Cell-specific toxicity of short-term JUUL aerosol exposure to human bronchial epithelial cells and murine macrophages exposed at the air-liquid interface

Backgroud JUUL, an electronic nicotine delivery system (ENDS), which first appeared on the US market in 2015, controled more than 75% of the US ENDS sales in 2018. JUUL-type devices are currently the most commonly used form of ENDS among youth in the US. In contrast to free-base nicotine contained in cigarettes and other ENDS, JUUL contains high levels of nicotine salt (35 or 59 mg/mL), whose cellular and molecular effects on lung cells are largely unknown. In the present study, we evaluated the in vitro toxicity of JUUL creme brulee-flavored aerosols on 2 types of human bronchial epithelial cell lines (BEAS-2B, H292) and a murine macrophage cell line (RAW 264.7). Methods Human lung epithelial cells and murine macrophages were exposed to JUUL creme brulee-flavored aerosols at the air-liquid interface (ALI) for 1-h followed by a 24-h recovery period. Membrane integrity, cytotoxicity, extracellular release of nitrogen species and reactive oxygen species, cellular morphology and gene expression were assessed. Results Creme brulee-flavored aerosol contained elevated concentrations of benzoic acid (86.9 mu g/puff), a well-established respiratory irritant. In BEAS-2B cells, creme brulee-flavored aerosol decreased cell viability (>= 50%) and increased nitric oxide (NO) production (>= 30%), as well asiNOSgene expression. Creme brulee-flavored aerosol did not affect the viability of either H292 cells or RAW macrophages, but increased the production of reactive oxygen species (ROS) by >= 20% in both cell types. While creme brulee-flavored aerosol did not alter NO levels in H292 cells, RAW macrophages exposed to creme brulee-flavored aerosol displayed decreased NO (>= 50%) and down-regulation of theiNOSgene, possibly due to increased ROS. Additionally, creme brulee-flavored aerosol dysregulated the expression of several genes related to biotransformation, inflammation and airway remodeling, includingCYP1A1,IL-6, andMMP12in all 3 cell lines. Conclusion Our results indicate that creme brulee-flavored aerosol causes cell-specific toxicity to lung cells. This study contributes to providing scientific evidence towards regulation of nicotine salt-based products.