E-cigarette flavored pods induce inflammation, epithelial barrier dysfunction, and DNA damage in lung epithelial cells and monocytes

Abstract

E-cigarette flavored pods are increasing in use among young adults. Although marketed as a safer alternative to conventional cigarettes, the health effects of e-cigarette flavored pods are unknown. We hypothesized that e-cigarette flavored pods would cause oxidative stress, barrier dysfunction, and an inflammatory response in monocytes and lung epithelial cells. JUUL pod flavors (Fruit Medley, Virginia Tobacco, Cool Mint, Crème Brulee, Cool Cucumber, Mango, and Classic Menthol) and similar pod flavors (Just Mango-Strawberry Coconut and Caffé Latte) were tested. These pod flavors generated significant amounts of acellular ROS and induced significant mitochondrial superoxide production in bronchial epithelial cells (16-HBE). Lung epithelial cells (16-HBE, BEAS-2B) and monocytes (U937) exposed to various pod aerosols resulted in increased inflammatory mediators, such as IL-8 or PGE₂. JUUL pod flavors, Crème Brulee and Cool Cucumber, caused epithelial barrier dysfunction in 16-HBE cells. Moreover, tested flavors also showed DNA damage upon exposure in monocytes. We determined the chemical constituents present in various flavors. Our data suggest that these constituents in flavored pods induce oxidative stress, inflammation, epithelial barrier dysfunction, and DNA damage in lung cells. These data provide insights into the regulation of e-cigarette flavored pods, as well as constituents in these flavors.

Figure 1

Cell-free ROS generated by JUUL pod flavors. JUUL pods containing Fruit Medley, Virginia Tobacco, Cool Mint, Cool Cucumber, Mango, and Classic Menthol flavors and 3R4F research cigarettes were bubbled through DCFH fluorescent dye. Filtered air was bubbled as a negative control comparison group. Five, ten, and fifteen puffs were generated of these flavors each puff with three-second puff durations. ROS levels are expressed in H₂O₂ µM equivalents. ^ap < 0.001 compared to 5 puff air control, ^bp < 0.001 compared to 10 puff air control, and ^cp < 0.001 compared to 15 puff air control, 3R4F research cigarettes were not analyzed; **p < 0.01, and ***p < 0.001 vs. 5 puffs ^#p < 0.001 vs. 10 puffs. Two wells per group with repeated measurements in pooled samples.

Figure 2

JUUL pods and other pod flavors induce the production of mitochondrial superoxide in lung epithelial cells. 16-HBE cells with 20,000 cells per transwell were grown to 80% confluency in complete media. After serum deprivation (1% FBS) the wells were exposed to three-sessions (each session 66 puffs) of JUUL pods and other pod aerosols with equal intervals between each session. Aerosolized flavors included JUUL pods (Classic Menthol and Cool Cucumber) and other pods (Just Mango -Strawberry Coconut and Caffé Latte). Air group and H₂O₂ (600 μM) were used as assay controls. After the last exposure, cells were collected and pooled for each treatment group (n = 3–6 pooled transwells per group) and stained with MitoSOX red and annexin V, and flow-cytometry analysis was performed. Representative scatter plots per treatment group were shown.

Figure 3

JUUL pods and other pod flavors induce the production of IL-8 in lung epithelial cells and monocytes. (A) 20,000 16-HBE cells grown in 24-well transwell plates exposed to three-sessions (each session 66 puffs) of JUUL pods and other pod aerosols with equal intervals between sessions. Aerosolized flavors were JUUL pods (Cool Cucumber and Classic Menthol) and other pod flavors (Just Mango -Strawberry Coconut and Caffé Latte). After the last exposure, IL-8 was measured in the conditioned media. *p < 0.05 vs. Control. N = 3 wells per group. (B) Approximately 300,000 U937 cells were cultured in a 12-well plate in complete medium. Cells were serum-deprived (1% FBS) for 24 hours and treated with 0.5% JUUL pods and other pod flavors. Treated flavors included JUUL pods (Cool Cucumber and Classic Menthol) and other pod flavors (Just Mango-Strawberry Coconut and Caffé Latte). Twenty-four hours later, IL-8 levels in the conditioned media were determined. *p < 0.05 vs. Control.

Figure 4

JUUL pods and other pod flavors induce the production of PGE₂ in lung epithelial cells and monocytes. (A) U937 with 300,000 cells per well were treated with 0.5% JUUL pods and other pod flavors in serum-deprived medium. Flavors included JUULpods (Cool Cucumber and Classic Menthol) and other pod flavors (Just Mango-Strawberry Coconut and Caffé Latte). Prostaglandin E₂ was measured in conditioned media. **p < 0.01 vs. control. (N = 2 treated wells per group with repeated experiments). (B) Approximately 325,000 BEAS-2B cells were cultured in 6-well plates and exposed to JUUL pods and other pod flavors in serum-deprived medium (1% FBS). Three sessions of 66-puffs were generated by Scireq Inexpose system. Aerosolized flavors included JUUL pods (Cool Cucumber and Classic Menthol) and other pods (Just Mango - Strawberry Coconut and Caffé Latte). Prostaglandin E₂ was measured in conditioned media. *p < 0.05, **p < 0.01 vs. control. N = 2–6 treated wells per group. (C) 20,000 16-HBE cells cultured in 24-well transwell inserts and exposed to JUUL pods and other pod flavors in serum-deprived medium (1%). Three sessions of 66-puffs were generated by Scireq Inexpose system. Flavors included JUUL pods (Cool Cucumber and Classic Menthol) and other pods (Just Mango-Strawberry Coconut and Caffé Latte). Prostaglandin E₂ was measured in conditioned media. N = 3–12 treated wells per group with repeated experiments.

Figure 5

JUUL pods and other pod flavors produce differential effects on inflammatory mediators and growth factors in lung epithelial cells. (A) BEAS-2B cells were grown and exposed to three sessions (66 puffs per session) of JUUL pods and other pod aerosol with equal intervals between sessions. Aerosolized flavors were JUUL pods (Cool Cucumber and Classic Menthol) and other pods (Just Mango-Strawberry Coconut and Caffé Latte). The air control group was used as the negative control. After the last exposure condition media was measured using Luminex for inflammatory cytokines and growth factors. *p < 0.05, **p < 0.01 vs. control, N = 2–6 wells per group. (B) 16-HBE cells were grown and exposed to three sessions (66 puffs per session) of JUUL pods and other pod aerosols with equal intervals between sessions. Aerosolized flavors were JUUL pods (Cool Cucumber and Classic Menthol) and other pods (Just Mango-Strawberry Coconut and Caffé Latte). The air group was used as the negative control. After the last exposure condition media was measured using Luminex for inflammatory cytokines and growth factors. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control, N = 3–12 treated wells per group.

Figure 6

JUUL pod flavors affects barrier function in epithelial cells. (A) 20,000 16-HBE cells grown in 24-well transwell plates exposed to three-sessions (each 66 puffs) of JUUL pod flavor cool cucumber with equal intervals of 12 hours between sessions. After the final exposure, the resistance was measured by EVOM2 device. ^$$$P < 0.001 vs. H₂O₂ and, **p < 0.01**p < 0.01, and ***p < 0.001 vs. Control. N = 3–12 wells per group. (B) 20,000 16-HBE cells were cultured in 24-well transwell plates. Designated wells were treated H₂O₂ (600 μM) for comparison control. JUUL pod, Crème Brulee, was aerosolized using Scireq Inexpose system. Cells were exposed to 66 puffs of Crème Brulee for 30 minutes each day for three consecutive days. Twenty-four hours post-exposure the voltage was measured by EVOM2 device. ***p < 0.001 vs. Control. N = 3–12/group.

Figure 7

DNA damage by pod flavor liquids. Approximately 300,000 U937 cells cultured in a 12-well plate were treated with 0.5% of selected JUUL pod and other pod flavors, Cool Cucumber, Classic Menthol, Just Mango (Strawberry Coconut), and Caffé Latte. H₂O₂ treatment as a comparison group. After 24 hours, cells were prepared for Comet Assay. Fluorescent images were captured to visualize DNA damage. Calculated percent changes in olive moments were presented compared to the untreated control. N = 6 images per sample.