Scientific rigor and reproducibility
We used a rigorous and unbiased approach throughout the experimental plans and data analyses to ensure that our data are reproducible along with full and detailed reporting of both methods and analyzed data. All the key biological and chemical resources that are used in this study were validated and authenticated and are of scientific standard from commercial sources. Our results adhere to NIH standards of reproducibility and scientific rigor.
Ethical approval: Institutional biosafety approvals
All experiments performed in this study were approved and in accordance with the University of Rochester Institutional Biosafety Committee. Cell lines (16-HBE, BEAS2B, U937) were procured from ATCC, USA. No ethical approval is required for these cell lines.
Procurement of JUUL pods and similar pod flavors
JUUL pod flavors, “Fruit Medley”, “Virginia Tobacco”, “Cool Mint”, Crème Brulee”, “Cool Cucumber”, “Mango”, and “Classic Menthol” with 5% nicotine were purchased from the JUUL online store as well as the local retail store. Other pod flavors, “Just Mango (Strawberry Coconut)” (nicotine concentration unlisted) by LCF labs and “Caffé Latte” by Eonsmoke labs with 6% nicotine, were purchased from local vape shops in Rochester, NY.
Qualitative analysis of the composition of JUUL pod constituents
JUUL pod flavors (Fruit Medley, Classic Menthol, Cool Mint, Crème Brulee, Cool Cucumber, and Virginia Tobacco) were diluted 100X into spectral grade methanol and injected into the gas chromatograph (GC) with a mass spectrometer (MS) detector (Agilent 7890 A gas chromatograph with 5975 MSD detector). The system used helium as the carrier gas, flowing 1.2 mL/min through an Agilent Technologies column (HP-5MS, 30 m × 0.250 mm, 0.25 μm, 19091S-433). The oven program initiated at 60 °C for 4 minutes, ramped to 150 °C over 2 minutes at 70 °C/min, spiked to 250 °C and then ramped again to 285 °C over 5 minutes at 25 °C/min. The total run time was almost 18 minutes, and the injection volume was 1 µL from a 10 µL syringe. The samples were analyzed by electron impact ionization in positive ion mode with a mass range of 50–550 m/z, with the source temperature at 230 °C and the quadrupole at 150 °C. Data analysis was performed using Agilent ChemStation software, with ion scans searched against the NIST database for identification. Retention times with the percentage probability of the constituents found are listed in Supplementary Table S1.
Assessment of acellular ROS
For the acellular ROS assessment, 2′7′dichlorofluoroscien diacetate (DCFH-DA) dye (Calbiochem, CA, catalog #287810) was prepared by mixing 5 mM DCFH-DA with 0.01N NaOH and followed by the addition of 25 mM phosphate buffer and HRP-streptavidin thirty minutes later. JUUL pod flavor vapors from the seven pod flavors were bubbled in the DCFH dye by a SCIREQ air pump following a puff profile of three puffs per minute lasting for three seconds with roughly 17s intervals at a flow rate of 1.6 L/min. For each pod, there were five, ten, or fifteen puffs bubbled through the DCFH dye. Using a standard curve generated from various H2O2 concentrations, produced ROS concentrations were obtained in μM H2O2 equivalents for each flavor.
Cell culture and flavor exposure/treatment
Human bronchial epithelial cells, 16-HBE cell line
16-HBE at 20,000 cells per well were cultured in 24-well transwell inserts (Corning, #3470) in complete media (DMEM media with 10% FBS, 15 mM HEPES, 1% Pen/strep, and 0.2% amphotericin B). At 80% confluency, the cells were serum-deprived at 1% FBS for 12-hours followed by three-sessions of aerosol exposure to Cool Cucumber, Classic Menthol, Just Mango (Strawberry Coconut), and Caffé Latte flavors (refer to the in vitro exposure section for more details on exposure methods). Treatment with H2O2 (600 μM) was used as a comparison positive control. Immediately after the aerosol exposure, the cells were used for the barrier dysfunction assessment and MitoStress assay. The conditioned media was stored at −80 °C for cytokine assessment with ELISA and Luminex.
Human bronchial epithelial cells, BEAS-2B cell line
BEAS-2B cells were cultured in 6-well plates with approximately 325,000 cells per well in complete media in DMEM: F12 complete media with 5% FBS, 1% pen/strep, and 15 mM HEPES. At 80% confluency, the cells were serum-deprived at 1% FBS for 12-hours followed by three sessions of aerosol exposure to Cool Cucumber, Classic Menthol, Just Mango (Strawberry Coconut), and Caffé Latte flavors (refer to the in vitro exposure section for more details on exposure methods). Tumor necrosis factor alpha (TNF-α) (10 ng/mL) treatment was used as a comparison positive control. Twenty-four hours post aerosol exposure, the conditioned media were collected and stored at −80 °C for cytokine assessment with ELISA and Luminex multiplex assay.
Monocytes, U937 cell line
U937 cells (approx. 300,000) were cultured in 12-well plates in complete RPMI 1640 media (ATCC, 30-2001) with 10% FBS complete with 1% pen/strep. Approximately 12-hours before treatments cells were serum-deprived at 1% FBS. Subsequently, designated wells were directly treated with Cool Cucumber, Classic Menthol, Just Mango (Strawberry Coconut), Caffé Latte flavor liquids at 0.5% and H2O2 (100 µM) as a positive control. Twenty-four hours post-exposure the conditioned media was used for cytokine analysis by ELISA and Luminex, and the cells were used for DNA damage assessment by Comet assay.
In-vitro aerosol exposure system
JUUL pod device was connected to one of the pumps of the Scireq Inexpose e-cig exposure system (Scireq, Monreal). The mouthpiece of the JUUL device was then connected to an Enzyscreen chamber (Enzyscreen, Netherlands). For each exposure session, a cell culture plate was placed inside the Enzyscreen chamber and the vapors were released into the chamber. (66 puffs during 22 minutes with a three second puff duration at 1.6 L/min flow rate and an interpuff interval of approximately 17s.). The aerosols were allowed to equilibrate for eight minutes post-exposure resulting in a total of 30 minutes per session before returning the cell culture plate to the incubator. Next, aerosol exposure sessions were performed at 12-hour intervals (Supplementary Fig. S1B).
Assessment of mitochondrial superoxide generation
Immediately after the last exposure with the selected flavor aerosol, the cells were trypsinized and stained with MitoSOX Red and Annexin V according to MitoStress kit protocol (Millipore, FCCH100109). For the flavors, Cool Cucumber, Classic Menthol, Just Mango (Strawberry Coconut), and Caffé Latte, six transwells were pooled together per flavor, and two pooled samples were run for each tested flavor. Sample acquisition and analysis were performed by the Guava Millipore Easycyte-8 instrument and its software, respectively.
IL-8 cytokine quantification by ELISA
Conditioned media from 16-HBE and U937 cells were collected after the specified incubation period post-treatment/exposure, and the IL-8 levels were quantified using ELISA kit (Invitrogen, CHC1303) according to the manufacturer’s instructions.
Prostaglandin E2 quantification by ELISA
Conditioned media from U937, BEAS-2B, and 16-HBE cells were collected after the specified incubation period post-treatment/exposure. A competitive ELISA assay was performed to quantify PGE2 levels according to the manufacturer’s protocol (Cayman, 514010).
Quantification of inflammatory mediators by Luminex
Conditioned media collected from BEAS-2B and U937 cells were assayed for inflammatory cytokines and growth factors by Bioplex Pro human cytokine 27-plex (BioRad, M500KCAF0Y). After the specified post-exposure/treatment incubation period, collected conditioned media was assayed for cytokines with Bioplex 3D system. Only the detectable mediators were plotted and compared with the untreated control group.
Epithelial barrier function assessment
After 16-HBE cell exposure to Crème Brulee, Classic Menthol, Just Mango (Strawberry Coconut), and Caffé Latte flavors, the barrier function was evaluated by EVOM2 (WPI instruments, FL). The transepithelial voltage and the resistance were measured three times in each transwell at different positions, and the average numbers were reported per transwell.
DNA damage assessment by JUUL pods and other pod flavors
Twenty four hours after monocytes (U937) being treated with JUUL pods and similar pod flavors, the cells were prepared for DNA damage assessment by Comet assay according to the manufacturer’s protocol (Trevigen, MD). Briefly, the alkaline procedure was followed and approximately 5000 cells per sample were used and stained with SYBR gold 1:10,000. EVOS imaging system was used to capture the fluorescence images. The images were inverted for the Opencomet analysis. The olive tail moment was obtained using Opencomet software for the representative images, and the percent change was calculated in comparison to the control group.
Statistical analysis
Statistical analysis of data was done using two-way ANOVA with a Tukey’s post-hoc test for all analyses of multiple groups with multiple variables and one-way ANOVA with Dunnett’s post-hoc test was used to analyze all comparison of multiple groups with a single variable using GraphPad Prism 8.0. Data are represented as mean ± SEM. Cells and conditioned media samples were pooled in some assays. Statistical significance was reported as *p < 0.05, **p < 0.01, and ***p < 0.001.
Disclaimer
The authors have nothing to claim or disclaim about any products used here to test their toxicological and biological effects. The authors have no personal interests or gains from the outcome of this study. The products tested are available commercially to consumers/users.
