Human bronchial
epithelial cell line (16HBE14o) |
2,5-dimethylpyrazine (chocolate, nutty flavor) Damascenone (apple, citrus, wine-like) Linalool (floral, spice) α-ionone (fruity, raspberry) Ethyl maltol (caramel) Furaneol (strawberry, sweet) Vanillin (vanilla) |
24-h direct exposure (flavoring chemical) |
No nicotine |
N/A |
2,5-Dimethylpyrazine reduces the physiological response to signaling molecules important in aspects of airway epithelial cell innate immunity. 2,5-Dimethylpyrazine increases airway epithelial ion conductance. 2,5-Dimethylpyrazine activates apical ion efflux via cystic fibrosis transmembrane conductance regulator (CFTR). Vanillin and 2,5-Dimethylpyrazine significantly altered airway epithelial cellular physiology indicative of cellular signaling events. Linalool, α-ionone, damascenone, ethyl maltol, and furaneol, no significant effects? |
(Sherwood and Boitano, 2016) |
| Immortalized human bronchial epithelial cells (16-HBE, BEAS-2B) |
Cool Cucumber Mango Strawberry coconut Classic Menthol Caffe Latte |
66 puffs during 22 minutes with a three-second puff duration at 1.6 L/min flow rate and an inter puff interval of approximately 17s. |
50 mg/mL nicotine |
No nicotine control |
Cool cucumber, classic menthol, caffe latte induced pro-inflammatory biomarkers (IL-1ß, IFN-γ, IL-17, etc.) depending on the cell line as 16-HBE and BEAS-2B present specific physiological differences. It is unclear whether these effects are in part influenced by nicotine content due to the lack of nicotine control samples. Classic Menthol induced the most significant mitochondrial ROS production in 16-HBE. Prostaglandin production was induced in BEAS-2B, not in 16-HBE, by strawberry coconut. IL-8 production was induced in 16-HBE, but not in BEAS-2B, by cool cucumber, classic menthol, strawberry coconut, and caffe latte. |
(Muthumalage et al. 2019) |
| Normal human bronchial epithelial (NHBE) cells |
|
24-h chemical exposure after media dilution in an ALI model |
No nicotine |
N/A |
Cytoskeletal and cilia processes among common genes (142 genes) were perturbed by both diacetyl and 2,3-pentanedione. The expression of multiple genes involved in cilia biogenesis was significantly downregulated by diacetyl and 2,3-pentanedione. The number of ciliated cells significantly decreased. |
(Park et al., 2019) |
| BEAS-2B and human pulmonary fibroblast (HPF) |
|
48-h chemical (diluted liquid) direct exposure |
No nicotine |
N/A |
Both flavor chemicals were highly cytotoxic following a MTT assay at concentrations 30 (menthol) and 100 times (ethyl maltol) lower than the highest concentrations in the refill fluids. BEAS-2B cells (IC50 = 0.15 mg/ml) were more sensitive to ethyl maltol than hPF (IC50 = 0.28 mg/ml). No difference in sensitivity to menthol between HPF and BEAS-2B. |
(Omaiye et al. 2019) |
| BEAS-2B |
|
30 min x 3 (Each one after 12-h) aerosol exposure |
5% nicotine
(Specific mg/ml of nicotine not specified) |
No nicotine control |
Menthol- mitochondrial effects: proton leakage, a decrease in coupling efficiency, and a reduction of complex I, II, and IV 24-h post-menthol exposure reduction of basal respiration, maximal respiration, spare capacity, and complex I. Tobacco had no significant effects on mitochondrial respiration, but immediately post final exposure increased complex I, IV, and V. Whether this effect is synergistic with nicotine was unclear. |
(Lamb, Muthumalage, and Rahman 2020) |
| Human pulmonary fibroblasts (HPF) |
|
48-h e-liquid direct exposure to 1% and 0.3% doses. |
Different concentrations of nicotine, but not specified. |
No nicotine control |
Nicotine concentration did not correlate with cytotoxicity in MTT assays. Cinnamon Ceylon cytotoxicity effect following MTT assay was dose-dependent. Cinnamaldehyde and 2-methoxycinnamaldehyde (2-MOCA) were the major components of cinnamon flavors. Cinnamaldehyde and 2-MOCA standards were highly cytotoxic in MTT assay. |
(Behar et al. 2014b) |
| Human lung fibroblasts (HPF) |
Bubblegum Butterscotch Caramel Butterfinger Menthol Artic Wisconsin frost Domestic JC Original French vanilla Vanilla Tahiti Tennessee cured Island Valencia Mint chocolate Swiss Dark Caramel Espresso Mercado Simply strawberry Arctic Menthol Summer Peach Black Cherry Chocolate truffle Cinnamon Ceylon |
48-h direct e-liquid exposure to 0.001%, 0.01%, 0.03%, 0.1%, 0.3%, and 1% v/v. |
0 to 24 mg/ml |
No nicotine alone control |
Most significant cytotoxicity following MTT assay was observed with cinnamon and menthol flavors which did not contain nicotine. Levels of nicotine were not correlated with the degree of MTT cytotoxicity. The humectants vegetable glycerin (VG) and propylene glycerin (PG) were non-cytotoxic for the hPF. |
(Bahl et al. 2012) |
| Human airway epithelial cells (H292) & human lung fibroblast cells (HFL-1 cell line) |
Tobacco Cinnamon roll Grape |
30 s interval with a 4 s pulse for different time durations 5, 10, and 15 minutes at ALI |
0, 6, 16, 18 and 24 mg |
No nicotine alone control present |
Tobacco and cinnamon roll-flavored e-liquid increased the secretion of inflammatory cytokines IL-6 and IL-8 in H292 and HFL-1. The addition of nicotine gave a striking, dose-dependent increase only in IL-8 secretion in HFL-1. |
(Lerner et al. 2015) |
| Normal human bronchial epithelial (NHBE) cells |
|
24-h direct e-liquid exposure |
Different dilutions (0.03125%, 0.0625%, 0.125%, 0.25%, 0.5%, 1%, 2% & 3% of 24 mg/ml e-liquids. |
No Nicotine control |
Only menthol elicited dose-dependent cytotoxic effects on activating G0/G1cycle arrest, decreasing glutathione concentration, activation of caspase 3/7 activity, increasing nuclear factor-kB (NF-kB), & increased mitochondrial mass. Tobacco, vanilla, and menthol decreased mitochondrial membrane potential dose-dependently. Glutathione content decreased after exposure to tobacco, blueberry, and vanilla, but only above 1%. No nicotine-free e-liquids were tested. Therefore, it is unclear whether these effects are due to flavors alone or a combination of flavor plus nicotine. Base liquids (PG/VG), with or without nicotine, and commercial, flavored, nicotine-containing e-liquids (CFs), had little or no effect on cell viability, cell count, mitochondrial mass, and potential and oxidative stress. |
(Czekala et al. 2019) |
| Pulmonary fibroblasts (HFL-1) |
|
24-h direct e-liquid exposure 0.1%, 0.25%, 0.5% & 1%. |
3 mg/ml
(No nicotine-free group included) |
Nicotine alone control present at 0.25% and 0.5%. |
A dose-dependent decrease in cell viability using the mixture of e-liquids. An increase in IL-8 release induced only by 0.25% flavor mix. Elevated senescence-associated beta-galactosidase (SA-β-gal) activity in a dose-response fashion. Transforming growth factor-β1 (TGF-β1) induced myofibroblast differentiation was inhibited by flavor mix, decreasing α-smooth muscle actin and fibronectin protein levels at only one concentration 0.5%. No significant nicotine effect was observed. |
(Lucas et al. 2020) |
| Human bronchial epithelial cell lines (BEAS-2B, H292) |
|
5s duration every 30s for 1-h to aerosol at ALI |
50 mg/mL |
No nicotine control |
Aerosol decreased cell viability (≥50%) of BEAS-2B cells alone and increased nitric oxide (NO) production (≥30%), as well as iNOS gene expression. In H292 cells, aerosol increased the production of reactive oxygen species (ROS).
Aerosol dysregulated expression of several genes related to biotransformation, inflammation, and airway remodeling, including CYP1A1, IL-6, and MMP12 in both cell lines. Because nicotine-free e-liquids were not investigated, it is unclear whether nicotine has any synergistic effects with Crème brûlée-flavor.
|
(Pinkston et al. 2020) |
| BEAS-2B, Human cystic fibrosis cell line (IB3-1), and Calu3 |
|
24-h exposure to condensates (50%, 25%, and 12.5% diluted with media) |
0, 6, 12 and 18 mg/mL |
No nicotine alone control |
Cherry- similarly cytotoxic to the C38 and BEAS-2B In BEAS-2B, tobacco- also cytotoxic following cell viability at all dilutions tested. Strawberry reduced cell viability in BEAS-2B cells was significantly greater than apple, cherry, and tobacco CALU-3 showed no cytotoxic response to any flavor. There was no nicotine concentration-dependent effect. |
(Leslie et al. 2017) |
| Human pulmonary fibroblasts (HPF), alveolar epithelial cells (A549) |
Vanilla Mint/Menthol Butterscotch Caramel Butterfinger |
E-fluid (0.001%, 0.01%, 0.03%, 0.1%, 0.3%, 1.0%) and aerosol exposures. For each batch of aerosol, 24 puffs were collected into 4 mL of culture medium |
18 mg/ml |
No nicotine control |
Butterfinger (creamy/buttery) and caramel aerosol were the most toxic in a dose-dependent fashion of all flavors following MTT assays. It is unclear whether the effects observed are due to nicotine plus as there are no nicotine controls.
|
(Behar et al., 2018) |
