Fig 1.
Refillable ENDS and Blu e-cigarettes.
(A) Refillable ENDS and Blu e-cigarettes (B) Clearomizer removed, outer wick shown covering top of heating element coil (Upper panel). Used wick removed showing darkened region that contacted heating coil (Middle panel). Heating coil wrapped around second wick (Lower panel). (C) Activating heating element on refillable ENDS with Clearomizer removed. Less than 1 second activation (Upper panel), 2 seconds activation (Middle panel), greater than 2 second’s activation (Lower panel). (D) Cartomizer casing removed after previous use (Upper panel). Poly-fill material with partially absorbed e-liquid wrapped around the core. Outer material removed exposing inner absorbent material tightly wrapped around heating element (Middle panel). Heating element exposed showing coil wrapped wick secured perpendicular to longer woven polymer tubing. A long thin fiber that was wrapped around the heating coil shows points of contact with coil wire (Lower panel).
Table 1.
ENDS e-liquids their trade name, flavor, and manufacturer information obtained from local retailers used in this study.
Fig 2.
OX/ROS in ENDS vapor. Aerosols or air-sham control drawn through DCFH OX/ROS indicator solution.
(A) Blu e-cigarette cartomizers; Classic tobacco or Magnificent menthol flavor e-cigs. Data are shown as mean ± SD (n = 3/group).* P < 0.05, *** P < 0.001 compared to air-sham control (B) eGo refillable vaporizer. Humectants; propylene glycol and glycerin. Commercial e-liquid refills; Vape Dudes Classic tobacco flavor. Data are shown as mean ± SEM (air, n = 15; propylene glycol, n = 23; glycerin, n = 21; Vape Dudes C. tobacco 0 mg nicotine, n = 7; Vape Dudes C. tobacco 24 mg nicotine, n = 3; Heating element, n = 9). *** P < 0.001 compared to air-sham control.
Table 2.
DCF fluorescence values obtained for refillable ENDS aerosols or ambient air alone drawn through DCFH in cell-free ROS assay.
Table 3.
State of the refillable ENDS heating element and its influence over successive use to generate OX/ROS in a cell-free ROS assay.
Fig 3.
E-liquid reactivity with DCFH exhibits differences between nicotine content and flavor additives.
(A) Commercially available e-liquids with different nicotine content. No nicotine (0 mg), low nicotine (6–12 mg) and high nicotine (16–24 mg). Data are shown as mean ± SD. *** P < 0.001. (B) Comparison of commercially available e-liquids, tobacco flavors (Tobacco, American tobacco, Classic tobacco 9x Tobacco, Marbo) versus non-tobacco flavors (Very berry, AMP, Mountain dew, Cinnamon roll, Grape vape, Cotton candy, Strawberry zing, Strawberry fields, Peaches n cream, Berry intense, Pineapple express, Melon mania, and Coconut). Data are shown as mean ± SD of n = 3, *** P < 0.001. Y-axis equal to DCF fluorescence Intensity Units (FIU).
Table 4.
DCF fluorescence of refillable e-liquids with different flavors and nicotine concentrations after addition of DCFH solution analyzed by a cell-free ROS assay.
Fig 4.
Addition of e-liquids to cell culture media induces morphological changes in human lung fibroblasts.
(A) HFL-1 cells grown to 95% confluence were treated with the following e-liquids; propylene glycol, glycerin, or Ecto American tobacco flavor for 24 hrs and then examined for morphological changes by phase-contrast microscopy. Treatment of HFL-1 with 1.0% CSE for 24 hrs included for comparison. Images captured at 20x magnification. Embedded images show expansion of defined area of monolayer as demarcated by dashed boxes. Representative images are shown (n = 3). Enlarged vacuolarized cells in expansion area or large circularized cells (solid arrow), and areas of lost cell-cell connection next to spindle formations (dashed arrow) within defined area vs control. (B) Average number of cells counted adjacently across a single diagonal of 3 defined areas placed randomly (dashed boxes within images). The direction of diagonal cell counts is based on cell orientation in each image. Data are shown as mean ± SD. *P < 0.05; **P < 0.01; and *** P < 0.001 as compared to untreated control culture in growth media.
Table 5.
Effect of e-liquids on HFL-1 cell viability in small 24-well culture area after 24 hours.
Fig 5.
Inflammatory mediators secreted by human lung fibroblasts (HFL-1) treated with e-liquids/humectants and human epithelial airway cells (H292) treated by air-liquid interface with e-cigarette aerosols.
(A) Levels of IL-8 release in conditioned media from HFL-1 cells treated for 24 hrs with 1% humectants or e-liquids or CSE were measured by ELISA. Data are shown as mean ± SD of n = 3. *** P < 0.001 compared to control cells maintained in media with 0.5% FBS. (B) H292 cells were exposed to Blu e-cigarette aerosols with a puff of 3–4 sec for 5, 10 and 15 min. After exposure, H292 cells were incubated at 37°C in 5% CO2 incubator for 16 hrs and levels of IL-8, and (C) IL-6 release in conditioned media were measured by ELISA. Data are shown as mean ± SD. *P < 0.05; **P < 0.01; and *** P < 0.001 as compared to air group (cells maintained in incubator).
Fig 6.
Air-liquid interface deposition of fluorescent substance on human bronchial airway epithelial cells.
Beas-2B cells exposed to Blu e-cigarette vapor with a puff of 3–4 sec for 15 min. After exposure cells were immediately collected and measured by flow cytometry. (A) Histogram showing increase in non-specific fluorescence in cells exposed to e-cig aerosols. (B) Average fluorescence for e-cig exposed cells versus air-sham control shown as Mean Fluorescence Intensity (MFI). Data are shown as mean ± SD, n = 3, * P< 0.05 compared to air-sham control cells.
Fig 7.
Acute e-cigarette aerosol exposure causes lung inflammation and pro-inflammatory response in mouse lungs.
WT Mice (C57BL/6J) were exposed to e-cigarette aerosol exposure (200 mg/m3 TPM) for 3 days and sacrificed 24 hrs after the last exposure. (A) At least 500 cells in the bronchoalveolar lavage fluid (BALF) were counted with hemocytometer to determine the number of macrophages and total cells on cytospin slides stained with Diff-Quik. (B) Levels of pro-inflammatory mediators MCP-1 and IL-6 were measured in BAL fluid obtained from room air and e-cig aerosol exposed mice (C57BL/6J). Data are shown as mean ± SD. *** P < 0.001 compared to air group mice (C) Cytokine/chemokine levels in BAL fluid from room air and e-cig aerosol exposed mice were also measure using Luminex multiplex assay. Data are shown as mean ± SEM, n = 3, *P < 0.05 compared to air group mice (C57BL/6J).
Fig 8.
Intracellular glutathione levels in mouse lung following acute e-cigarette aerosol exposure.
Mice were exposed to e-cig aerosol exposure (200 mg/m3 TPM) for 3 days and sacrificed immediately after the last exposure (3rd day after 5 hrs exposure). Levels of (A) Total glutathione. (B) glutathione disulfide GSSG. (C) Total glutathione to GSSG ratio and (D) GSSG to total glutathione ratio were measured in lung homogenates. Data are shown as mean ± SD (n = 3/group).* P < 0.05 compared to air group mice (C57BL/6J).