High‐Content Screening Comparison of 1R6F Cigarette Smoke Extract and Selected Heated Tobacco and Electronic Vapour Product Aerosol Extracts in Endothelial and Bronchial Epithelial Cell Models

ABSTRACT Cigarettes induce oxidative stress and DNA damage, processes that contribute to cardiovascular and pulmonary disease. High content screening (HCS) is an increasingly valuable tool for comparative toxicology because it enables simultaneous quantification of multiple mechanistic endpoints at single‐cell resolution. In this study, HCS was used to compare the biological activity of extracts from reference cigarette 1R6F smoke and aerosol extracts from one heated tobacco product and two electronic vapour products in human coronary artery endothelial cells and bronchial epithelial cells. Endpoints included reactive oxygen species generation, glutathione depletion, γH2AX induction as a marker of DNA double‐strand break signalling and NRF2 translocation as an indicator of oxidative stress signalling. 1R6F cigarette smoke extract showed the highest cytotoxic and genotoxic responses, whereas the selected HTP and EVP aerosol extracts generally showed lower potency, with higher minimum effective concentrations across several endpoints under the tested conditions. N ‐acetylcysteine (NAC) attenuated several responses, supporting oxidative stress as an important contributor to the observed cellular effects. Overall, under the tested in vitro conditions, extracts from the selected HTP and EVP products showed lower potency across several oxidative stress‐, genotoxicity‐ and cytotoxicity‐associated endpoints than 1R6F cigarette smoke extract. These findings support product‐specific comparative in vitro bioactivity assessment but do not directly establish reduced human health risk, product safety or clinical harm reduction. The study extends previous HCS work by integrating potency‐based comparisons, mechanistic interrogation using NAC and cross‐tissue evaluation.