Reactive Oxygen Species, Mitochondrial Membrane Potential, and Cellular Membrane Potential Are Predictors of E-Liquid Induced Cellular Toxicity

INTRODUCTION: The use of flavors in electronic cigarettes appeals to adults and never-smoking youth. Consumption has rapidly increased over the last decade, and in the U.S. market alone, there are over 8000 unique flavors. The U.S. Food and Drug Administration (FDA) has begun to regulate e-liquids, but many have not been tested, and their impact, both at the cellular level, and on human health remains unclear. METHODS: We tested e-liquids on the human cell line HEK293T and measured toxicity, mitochondrial membrane potential (ΔΨ (m)), reactive oxygen species production (ROS), and cellular membrane potential (V(m)) using high-throughput screening (HTS) approaches. Our HTS efforts included single-dose and 16-point dose–response curves, which allowed testing of ≥90 commercially available e-liquids in parallel to provide a rapid assessment of cellular effects as a proof of concept for a fast, preliminary toxicity method. We also investigated the chemical composition of the flavors via gas chromatography–mass spectrometry. RESULTS: We found that e-liquids caused a decrease in ΔΨ (m) and V(m) and an increase in ROS production and toxicity in a dose-dependent fashion. In addition, the presence of five specific chemical components: vanillin, benzyl alcohol, acetoin, cinnamaldehyde, and methyl-cyclopentenolone, but not nicotine, were linked with the changes observed in the cellular traits studied. CONCLUSION: Our data suggest that ΔΨ (m), ROS, V(m), and toxicity may be indicative of the extent of cell death upon e-liquid exposure. Further research on the effect of flavors should be prioritized to help policy makers such as the FDA to regulate e-liquid composition. IMPLICATIONS: E-liquid cellular toxicity can be predicted using parameters amenable to HTS. Our data suggest that ΔΨ (m), ROS, V(m), and toxicity may be indicative of the extent of cell death upon e-liquid exposure, and this toxicity is linked to the chemical composition, that is, flavoring components. Further research on the effect of flavors should be prioritized to help policy makers such as the FDA to regulate e-liquid composition.