Linking environmental concentrations to cytotoxic mechanisms in second-hand vapor exposure of electronic cigarette via an integrated approach for in vitro toxicity assessment.

Aerosols emitted from electronic cigarette (E-cig) and tobacco cigarette (T-cig) can remain suspended as second-hand vapor (SHV) and second-hand smoke (SHS), respectively, posing potential health hazards to bystanders. However, traditional in vitro toxicity studies are often based on inhalation dose for users rather than the lung deposition dose for bystanders, which limits the accuracy of SHV exposure toxicity assessment and understanding of the related health effects. To address these deficiencies, this study developed an approach that integrates three key steps: obtaining characteristics of SHV aerosols; calculating the inhaled deposition dose; and extrapolating the exposure concentration in the air-liquid interface (A-LI) system. This approach facilitates the determination of in vitro exposure doses based on estimated environmental concentration data. The actual exposure doses that maintained cell viability above 70% were 1 × 104 particles/cm3 for SHS and 1 × 105 particles/cm3 for SHV in the A-LI system, respectively. While the cytotoxicity induced by SHV was weaker than SHS exposure, the SHV exposure still caused cell damage and dysfunction. Gene enrichment analysis revealed that SHV and SHS exposure elicited distinct cellular regulatory pathways. For E-cig exposure, the MMP9 and FOS genes were dysregulated, which may be related to cellular inflammatory response. Furthermore, to validate transcriptomic results, the expression levels of the mentioned genes were confirmed by quantitative real-time PCR (Pearson correlation coefficient of 0.89). This study may provide an approach with comparable and traceable dose levels to assess inhalation toxicology of second-hand aerosol exposure for bystanders, and also offers the important scientific basis for E-cig risk assessment.