Abstract / Summary
Electronic cigarettes (ECs) are increasingly marketed as safer alternatives to conventional cigarettes, yet mounting evidence highlights that they are not without harm. In vitro air–liquid interface (ALI) models provide physiologically relevant systems to examine how EC aerosols affect lung epithelial cells. Previous studies have typically focussed on bronchial epithelial cells, leaving the alveolar region unexplored. In this study, we use the EC Aerosol Machine–Cell Chamber (ECAM-cell chamber) to expose NCI-H441 cells, a human alveolar type II-like cell line, cultured at the ALI to aerosols generated by a Geekvape L200 Aegis Legend EC device (50 W, 20 mL/min, 60 puffs). The exposure configurations consisted of distinct treatment formulations evaluating a 50:50 propylene glycol/vegetable glycerol (PG/VG) vehicle supplemented with combinations of free-base nicotine, nicotine salicylate salts, and tobacco flavour. Following a 24 h post-exposure incubation period, barrier integrity (transepithelial electrical resistance, TEER), cytotoxicity (lactate dehydrogenase, LDH), inflammation (interleukin-8 (IL-8) secretion by ELISA), and gene expression were evaluated compared to control-air. The PG/VG carrier solvents alone significantly reduced TEER and increased LDH and IL-8 secretion compared to air controls ( p < 0.001), indicating that these base matrices are not biologically inert. Co-exposure with freebase nicotine did not increase IL-8 relative to control-air baseline, while upregulated genes associated with water/ion transport and epithelial signalling, including AQP3 , CHRNA5 , SCNN1A , ATP1A1 , and ACE2 . In contrast, tobacco flavour and nicotine salts were associated with reduced TEER, elevated LDH and IL-8 release, and downregulation of these genes. These findings demonstrate that EC aerosols differentially affect ATII-like cells depending on nicotine formulation and flavouring. While free-base nicotine co-exposure limits the heavy cytokine release triggered by vehicle carrier solvent (PG/VG) and upregulates transport transcripts, nicotine salts and flavour additives were associated with greater physical epithelial injury and elevated inflammatory marker release.