Whole cigarette smoke condensates of heated tobacco products disrupt cell adhesion and induce anoikis in human bronchial epithelial cells

Heated tobacco products (HTPs) have emerged as alternatives to conventional cigarettes, with claims of reduced health risks. However, this notion remains controversial due to limited evidence, necessitating further investigation of their toxicological effects. This study examined the cytotoxic effects of HTP aerosols in parallel with cigarette smoke. Whole cigarette smoke condensates (WCSCs) were prepared from three commercially available HTPs (Lil, iQOS, and Glo) and 3R4F reference cigarettes, capturing both gas and particulate phases. Cytotoxicity was assessed in the human bronchial epithelial cell line BEAS-2B treated with 200–1200 µg/mL HTP-WCSCs or 40–240 µg/mL 3R4F-WCSC using four distinct assays including lactate dehydrogenase (LDH) leakage, neutral red uptake, formazan formation, and crystal violet staining assays. HTP-WCSCs exhibited lower overall cytotoxic potency than 3R4F-WCSC when normalized to total particulate matter or nicotine concentration. Notably, the LDH leakage assay consistently yielded lower toxicity estimates, likely due to non-lytic cell detachment. Despite the difference in cytotoxic potential, HTP-WCSCs and 3R4F-WCSC triggered similar cytotoxic responses; focal adhesion disruption, cell detachment, and subsequent anoikis, potentially mediated by Bit1. This detachment-induced cell death was associated with thiol depletion and reactive oxygen species generation. In vivo relevance of these findings was confirmed in the lungs of rats exposed to 30 mg/mL Lil-WCSC or 200 µg/L cigarette smoke via respiratory routes for 2 or 4 weeks, respectively. Despite quantitative differences in cytotoxicity, the mechanisms and patterns were consistent across all products. These results suggest that smoking, irrespective of product type, may compromise respiratory epithelial integrity through the induction of anoikis.