Comparison of different cytotoxicity assays to assess cigarette smoke and alternative tobacco products toxicity

Electronic Nicotine Delivery Systems (ENDS), i.e., electronic cigarettes (e-cigs) and Tobacco Heating Products (THPs), are rapidly growing in popularity. The marketing of these products is regulated by specific rules in the European Union and in the US, which permit their legal sales. Nonetheless, comprehensive quality and safety requirements for regulatory purposes are still under development. Cytotoxicity studies are an important initial step in appraising the potential toxicity of ENDS. The aim of the present study was to screen a battery of different in vitro cytotoxicity methods for the assessment of toxicity induced by ENDS. We evaluated different cytotoxicity assays, including neutral red uptake (NRU), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Annexin V apoptosis, High Content Screening (HCS) assays and Real Time Cell Analysis (RTCA), to compare two e-cigs (Vype ePen 3 and Vype eStick Maxx) and two THPs (IQOS and GLO™) with the 1R6F reference tobacco cigarette. Human bronchial epithelial cells (H292) were exposed to 1R6F smoke (5 puffs by HCI regime), ePen vapor (10 puffs by modified HCI regime), eStick vapor (25 puffs by CRM81 regime), IQOS vapor (7 puffs by HCI regime) and GLO vapor (8 puffs by HCI regime) at air-liquid interface. All tests showed reduced cell viability following 1R6F smoke exposure and slight or no reduction with ENDS at 24 hours compared to controls. In addition, Annexin V and RTCA exhibited a further significant reduction in cell viability following 1R6F exposure compared with other assays. Furthermore, Annexin V allowed to discriminate viable cells from those in early/late apoptosis. Finally, RTCA and HCS being time-resolved analyses allowed also to determine the kinetic dependency parameter for toxicity of smoke/vapor chemicals on cell viability. In conclusion, NRU assay may be considered a suitable test, especially when combined with a time-resolved test, for assessing the kinetic of cytotoxicity induced by these products.