In Vitro High-Throughput Toxicological Assessment of E-Cigarette Flavors in Human Bronchial Epithelial Cells and the role of TRPA1 in Cinnamon Flavor-Induced Toxicity

Abstract Background Electronic cigarettes (ECs) are considered a less hazardous alternative to tobacco smoking but are not harmless. Growing concerns about the safety profiles of flavors in e-liquids underpin the need for this study. Methods Here, we screened 53 nicotine-free flavored e-liquids (across 15 flavor categories) across a 3-point concentration range (0.25%, 0.5%, and 1% v/v) in a high-throughput fashion in human bronchial epithelial (HBEC-3KT) submerged cell cultures to identify ‘toxic hits’ using in vitro endpoint assays comprising cell count, cell viability, and lactate dehydrogenase (LDH). Results We observed significant, dose-dependent adverse effects only with cinnamon, vanilla tobacco, and hazelnut e-liquids compared to media-only control and PG/VG vehicle controls. Hence, we further analyzed these three flavors for their effects on HBEC-3KT proliferation, mitochondrial health, and oxidative stress. A significant decrease in cell proliferation after 36h was observed for each e-liquid toxic hit compared to media-only and PG/VG controls. Hazelnut (at all concentrations) and vanilla tobacco (1%) increased cytoplasmic reactive oxygen species (ROS) generation compared to media-only and PG/VG controls. Conversely, all three flavors at 0.5% and 1% significantly decreased mitochondrial membrane potential (MP) compared to PG/VG and media-only controls. We hypothesized that the cytotoxic effects of cinnamon flavor in e-liquids might be mediated via the transient protein receptor ankyrin subtype 1 (TRPA1); however, TRPA1 antagonist AP-18 (10 µM) did not mitigate these effects, and cinnamon significantly increased TRPA1 transcript levels. Therefore, pathways that mediate cinnamon’s cytotoxicity warrant further investigations. Conclusion This study could inform public health authorities on the relative health risks assessment following exposure to EC flavor ingredients.