JUUL E-liquids Research Articles

Pod-based e-liquids impair human vascular endothelial cell function.

Pod-based electronic (e-) cigarettes more efficiently deliver nicotine using a protonated formulation. The cardiovascular effects associated with these devices are poorly understood. We evaluated whether pod-based e-liquids and their individual components impair endothelial cell function. We isolated endothelial cells from people who are pod users (n = 10), tobacco never users (n = 7), and combustible cigarette users (n = 6). After a structured use, pod users had lower acetylcholine-mediated endothelial nitric oxide synthase (eNOS) activation compared with never users and was similar to levels from combustible cigarette users (overall P = 0.008, P = 0.01 pod vs never; P = 0.96 pod vs combustible cigarette). The effects of pod-based e-cigarettes and their constituents on vascular cell function were further studied in commercially available human aortic endothelial cells (HAECs) incubated with flavored JUUL e-liquids or propylene glycol (PG):vegetable glycerol (VG) at 30:70 ratio with or without 60 mg/mL nicotine salt for 90 min. A progressive increase in cell death with JUUL e-liquid exposure was observed across 0.0001-1% dilutions; PG:VG vehicle with and without nicotine salt induced cell death. A23187-stimulated nitric oxide production was decreased with all JUUL e-liquid flavors, PG:VG and nicotine salt exposures. Aerosols generated by JUUL e-liquid heating similarly decreased stimulated nitric oxide production. Only mint flavored e-liquids increased inflammation and menthol flavored e-liquids enhanced oxidative stress in HAECs. In conclusion, pod e-liquids and their individual components appear to impair endothelial cell function. These findings indicate the potential harm of pod-based devices on endothelial cell function and thus may be relevant to cardiovascular injury in pod type e-cigarette users.

Vaping /E‐liquid Exposure Causes Dysregulation of Neutrophil Extracellular Trap formation

IntroductionElectronic cigarettes (e‐cigarettes) vaporize e‐liquids containing propylene glycol and vegetable glycerine (PG/VG), nicotine, and flavors. The inhaled aerosol from e‐liquids may be potentially toxic or harmful. Although the short and long‐term health consequences of e‐cigarettes use are poorly understood, their use has risen steeply among adolescents and young adults. Neutrophils play an essential role in innate immune and are the first responders during infections. Thus, this study aims to test whether vaping can affect neutrophil function. We hypothesized that vaping e‐cigarettes and exposure to e‐liquids activate neutrophils, cause degranulation, and induce extracellular traps (NETs) through altered calcium signaling.Method and MaterialsNeutrophils were isolated from human blood samples of non‐vapor control (n=9) and e‐cigarette users (n=5) and studied within 2 hours. To measure changes in cytosolic calcium, neutrophils were loaded with Fluo‐4. Intracellular calcium responses were monitored on a Tecan multi‐plate reader following stimulation by 1 µM thapsigargin and 1 µM fMLP/100ng per ml LPS. In addition, since elevations in intracellular calcium trigger neutrophil activation and degranulation, elastase activity in the media samples was measured using a specific fluorogenic substrate (MAA‐3133, © vivitide, LLC.) at excitation/emission wavelengths 380/460 nm. To study NET formation, neutrophils were labeled with SYTOX Green dye and then exposed to different concentrations of Juul e‐liquids (menthol, classic tobacco flavors) and Bidi Stick flavors (Arctic, Gold, and Solar). Data acquisition was performed with Cytation‐5 Cell Imaging Plate Reader, then analysis and representative images were captured by confocal microscopy.ResultsNeutrophils from e‐cigarette users (n=5 vapers) showed significantly (P < 0.001) increased intracellular calcium responses to thapsigargin and fMLP/LPS treatments compared to the non‐user controls (n=9). Additionally, vapers' neutrophils also secreted more elastase (P < 0.001) compared to the control non‐users, indicating that e‐cigarette users may be susceptible to increased systemic protease activity. Our data also indicated that Juul and Bidi Stick e‐liquids stimulated neutrophils to induce NETs in a dose‐dependent manner. Furthermore, the study revealed that Juul e‐liquid menthol flavor with 5% nicotine could significantly (P < 0.001) release NETs than equivalent nicotine ± benzoic acid alone.SummaryCirculating neutrophils are normally quiescent. However, our data indicated that circulating neutrophils from vapers were abnormally active and degranulated by released elastase. Additionally, e‐cigarette liquids dysregulated neutrophils by increasing NETs production. Overall, we speculate that vaping imbalanced innate immune system and may lead to abnormal responses to the infection, inappropriate inflammatory responses, and adverse health effects.

Differential impact of JUUL flavors on pulmonary immune modulation and oxidative stress responses in male and female mice.

JUUL is a popular e-cigarette brand that manufactures e-liquids in a variety of flavors, such as mango and mint. Despite their popularity, the pulmonary effects of flavored JUUL e-liquids that are aerosolized and subsequently inhaled are not known. Therefore, the purpose of this study was to evaluate if acute exposure to JUUL e-cigarette aerosols in three popular flavors elicits an immunomodulatory or oxidative stress response in mice. We first developed a preclinical model that mimics human use patterns of e-cigarettes using 1puff/min or 4puffs/min exposure regimes. Based on cotinine levels, these exposures were representative of light/occasional and moderate JUUL users. We then exposed C57BL/6 mice to JUUL e-cigarette aerosols in mango, mint, and Virginia tobacco flavors containing 5% nicotine for 3days, and assessed the inflammatory and oxidative stress response in the lungs and blood. In response to the 1puff/min regime (light/occasional user), there were minimal changes in BAL cell composition or lung mRNA expression. However, at 4puffs/min (moderate user), mint-flavored JUUL significantly increased lung neutrophils, while mango-flavored JUUL significantly increased Tnfα and Il13 mRNA in the lungs. Both the 1- and 4puffs/min regimes significantly increased oxidative stress markers in the blood, indicating systemic effects. Thus, JUUL products are not inert; even short-term inhalation of flavored JUUL e-cigarette aerosols differentially causes immune modulation and oxidative stress responses.

Electronic Cigarette Solvents, JUUL E-Liquids, and Biomarkers of Exposure: In Vivo Evidence for Acrolein and Glycidol in E-Cig-Derived Aerosols.

Despite the increasing popularity of e-cigarettes, their long-term health effects remain unknown. In animal models, exposure to e-cigarette has been reported to result in pulmonary and cardiovascular injury, and in humans, the acute use of e-cigarettes increases heart rate and blood pressure and induces endothelial dysfunction. In both animal models and humans, cardiovascular dysfunction associated with e-cigarettes has been linked to reactive aldehydes such as formaldehyde and acrolein generated in e-cigarette aerosols. These aldehydes are known products of heating and degradation of vegetable glycerin (VG) present in e-liquids. Here, we report that in mice, acute exposure to a mixture of propylene glycol:vegetable glycerin (PG:VG) or to e-cigarette-derived aerosols significantly increased the urinary excretion of acrolein and glycidol metabolites—3-hydroxypropylmercapturic acid (3HPMA) and 2,3-dihydroxypropylmercapturic acid (23HPMA)—as measured by UPLC-MS/MS. In humans, the use of e-cigarettes led to an increase in the urinary levels of 23HPMA but not 3HPMA. Acute exposure of mice to aerosols derived from PG:13C3-VG significantly increased the 13C3 enrichment of both urinary metabolites 13C3-3HPMA and 13C3-23HPMA. Our stable isotope tracing experiments provide further evidence that thermal decomposition of vegetable glycerin in the e-cigarette solvent leads to generation of acrolein and glycidol. This suggests that the adverse health effects of e-cigarettes may be attributable in part to these reactive compounds formed through the process of aerosolizing nicotine. Our findings also support the notion that 23HPMA, but not 3HPMA, may be a relatively specific biomarker of e-cigarette use.

Abstract 10999: Pod-Based E-liquids Induce Vascular Endothelial Cell Dysfunction

Introduction: Pod-based e-cigarettes including JUUL represent the most commonly used product type and more efficiently deliver nicotine; however, their cardiovascular effects are poorly understood. Methods: We evaluated endothelial function (cell viability by TUNEL and nitric oxide bioavailability using DAF-2DA) in human aortic endothelial cells (HAECs) exposed for 90 minutes to one of four JUUL pod flavors (Menthol, Mint, Mango and Virginia Tobacco) or constituents propylene glycol (PG)/vegetable glycerol (VG) at 30:70 ratio with and without nicotine salt, as well as their heated particle fractions separated with a mini-MOUDI impactor. In healthy young adults, we assessed endothelial cell function in nonusers of tobacco products, pod users and combustible cigarette users for eNOS activation. Results: We observed significant increases in cell death with JUUL e-liquid flavors across 0.0001-1% dilutions. PG/VG vehicle alone and with nicotine salt induced cell death to similar levels as the JUUL e-liquids. All JUUL e-liquids at 0.0001% dilution impaired A23187-stimulated nitric oxide production. Exposure to particle fractions of JUUL e-liquids at 0.001% dilution also reduced A23187-stimulated nitric oxide production. Endothelial cells from pod users showed reduced eNOS activation compared with nonusers and similar to reduced levels in combustible cigarette users (figure). Conclusions: Our data suggest endothelial toxicity of pod-based e-liquids and their constituents, including in young adult users.

A Rapid and Sensitive Chemical Screening Method for E-Cigarette Aerosols Based on Runtime Cavity Ringdown Spectroscopy

Growing demand of Juul and other electronic cigarettes, despite critical knowledge gaps about their chemical composition, has led to concerns regarding their potential health effects. We introduce a novel analytical approach, runtime cavity ringdown spectroscopy (rtCRDS) for rapid detection of oxidative products in e-cigarette aerosols, to facilitate the study of aerosol from a single puff of e-liquid. We report a systematic investigation of three flavors of commercial Juul pods (Virginia tobacco, mango, and menthol) and known commercial e-liquid ingredients (propylene glycol (PG), vegetable glycerin (VG), nicotine, ethyl maltol, benzoic acid, and nicotine benzoate) vaped using Juul devices. Juul e-liquids and neat chemical additives spiked into a 30:70 PG/VG solution were vaped and their aerosols were collected in 1-L Tedlar gas bags and analyzed using rtCRDS. Acetaldehyde, formaldehyde, and acetone were identified as primary oxidative products in aerosolized PG/VG. Ethanol was detected as a major constituent of the three commercial Juul flavors. Spectral intensities of carbonyl compounds increased with the addition of spikes, benzoic acid, ethyl maltol, and nicotine to PG/VG, suggesting that oxidative product generation increases with common additives. The method of direct, rapid analysis of e-cig aerosols introduced here can be used to complement traditional methods in vaping exposures.

JUUL e-liquid exposure elicits cytoplasmic Ca2+ responses and leads to cytotoxicity in cultured airway epithelial cells

RationaleThe popularity of new and emerging tobacco products such as E-cigarettes (E-cigs) is rapidly expanding worldwide. However, uncertainties surrounding the potential health consequences due to the use of such products exist and warrant further study. MethodsCultured A549 and Calu-3 airway epithelia were exposed to three out of the eight types of JUUL brand e-liquids (“Mint”, “Virginia Tobacco” and “Menthol”, all containing 3% nicotine at 1% and 3% (vol/vol) dilutions) and assessed for viability using a resazurin-based assay. Intracellular Ca2+ levels were measured using fluorescent indicators and pro-inflammatory cytokine levels were monitored by quantitative PCR (qPCR). Cultures were also analyzed by flow cytometry to evaluate apoptotic markers and cell viability. ResultsExposing the airway epithelial cells to the flavored JUUL e-liquids led to significant cytotoxicity, with the “Mint” flavor being the overall most cytotoxic. The “Mint” flavored e-liquid also led to significant elevations in intracellular Ca2+ and upregulation of the pro-inflammatory cytokine IL-6 and early apoptotic marker Annexin V. ConclusionsJUUL e-liquid challenge resulted in a loss of airway epithelial cell viability, induced pro-inflammatory responses and eventually caused apoptosis.

Abstract 17343: E-cigarette Aerosol Exposure Acutely Alters Cardiac Conduction and Autonomic Balance and Induces Arrhythmia in Mice

Background: E-cigarette (e-cig) use has rapidly increased, especially among youth. Vaping has been linked to adverse cardiopulmonary effects, but the full extent of effects remains unknown. Several constituents in e-cigs may increase cardiac risk partly by disturbing cardiac electrophysiology and the autonomic nervous system. Hypothesis: E-cig aerosols will differentially induce pro-arrhythmic changes in cardiac conduction and autonomic balance in mice depending on the presence of nicotine and flavors. Methods: Electrocardiograms (ECGs) were collected by telemetry in 5 healthy male mice (C57BL/6) exposed for 6 hours to clean air or e-cig aerosols (9-minute puff sessions every 18 minutes) from JUUL e-liquids (Va. Tobacco, Mango, or Menthol at ≈5% nicotine benzoate), or a nicotine-free mixture of propylene glycol and vegetable glycerin solvents (PG:VG, 30:70 ratio). ECG morphology, heart rate variability (HRV), and arrhythmias were analyzed by mixed models with P < 0.05 (vs. Air) for all effects. Results: PG:VG increased high grade supraventricular block arrhythmias (121±45 events/hour) relative to Air (0±0 events/hour) and decreased heart rate (HR, -25±8 beats/min), whereas aerosols from all nicotine-containing e-liquids increased HR and decreased HRV, suggesting sympatho-excitation. However, these effects were significantly attenuated for Mango relative to Menthol and Va. Tobacco. As well, only PG:VG and Mango increased ventricular premature beats (VPBs, 9.6±3.7 and 5.5±1.2 events/hour, ±SEM) relative to Air (1.1±0.24 events/hour). VPBs correlated with changes in standard deviation of RR and mean HR from pre-exposure (Spearman’s r : 0.51 and -0.27, P < 0.0001). Prolonged repolarization (QTc) correlated with VPBs during exposures to PG:VG ( r = 0.38, P = 0.046), but not nicotine-containing e-liquids ( r = 0.15, P = 0.38). Conclusions: E-cigs may increase risk for cardiac arrhythmia through e-liquid solvents, which when thermally aerosolized generate toxic aldehydes and particulates. Nicotine and flavor chemicals may modify the cardiac and autonomic impacts of e-cigs. Further studies are needed to determine how e-cig aerosols induce cardiac arrhythmia and whether these effects translate to cardiac morbidity and mortality in humans.

Cellular effects of nicotine salt-containing e-liquids.

"Pod-based" e-cigarettes such as JUUL are currently the most prevalent electronic nicotine delivery systems (ENDS) in the United States. JUUL-type ENDS utilize nicotine salts protonated with benzoic acid rather than freebase nicotine. However, limited information is available on the cellular effects of these products. Cytoplasmic Ca2+ is a universal second messenger that controls many cellular functions including cell growth and cell death. Of note, dysregulation of cell Ca2+ homeostasis has been linked with several disease processes including autoimmune disease and several types of cancer. We exposed HEK293T cells and THP-1 macrophage-like cells to different JUUL e-liquids. We evaluated their effects on cellular viability and Ca2+ signaling by measuring fluorescence from calcein-AM/propidium iodide and Fluo-4, respectively. E-liquid autofluorescence was used to look for e-liquid permeation into cells. To identify the mechanisms behind the Ca2+ responses, different inhibitors of Ca2+ channels and phospholipase C signaling were used. JUUL e-liquids caused significant cytotoxic effects, with "Mint" flavor being the most cytotoxic. The Mint flavored e-liquid also caused a significant elevation in cytoplasmic Ca2+ . Using autofluorescence, the permeation of JUUL e-liquids into live cells was confirmed, indicating that intracellular organelles are directly exposed to e-liquids. Further studies identified the endoplasmic reticulum as being the source of e-liquid-induced changes in cytoplasmic Ca2+ . Nicotine salt-based e-liquids cause cytotoxicity and elevate cytoplasmic Ca2+ , indicating that they can exert biological effects beyond what would be expected with nicotine alone. These effects are flavor-dependent, and we propose that flavored e-liquids be reassessed for potential lung toxicity.

Trendy e-cigarettes enter Europe: chemical characterization of JUUL pods and its aerosols

The popularity and the high nicotine content of the American pod e-cigarette JUUL have raised many concerns. To comply with European law, the nicotine concentration in the liquids of the European version, which has been recently released on the market, is limited to below 20 mg/mL. This limit can possibly be circumvented by technological adjustments that increase vaporization and consequently, elevate nicotine delivery. In this study, we compare vapor generation and nicotine delivery of the initial European version, a modified European version, and the original American high-nicotine variant using a machine vaping set-up. Additionally, benzoic acid and carbonyl compounds are quantified in the aerosol. Further, concentrations of nicotine, benzoic acid, propylene glycol, and glycerol, along with the density and pH value of JUUL e-liquids have been assessed. Whereas the initial European version did not compensate for the low nicotine content in the liquid, we provide evidence for an increased vaporization by the modified European version. As a consequence, nicotine delivery per puff approximates the American original. Notably, this is not associated with an increased generation of carbonyl compounds. Our data suggest a similar addictiveness of the enhanced European version and the original American product.

Free Radical, Carbonyl, and Nicotine Levels Produced by Juul Electronic Cigarettes.

Free radicals and carbonyls produced by electronic cigarettes (e-cigs) have the potential to inflict oxidative stress. Recently, Juul e-cigs have risen drastically in popularity; however, there is no data on nicotine and oxidant yields from this new e-cig design. Aerosol generated from four different Juul flavors was analyzed for carbonyls, nicotine, and free radicals. The e-liquids were analyzed for propylene glycol (PG) and glycerol (GLY) concentrations. To determine the effects of e-liquid on oxidant production, Juul pods were refilled with nicotine-free 30:70 or 60:40 PG:GLY with or without citral. No significant differences were found in nicotine (164 ± 41 µg/puff), free radical (5.85 ± 1.20 pmol/puff), formaldehyde (0.20 ± 0.10 µg/puff), and acetone (0.20 ± 0.05 µg/puff) levels between flavors. The PG:GLY ratio in e-liquids was ~30:70 across all flavors with GLY being slightly higher in tobacco and mint flavors. In general, when Juul e-liquids were replaced with nicotine-free 60:40 PG:GLY, oxidant production increased up to 190% and, with addition of citral, increased even further. Juul devices produce free radicals and carbonyls, albeit, at levels substantially lower than those observed in other e-cig products, an effect only partially because of a low PG:GLY ratio. Nicotine delivery by these devices was as high as or higher than the levels previously reported from cigarettes. These findings suggest that oxidative stress and/or damage resulting from Juul use may be lower than that from cigarettes or other e-cig devices; however, the high nicotine levels are suggestive of a greater addiction potential.

E-cigarettes’ chemistry may explain their popularity among teens

Although children under the age of 18 can’t legally buy e-cigarettes in most states, the devices have exploded in popularity among teens. According to a 2017 survey conducted by the University of Michigan, almost one-third of high school seniors have used an e-cigarette within the past year. One device, offered by Juul Labs, has risen above hundreds of competitors, capturing 54% of the overall e-cigarette market in just three years. Shaped like a sleek thumb drive, the device can be used discreetly and delivers a nicotine hit by vaporizing liquids containing the drug in flavors such as Cool Mint and Creme Brulee. To probe the possible chemical underpinnings of e-cigarettes’ popularity, chemists at Portland State University analyzed 11 e-cigarette liquids, or e-liquids, to measure the amounts of two forms of nicotine—protonated nicotine and free-base nicotine (shown). In particular, the team found that Juul e-liquids contained mostly protonated nicotine, which may