IL-4, IL-8, and VEGF while also decreasing IP-10, MIP-1β, and TARC levels in the basolateral supernatant of hNECs from nonsmokers. The hNECs from smoker had the most differentially secreted number of cytokines with the nicotine salt exposure, which caused increased levels of IFN-γ, IL-10, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-8, TNF-α, and VEGF and decreased levels of GM-CSF, IP-10, MCP-1, and TARC (Fig. 4B and Supplemental Table S2).

Figure 4.

Figure 4.

Open in a new tab

Altered cytokine levels from different forms of nicotine, freebase nicotine, and nicotine salt. Unbiased clustering of raw data averages of detectable cytokines from the basolateral supernatant of hNECs from nonsmokers and smokers 24 h postexposure to e-cig generated aerosol from PG:GLY with freebase nicotine or nicotine salt (A). Venn diagram of significantly altered cytokines from respective air controls (B). Nonsmokers n = 8, smokers n = 7. free, freebase nicotine; hNECs, human nasal epithelial cells; NS, nonsmoker; salt, nicotine salt.

Any potential cytotoxicity caused by the e-cig generated aerosol exposures was measured by LDH released into the basolateral supernatant of exposed hNECs 24 h postexposure. There were no significant increases in LDH release with any of the e-cig generated aerosol from exposures as compared to the air control (Supplemental Fig. S3).

DISCUSSION

We used primary differentiated airway epithelial cells to determine whether cells from smokers and nonsmokers showed differential responses to e-cig generated aerosols. The majority of adult e-cig users are former or current smokers (37) with a small percentage of e-cig users that were never smokers. However, among young adult (18–24 yr old) e-cig users, 40% are nonsmokers (43). This is especially relevant because the prevalence of e-cig use among U.S. adults increased from 2017 to 2018, predominantly among 18–24 yr olds (25), suggesting that non-smoker-initiated e-cig use is rising. By exposing hNECs from nonsmokers and smokers, we are aiming to mimic how nonsmokers, who begin to use e-cigs, and smokers, who are switching to e-cigs, respond to the exposure. Our data indicate that hNECs from nonsmokers, when exposed to the e-cig generated aerosols from humectants, have an elevated mucin response, which is not seen in hNECs from smokers (Fig. 1). In contrast, hNECs from smokers when exposed to the e-cig generated aerosols from humectants produce more pro-inflammatory cytokines, which was less robust in hNECs from nonsmokers (Fig. 2 and Supplemental Table S1). Furthermore, differential humectant responses are largely driven by the e-cig generated aerosol from GLY and not PG (Figs. 1 and 2). In addition, we were able to expose hNECs to e-cig generated PG:GLY with freebase nicotine or nicotine salt, which caused immensely different mucin and cytokine secretions from hNECs (Figs. 3 and 4).

Mucins are the major macromolecular component of mucus, which serves an important role in protecting the airways from inhaled particulates or pathogens. MUC5AC and MUC5B are the two main secreted mucin glycoproteins in the airways and are vital in forming the gel characteristic of mucus produced in the respiratory tree (36). These secreted mucins are typically upregulated in asthma or smoking-related diseases, such as chronic obstructive pulmonary disease (COPD) (35, 44). Our data demonstrate that the e-cig generated aerosols from the humectants increase MUC5AC secretions in hNECs from nonsmokers (Fig. 1, A and B). This is in agreement with previous studies that have shown increased concentrations of MUC5AC in the induced sputum and bronchial brushings of current e-cig users (45, 46). In addition, human bronchial epithelial cells, which have been exposed to e-cig generated aerosol from PG:GLY, have also shown increases in MUC5AC levels (45). Our data indicate that this increase of MUC5AC secretions in hNECs from nonsmokers is largely driven by the e-cig generated aerosol from GLY and not PG (Fig. 1, A and B). One of the main thermal degradation products of GLY from e-cigs is acrolein, which has been previously shown to increase goblet cell number, mucin secretion, and specifically Muc5ac gene expression in the airways of exposed rats (47). Furthermore, acrolein has been shown to specifically increase MUC5AC gene expression without increasing MUC5B gene expression in human lung carcinoma cells (48). Interestingly, inhalational exposures in rats with nebulized PG have been shown to increase mucin-stained goblet cells, which was not measured in this study (20). This is supported by human data, showing that nonsmokers who use e-cigs are more likely to have increased phlegm/sputum production (49) and adolescents who use e-cigs have increased rates of chronic bronchitis symptoms, which includes increased phlegm production (50).

Interestingly, although the e-cig generated aerosol from GLY did not cause an increase in MUC5AC levels in hNECs from smokers, it did cause elevated pro-inflammatory cytokine secretion in these cells. When hNECs from smokers were exposed to e-cig generated aerosol from GLY, it caused an increase in IL-8, IL-6, VEGF, and IL-2 release, which was not seen in hNECs from nonsmokers (Supplemental Table S1). Similarly, other in vitro exposures of airway epithelial cells with the e-cig generated aerosols from PG and GLY have also shown increased pro-inflammatory cytokine release including IL-6 (23, 24) and IL-8 (24, 51). It should be noted that although the e-cig generated aerosol from GLY did not cause increases in IL-6 and IL-8 in hNECs from nonsmokers, the e-cig generated aerosol from the GLY exposure did increase levels of IL-1β, IL-15, and IL-10 in both hNECs from nonsmokers and smokers (Supplemental Table S1). Overall, the pro-inflammatory response to the e-cig generated aerosol from the humectants is largely driven by GLY with the biggest responses seen in hNECs from smokers.

PG and GLY can be found in most of all e-liquids with nicotine being another common ingredient used in e-cigs (2). There are currently two different forms of nicotine on the market, freebase nicotine and nicotine salt. Freebase nicotine had been the most common form of nicotine used in e-cigs, but with the introduction of nicotine salts with JUUL there are several e-cig companies now offering nicotine salts in their products (17, 52). Nicotine salts are formed when an organic acid such as benzoic, lactic, levulinic, tartaric, malic, or salicylic is added to nicotine and protonates the nicotine (15). The nicotine salt used in this study was found to contain lactic acid (Supplemental Fig. S2), which is naturally produced by the body and can serve as a metabolic substrate by airway epithelial cells (53). When lactic acid protonates nicotine, it becomes lactate and the nicotine undergoes extensive but incomplete nicotine protonation. The presence of the acid lowers the pH of the e-liquid, thereby decreasing the harshness perceived by the user when inhaling the e-cig-derived aerosol (52). This has allowed e-cig companies to increase the concentration of nicotine from 36 to 50 mg/mL and even up to 67 mg/mL (17, 54, 55).

We decided to test the effects of the freebase nicotine and nicotine salt in the context of e-cig generated aerosol, comparing the effects on both mucin secretion and cytokine levels, since nicotine has been shown to promote mucin secretion (56) and has anti-inflammatory effects (57). In primary human bronchial epithelial cells, nicotine exposure can increase mucin secretion (56) and our data show that the e-cig generated aerosol from PG:GLY with freebase nicotine increased both MUC5AC and MUC5B secretions in hNECs from nonsmokers but not in hNECs derived from current smokers (Fig. 3, A–C). However, when hNECs from nonsmokers are exposed to e-cig generated aerosol from PG:GLY with nicotine salt, no changes in mucin secretions were observed (Fig. 3, D–F). Nicotine is also typically considered anti-inflammatory (57–59) and the e-cig generated aerosol from PG:GLY with freebase nicotine decreased levels of IP-10, MCP-1, TARC, and GM-CSF in hNECs from nonsmokers, whereas the nicotine salt exposure increased levels of IL-1β, IL-2, and IL-4 in these same cells. Furthermore, exposure to e-cig generated aerosol from PG:GLY with nicotine salt increased IFN-γ, IL-10, IL-13, IL-1β, IL-2, IL-4, IL-8, TNF-α, and VEGF levels in hNECs from smokers, which was not seen in hNECs from smokers exposed to e-cig generated aerosol from PG:GLY with freebase nicotine (Supplemental Table S2). As illustrated in the summary Venn diagram in Fig. 4B, thymus and activation-regulated chemokine (TARC) is the only cytokine whose protein levels were decreased in response to e-cig generated aerosols with freebase nicotine or nicotine salt in nonsmokers and smokers as compared to their respective air controls. TARC is expressed in the thymus and by several cell types including bronchial and nasal epithelial cells (60, 61). It can induce chemotaxis of Th2 lymphocytes and is thought to play a significant role in allergic and asthmatic diseases (60). The fact that it is decreased in all of the nicotine e-cig exposures in hNECs from nonsmokers and smokers raises interesting questions regarding the role that e-cigs may play in allergic responses.

These data pose the question whether other nicotine salts using different acids cause similar or different effects. It is also possible that the presence of an organic acid might be prompting the formation of different or more thermal degradation products (62). It is known that the thermal degradation products of PG and GLY without the nicotine salts include free radicals (22, 63), reactive oxygen species (64), and aldehydes such as formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, and methylgloxal (21, 65). These thermal degradation products can have adverse effects on the airway epithelium. The potential effect of an organic acid on the formation of thermal degradation products will require further chemical characterization studies. Our data underscore the notion that the two forms of nicotine, freebase and salt, and the various organic acids used to generate nicotine salts are likely to have different effects on the exposed airways, highlighting the need for additional research concerning the effects of nicotine salts. Our data underscore the notion that the two forms of nicotine, freebase and salt, and the various organic acids used to generate nicotine salts are likely to have different effects on the exposed airways, highlighting the need for additional research concerning the effects of nicotine salts.

A relevant deposited dose with in vitro e-cig studies has not yet been established. It is currently estimated that 15%–45% of the e-cig generated aerosol is deposited in the airways (66). This number reflects aerosol deposition across the entire airways. However, it is possible that, similar to other inhaled pollutants, deposition is much more concentrated in certain “hot spots,” which is further influenced by pre-existing diseases (67). Hence, although the in vitro aerosol deposition is likely higher than what epithelial cells would encounter in the airways, heterogeneity of aerosol deposition may result in high deposition areas.

In this study, we set out to determine whether airway epithelial cells from nonsmokers and smokers respond differently to e-cig generated aerosols. Our data demonstrate that hNECs from nonsmokers increase mucin release, whereas hNECs from smokers have a robust pro-inflammatory response when exposed to e-cig generated aerosols. What these data also illustrate is that independent of flavorings, the humectants and nicotine exert their own effects on airway epithelial cells. Hence, although the flavorings are important in e-cig youth initiation (68) and induce adverse effects (6, 12, 13), it is important to understand the biological effects caused by e-liquid components that are ubiquitous to all e-cigs, such as the humectants and nicotine. As e-cig use is increasing among young adults, many of whom are nonsmokers (25, 38), as well as touted as a potential cessation aid in smokers (37), our data indicate that the biological effects of e-cig use are likely to be different in these groups and are further dependent on what type of nicotine used.

SUPPLEMENTAL DATA

Supplemental Figs. S1–S3 and Supplemental Tables S1 and S2: https://doi.org/10.6084/m9.figshare.13118345.

GRANTS

The work was supported in part by the National Institutes of Health (NIH) Grants P50 HL120100, R01HL139369, 3R01HL139369-01S1, T32 ES007126, and P01 HL 108808-06. Research reported in this publication was in part supported by NIH and the FDA Center for Tobacco Products (CTP).

DISCLAIMERS

The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the FDA.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Y.-N.H.E. and I.J. conceived and designed research; Y.-N.H.E., C.B.M., and Y.C. performed experiments; Y.-N.H.E., C.B.M., and Y.C. analyzed data; Y.-N.H.E. and M.E.R. interpreted results of experiments; Y.-N.H.E. and M.E.R. prepared figures; Y.-N.H.E. drafted manuscript; Y.-N.H.E., C.M.B, Y.C., M.E.B., J.D.S., and I.J. edited and revised manuscript; Y.-N.H.E., C.M.B., Y.C., M.E.B., J.D.S., C.E., and I.J. approved final version of manuscript.

REFERENCES

  • 1.Brown CJ, Cheng JM. Electronic cigarettes: product characterisation and design considerations. Tob Control 23: ii4–ii10, 2014. doi: 10.1136/tobaccocontrol-2013-051476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Hutzler C, Paschke M, Kruschinski S, Henkler F, Hahn J, Luch A. Chemical hazards present in liquids and vapors of electronic cigarettes. Arch Toxicol 88: 1295–1308, 2014. doi: 10.1007/s00204-014-1294-7. [DOI] [PubMed] [Google Scholar]
  • 3.Zhu S-H, Sun JY, Bonnevie E, Cummins SE, Gamst A, Yin L, Lee M. Four hundred and sixty brands of e-cigarettes and counting: implications for product regulation. Tob Control 23: iii3–iii9, 2014. doi: 10.1136/tobaccocontrol-2014-051670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Allen JG, Flanigan SS, LeBlanc M, Vallarino J, MacNaughton P, Stewart JH, Christiani DC. Flavoring chemicals in e-cigarettes: diacetyl, 2,3-pentanedione, and acetoin in a sample of 51 products, including fruit-, candy-, and cocktail-flavored e-cigarettes. Environ Health Perspect 124: 733–739, 2016. doi: 10.1289/ehp.1510185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Behar RZ, Luo W, Lin SC, Wang Y, Valle J, Pankow JF, Talbot P. Distribution, quantification and toxicity of cinnamaldehyde in electronic cigarette refill fluids and aerosols. Tob Control 25: ii94–ii102, 2016. doi: 10.1136/tobaccocontrol-2016-053224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Clapp PW, Lavrich KS, van Heusden CA, Lazarowski ER, Carson JL, Jaspers I. Cinnamaldehyde in flavored e-cigarette liquids temporarily suppresses bronchial epithelial cell ciliary motility by dysregulation of mitochondrial function. Am J Physiol Lung Cell Mol Physiol 316: L470–L486, 2019. doi: 10.1152/ajplung.00304.2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Clapp PW, Pawlak EA, Lackey JT, Keating JE, Reeber SL, Glish GL, Jaspers I. Flavored e-cigarette liquids and cinnamaldehyde impair respiratory innate immune cell function. Am J Physiol Lung Cell Mol Physiol 313: L278–L292, 2017. doi: 10.1152/ajplung.00452.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Janice G, K. SI, Robert F, R. SE, E. FA, Risa R, Todd P, Irfan R. Inflammatory response and barrier dysfunction by different e-cigarette flavoring chemicals identified by gas chromatography–mass spectrometry in e-liquids and e-vapors on human lung epithelial cells and fibroblasts. Appl Vitr Toxicol 3: 28–40, 2017. doi: 10.1089/aivt.2016.0030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Lerner CA, Sundar IK, Yao H, Gerloff J, Ossip DJ, McIntosh S, Robinson R, Rahman I. Vapors produced by electronic cigarettes and e-juices with flavorings induce toxicity, oxidative stress, and inflammatory response in lung epithelial cells and in mouse lung. PLoS One 10: e0116732, 2015. doi: 10.1371/journal.pone.0116732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Li J, Newcombe R, Walton D. The prevalence, correlates and reasons for using electronic cigarettes among New Zealand adults. Addict Behav 45: 245–251, 2015. doi: 10.1016/j.addbeh.2015.02.006. [DOI] [PubMed] [Google Scholar]
  • 11.Park HR, O'Sullivan M, Vallarino J, Shumyatcher M, Himes BE, Park JA, Christiani DC, Allen J, Lu Q. Transcriptomic response of primary human airway epithelial cells to flavoring chemicals in electronic cigarettes. Sci Rep 9: 1400, 2019. doi: 10.1038/s41598-018-37913-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Rowell TR, Reeber SL, Lee SL, Harris RA, Nethery RC, Herring AH, Glish GL, Tarran R. Flavored e-cigarette liquids reduce proliferation and viability in the CALU3 airway epithelial cell line. Am J Physiol Lung Cell Mol Physiol 313: L52–L66, 2017. doi: 10.1152/ajplung.00392.2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Sherwood CL, Boitano S. Airway epithelial cell exposure to distinct e-cigarette liquid flavorings reveals toxicity thresholds and activation of CFTR by the chocolate flavoring 2,5-dimethypyrazine. Respir Res 17: 57, 2016. doi: 10.1186/s12931-016-0369-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Huang J, Duan Z, Kwok J, Binns S, Vera LE, Kim Y, Szczypka G, Emery SL. Vaping versus JUULing: how the extraordinary growth and marketing of JUUL transformed the US retail e-cigarette market. Tob Control 28: 146–151, 2019. doi: 10.1136/tobaccocontrol-2018-054382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Harvanko AM, Havel CM, Jacob P, Benowitz NL. Characterization of nicotine salts in 23 electronic cigarette refill liquids. Nicotine Tob Res 22: 1239–1243, 2020. doi: 10.1093/ntr/ntz232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Lisko JG, Tran H, Stanfill SB, Blount BC, Watson CH. Chemical composition and evaluation of nicotine, tobacco alkaloids, ph, and selected flavors in e-cigarette cartridges and refill solutions. Nicotine Tob Res 17: 1270–1278, 2015. doi: 10.1093/ntr/ntu279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Jackler RK, Ramamurthi D. Nicotine arms race: JUUL and the high-nicotine product market. Tob Control 28: 623–628, 2019.doi: 10.1136/tobaccocontrol-2018-054796. [DOI] [PubMed] [Google Scholar]
  • 18.Phillips B, Titz B, Kogel U, Sharma D, Leroy P, Xiang Y, Vuillaume G, Lebrun S, Sciuscio D, Ho J, Nury C, Guedj E, Elamin A, Esposito M, Krishnan S, Schlage WK, Veljkovic E, Ivanov NV, Martin F, Peitsch MC, Hoeng J, Vanscheeuwijck P. Toxicity of the main electronic cigarette components, propylene glycol, glycerin, and nicotine, in Sprague-Dawley rats in a 90-day OECD inhalation study complemented by molecular endpoints. Food Chem Toxicol 109: 315–332, 2017. doi: 10.1016/j.fct.2017.09.001 [DOI] [PubMed] [Google Scholar]
  • 19.Renne RA, Wehner AP, Greenspan BJ, Deford HS, Ragan HA, Westerberg RB, Buschbom RL, Burger GT, Hayes AW, Suber RL, Mosberg AT. 2-week and 13-week inhalation studies of aerosolized glycerol in rats. Inhal Toxicol 4: 95–111, 1992. doi: 10.3109/08958379209145307. [DOI] [Google Scholar]
  • 20.Suber RL, Deskin R, Nikiforov I, Fouillet X, Coggins CRE. Subchronic nose-only inhalation study of propylene glycol in Sprague-Dawley rats. Food Chem Toxicol 27: 573–583, 1989. doi: 10.1016/0278-6915(89)90016-1. [DOI] [PubMed] [Google Scholar]
  • 21.Sleiman M, Logue JM, Montesinos VN, Russell ML, Litter MI, Gundel LA, Destaillats H. Emissions from electronic cigarettes: key parameters affecting the release of harmful chemicals. Environ Sci Technol 50: 9644–9651, 2016. doi: 10.1021/acs.est.6b01741. [DOI] [PubMed] [Google Scholar]
  • 22.Bitzer ZT, Goel R, Reilly SM, Foulds J, Muscat J, Elias RJ, Richie JP. Effects of solvent and temperature on free radical formation in electronic cigarette aerosols. Chem Res Toxicol 31: 4–12, 2018. doi: 10.1021/acs.chemrestox.7b00116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Anthérieu S, Garat A, Beauval N, Soyez M, Allorge D, Garçon G, Lo-Guidice J-M. Comparison of cellular and transcriptomic effects between electronic cigarette vapor and cigarette smoke in human bronchial epithelial cells. Toxicol Vitr 45: 417–425, 2017. doi: 10.1016/j.tiv.2016.12.015. [DOI] [PubMed] [Google Scholar]
  • 24.Escobar Y-NH, Nipp G, Cui T, Petters SS, Surratt JD, Jaspers I. In vitro toxicity and chemical characterization of aerosol derived from electronic cigarette humectants using a newly developed exposure system. Chem Res Toxicol 33: 1677–1688, 2020. doi: 10.1021/acs.chemrestox.9b00490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Bao W, Liu B, Du Y, Snetselaar LG, Wallace RB. Electronic cigarette use among young, middle-aged, and older adults in the United States in 2017 and 2018. JAMA Intern Med 180: 313–314, 2020. doi: 10.1001/jamainternmed.2019.4957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Wang TW, Neff LJ, Park-Lee E, Ren C, Cullen KA, King BA. E-cigarette use among middle and high school students—United States, 2020. Morb Mortal Wkly Rep 69: 1310–1312, 2020. doi: 10.15585/mmwr.mm6937e1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Ayers JW, Leas EC, Allem J-P, Benton A, Dredze M, Althouse BM, Cruz TB, Unger JB. Why do people use electronic nicotine delivery systems (electronic cigarettes)? A content analysis of Twitter, 2012-2015. PLoS One 12: e0170702–e0170702, 2017. doi: 10.1371/journal.pone.0170702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Hummel K, Hoving C, Nagelhout GE, de Vries H, van den Putte B, Candel MJJM, Borland R, Willemsen MC. Prevalence and reasons for use of electronic cigarettes among smokers: findings from the International Tobacco Control (ITC) Netherlands Survey. Int J Drug Policy 26: 601–608, 2015. doi: 10.1016/j.drugpo.2014.12.009. [DOI] [PubMed] [Google Scholar]
  • 29.Rager JE, Bauer RN, Müller LL, Smeester L, Carson JL, Brighton LE, Fry RC, Jaspers I. DNA methylation in nasal epithelial cells from smokers: identification of ULBP3-related effects. Am J Physiol Lung Cell Mol Physiol 305: L432–L438, 2013. doi: 10.1152/ajplung.00116.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Spira A, Beane J, Shah V, Liu G, Schembri F, Yang X, Palma J, Brody JS. Effects of cigarette smoke on the human airway epithelial cell transcriptome. Proc Natl Acad Sci USA 101: 10143–10148, 2004. doi: 10.1073/pnas.0401422101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Jaspers I, Horvath KM, Zhang W, Brighton LE, Carson JL, Noah TL. Reduced expression of IRF7 in nasal epithelial cells from smokers after infection with influenza. Am J Respir Cell Mol Biol 43: 368–375, 2010. doi: 10.1165/rcmb.2009-0254OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Speizer FE, Tager IRAB. Epidemiology of chronic mucus hypersecretion and obstructive airways disease. Epidemiol Rev 1: 124–142, 1979. doi: 10.1093/oxfordjournals.epirev.a036206. [DOI] [PubMed] [Google Scholar]
  • 33.Strzelak A, Ratajczak A, Adamiec A, Feleszko W. Tobacco smoke induces and alters immune responses in the lung triggering inflammation, allergy, asthma and other lung diseases: a mechanistic review. Int J Environ Res Public Health 15: 1033, 2018. doi: 10.3390/ijerph15051033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Kuschner WG, D'Alessandro A, Wong H, Blanc PD. Dose-dependent cigarette smoking-related inflammatory responses in healthy adults. Eur Respir J 9: 1989–1994, 1996. doi: 10.1183/09031936.96.09101989. [DOI] [PubMed] [Google Scholar]
  • 35.Kesimer M, Ford AA, Ceppe A, Radicioni G, Cao R, Davis CW, Doerschuk CM, Alexis NE, Anderson WH, Henderson AG, Barr RG, Bleecker ER, Christenson SA, Cooper CB, Han MK, Hansel NN, Hastie AT, Hoffman EA, Kanner RE, Martinez F, Paine 3rd R, Woodruff PG, O’Neal WK, Boucher RC. Airway mucin concentration as a marker of chronic bronchitis. N Engl J Med 377: 911–922, 2017. doi: 10.1056/NEJMoa1701632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Ma J, Rubin BK, Voynow JA. Mucins, mucus, and goblet cells. Chest 154: 169–176, 2018. doi: 10.1016/j.chest.2017.11.008. [DOI] [PubMed] [Google Scholar]
  • 37.Owusu D, Huang J, Weaver SR, Pechacek TF, Ashley DL, Nayak P, Eriksen MP. Patterns and trends of dual use of e-cigarettes and cigarettes among U.S. adults, 2015–2018. Prev Med Reports 16: 101009, 2019. doi: 10.1016/j.pmedr.2019.101009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.QuickStats: Cigarette Smoking Status Among Current Adult E-cigarette Users, by Age Group. National Health Interview Survey, United States, 2015. MMWR Morb Mortal Wkly Rep 65: 1177, 2016. doi: 10.15585/mmwr.mm6542a7. [DOI] [PubMed] [Google Scholar]
  • 39.Müller L, Brighton LE, Carson JL, Fischer WA, Jaspers I. Culturing of human nasal epithelial cells at the air liquid interface. J Vis Exp 80: 50646, 2013. doi: 10.3791/50646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Ramsey KA, Rushton ZL, Ehre C. Mucin agarose gel electrophoresis: western blotting for high-molecular-weight glycoproteins. J Vis Exp 112: e54153, 2016. doi: 10.3791/54153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Kolde R. pheatmap: pretty heatmaps. R package version 1.0.12. (Online). 2019. https://cran.r-project.org/package=pheatmap
  • 42.R Core Team. A language and environment for statistical computing, 2008. https://www.r-project.org [9 February 2021].
  • 43.Villarroel MA, Cha AE, Vahratian A. Electronic cigarette use among U.S. adults, 2018. NCHS Data Brief, no 365. Hyattsville, MD: National Center for Health Statistics, 2020, p. 1–8. [PubMed] [Google Scholar]
  • 44.Bonser LR, Erle DJ. Airway mucus and asthma: the role of MUC5AC and MUC5B. J Clin Med 6: 112, 2017. doi: 10.3390/jcm6120112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Ghosh A, Coakley RC, Mascenik T, Rowell TR, Davis ES, Rogers K, Webster MJ, Dang H, Herring LE, Sassano MF, Livraghi-Butrico A, Van Buren SK, Graves LM, Herman MA, Randell SH, Alexis NE, Tarran R. Chronic e-cigarette exposure alters the human bronchial epithelial proteome. Am J Respir Crit Care Med 198: 67–76, 2018. doi: 10.1164/rccm.201710-2033OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Reidel B, Radicioni G, Clapp PW, Ford AA, Abdelwahab S, Rebuli ME, Haridass P, Alexis NE, Jaspers I, Kesimer M. E-cigarette use causes a unique innate immune response in the lung, involving increased neutrophilic activation and altered mucin secretion. Am J Respir Crit Care Med 197: 492–501, 2018. doi: 10.1164/rccm.201708-1590OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Borchers MT, Wert SE, Leikauf GD. Acrolein-induced MUC5ac expression in rat airways. Am J Physiol Lung Cell Mol Physiol 274: L573–L581, 1998. doi: 10.1152/ajplung.1998.274.4.L573. [DOI] [PubMed] [Google Scholar]
  • 48.Borchers MT, Carty MP, Leikauf GD. Regulation of human airway mucins by acrolein and inflammatory mediators. Am J Physiol Lung Cell Mol Physiol 276: L549–L555, 1999. doi: 10.1152/ajplung.1999.276.4.L549. [DOI] [PubMed] [Google Scholar]
  • 49.Hedman L, Backman H, Stridsman C, Bosson JA, Lundbäck M, Lindberg A, Rönmark E, Ekerljung L. Association of electronic cigarette use with smoking habits, demographic factors, and respiratory symptoms. JAMA Netw open 1: e180789–e180789, 2018. doi: 10.1001/jamanetworkopen.2018.0789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.McConnell R, Barrington-Trimis JL, Wang K, Urman R, Hong H, Unger J, Samet J, Leventhal A, Berhane K. Electronic cigarette use and respiratory symptoms in adolescents. Am J Respir Crit Care Med 195: 1043–1049, 2017. doi: 10.1164/rccm.201604-0804OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Iskandar AR, Zanetti F, Marescotti D, Titz B, Sewer A, Kondylis A, Leroy P, Belcastro V, Torres LO, Acali S, Majeed S, Steiner S, Trivedi K, Guedj E, Merg C, Schneider T, Frentzel S, Martin F, Ivanov NV, Peitsch MC, Hoeng J. Application of a multi-layer systems toxicology framework for in vitro assessment of the biological effects of classic tobacco e-liquid and its corresponding aerosol using an e-cigarette device with MESHTM technology. Arch Toxicol 93: 3229–3247, 2019. doi: 10.1007/s00204-019-02565-9. [DOI] [PubMed] [Google Scholar]
  • 52.Duell AK, Pankow JF, Peyton DH. Nicotine in tobacco product aerosols: ‘it’s déjà vu all over again.’ Tob Control 29: 656–662, 2020. doi: 10.1136/tobaccocontrol-2019-055275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Lottes RG, Newton DA, Spyropoulos DD, Baatz JE. Lactate as substrate for mitochondrial respiration in alveolar epithelial type II cells. Am J Physiol Lung Cell Mol Physiol 308: L953–L961, 2015. doi: 10.1152/ajplung.00335.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Goniewicz ML, Boykan R, Messina CR, Eliscu A, Tolentino J. High exposure to nicotine among adolescents who use Juul and other vape pod systems (‘pods’). Tob Control 28: 676–677, 2019. doi: 10.1136/tobaccocontrol-2018-054565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Talih S, Salman R, El-Hage R, Karam E, Karaoghlanian N, El-Hellani A, Saliba N, Shihadeh A. Characteristics and toxicant emissions of JUUL electronic cigarettes. Tob Control 28: 678–680, 2019. doi: 10.1136/tobaccocontrol-2018-054616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Gundavarapu S, Wilder JA, Mishra NC, Rir-Sima-Ah J, Langley RJ, Singh SP, Saeed AI, Jaramillo RJ, Gott KM, Peña-Philippides JC, Harrod KS, McIntosh JM, Buch S, Sopori ML. Role of nicotinic receptors and acetylcholine in mucous cell metaplasia, hyperplasia, and airway mucus formation in vitro and in vivo. J Allergy Clin Immunol 130: 770–780.e11, 2012. doi: 10.1016/j.jaci.2012.04.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Li Q, Zhou X, Kolosov VP, Perelman JM. Nicotine suppresses inflammatory factors in HBE16 airway epithelial cells after exposure to cigarette smoke extract and lipopolysaccharide. Transl Res 156: 326–334, 2010. [Erratum in Transl Res 157: 322, 2011]. doi: 10.1016/j.trsl.2010.09.001. [DOI] [PubMed] [Google Scholar]
  • 58.Mabley J, Gordon S, Pacher P. Nicotine exerts an anti-inflammatory effect in a murine model of acute lung injury. Inflammation 34: 231–237, 2011. doi: 10.1007/s10753-010-9228-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Mishra NC, Rir-Sima-Ah J, Langley RJ, Singh SP, Peña-Philippides JC, Koga T, Razani-Boroujerdi S, Hutt J, Campen M, Kim KC, Tesfaigzi Y, Sopori ML. Nicotine primarily suppresses lung Th2 but not goblet cell and muscle cell responses to allergens. J Immunol 180: 7655–7663, 2008. doi: 10.4049/jimmunol.180.11.7655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Sandoval-López G, Teran LM. TARC: novel mediator of allergic inflammation. Clin Exp Allergy 31: 1809–1812, 2001. doi: 10.1046/j.1365-2222.2001.01268.x. [DOI] [PubMed] [Google Scholar]
  • 61.Terada N, Nomura T, Kim WJ, Otsuka Y, Takahashi R, Kishi H, Yamashita T, Sugawara N, Fukuda S, Ikeda-Ito T, Konno A. Expression of C-C chemokine TARC in human nasal mucosa and its regulation by cytokines. Clin Exp Allergy J Br Soc Allergy Clin Immunol 31: 1923–1931, 2001. doi: 10.1046/j.1365-2222.2001.01152.x. [DOI] [PubMed] [Google Scholar]
  • 62.Groll GEORGE, Hearne HPA. Process of converting a polyhydric alcohol to a carbonyl compound (Online). Shell, Dev. US Patent US73266034A. May 23, 1936. https://www.freepatentsonline.com/2042224.html.
  • 63.Goel R, Durand E, Trushin N, Prokopczyk B, Foulds J, Elias RJ, Richie JP. Highly reactive free radicals in electronic cigarette aerosols. Chem Res Toxicol 28: 1675–1677, 2015. doi: 10.1021/acs.chemrestox.5b00220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Son Y, Mishin V, Laskin JD, Mainelis G, Wackowski OA, Delnevo C, Schwander S, Khlystov A, Samburova V, Meng Q. Hydroxyl radicals in e-cigarette vapor and e-vapor oxidative potentials under different vaping patterns. Chem Res Toxicol 32: 1087–1095, 2019. doi: 10.1021/acs.chemrestox.8b00400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Talih S, Salman R, Karaoghlanian N, El-Hellani A, Saliba N, Eissenberg T, Shihadeh A. “Juice Monsters”: sub-ohm vaping and toxic volatile aldehyde emissions. Chem Res Toxicol 30: 1791–1793, 2017. doi: 10.1021/acs.chemrestox.7b00212. [DOI] [PubMed] [Google Scholar]
  • 66.Sosnowski TR, Kramek-Romanowska K. Predicted deposition of e-cigarette aerosol in the human lungs. J Aerosol Med Pulm Drug Deliv 29: 299–309, 2016. doi: 10.1089/jamp.2015.1268. [DOI] [PubMed] [Google Scholar]
  • 67.Bennett WD, Xie M, Zeman K, Hurd H, Donaldson S. Heterogeneity of particle deposition by pixel analysis of 2D gamma scintigraphy images. J Aerosol Med Pulm Drug Deliv 28: 211–218, 2015. doi: 10.1089/jamp.2013.1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Ambrose BK, Day HR, Rostron B, Conway KP, Borek N, Hyland A, Villanti AC. Flavored tobacco product use among US youth aged 12-17 years, 2013-2014. JAMA 314: 1871–1873, 2015. doi: 10.1001/jama.2015.13802. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from American Journal of Physiology - Lung Cellular and Molecular Physiology are provided here courtesy of American Physiological Society

RESOURCES