Heated cigarette smoke extract induces oxidative stress and remodels lipid signatures of the extracellular vesicle released by bronchial epithelial cells

Heated tobacco products (HTPs) are increasingly used as alternatives to conventional cigarettes, but their biological effects remain debated. Here, we investigated the impact of heated cigarette smoke extract (hCSE) on human bronchial epithelial BEAS-2B cells and their released extracellular vesicles (EVs), comparing the results with those previously reported for burned CSE (bCSE). Exposure to hCSE reduces in a dose-dependent manner the cell viability and induces oxidative stress, as evidenced by the accumulation of carbonylated proteins and the upregulation of antioxidant enzymes, although at higher doses than bCSE. Cell exposure to hCSE concentration capable of inducing oxidative stress without markedly reducing cell viability, triggered the release of EVs carrying elevated levels of carbonylated proteins. This suggests that EVs may partially mediate the removal of oxidized proteins as a cellular defence mechanism. Lipid analysis revealed that hCSE modified phospholipid composition of EVs, showing increasing levels of lipid species containing polyunsaturated fatty acids. High levels of saturated lipid species have been previously observed in EVs released by bCSE-treated cells. Notably, these distinct lipid signatures could, if validated by further investigations, become a useful marker for distinguishing the effects induced by different types of smoke. In summary, these findings indicate that hCSE can exert harmful effects, and support the role of EVs as potential markers of smoke-related cellular damage associated with different types of smoke. Results also suggest further studies to assess whether the lipidomic changes induced by bCSE and hCSE have functional consequences on the biological activity of EVs, and whether the oxidative alterations of EVs may contribute to the propagation of hCSE-induced cell damage.