High-throughput toxicological assessment of heated tobacco product aerosols using RNA sequencing-based...

Heated tobacco products (HTPs) operate without combustion and are designed to reduce exposure to harmful constituents compared to cigarettes. Within the HTP category, rapid consumer-led technological development has resulted in a growing diversity of heating approaches across products, including the incorporation of multiple heating modes within individual devices. In this study, to gain deeper insight into the molecular mechanisms underlying biological responses to HTP aerosols, high-throughput transcriptomics was used to evaluate acute transcriptomic responses in human bronchial epithelial cells exposed to the extract of aerosols from multiple HTPs across all operable heating modes. Eleven emission constituents, including nine cigarette smoke constituents proposed for mandated reduction by the World Health Organization and combustion markers defined by British Standards Institution, were analyzed. HTP aerosols contained fewer detectable constituents and lower levels of measurable analytes than smoke from the reference cigarette (1R6F), confirming reduced combustion-related toxicants and non-combustion operation. Consistent with these chemical differences, HTP aerosols elicited weaker cytotoxic and transcriptomic responses than 1R6F smoke at comparable concentrations. While concentration-response modeling identified comparable transcriptomic perturbation patterns between HTP aerosols and 1R6F smoke, transcriptomic amplitudes were uniformly lower in response to HTP aerosols and no product-specific pathways were detected under the tested conditions. Transcriptomic points of departure (PoD) demonstrated greater sensitivity than cytotoxicity-based PoD, supporting their utility for evaluating the potential toxicological effects of HTPs at lower exposure levels than those identified by conventional cytotoxicity endpoints.