Toxicological effects of long-term continuous exposure to ambient air on human bronchial epithelial Calu-3 cells exposed at the air-liquid interface

Air pollution significantly contributes to the global burden of respiratory and cardiovascular diseases. While single source/compound studies dominate current research, long-term, multi-pollutant studies are crucial to understanding the health impacts of environmental aerosols. Our study aimed to use the first air-liquid interface (ALI) aerosol exposure system adapted for long-term in vitro exposures for ambient air in vitro exposure. The automated exposure system was adapted to enable long-term cell exposure. ALI human bronchial epithelial cells (Calu-3) were continuously exposed for 72 h to the ambient air from a European urban area (3 independent exposures). Experimental evaluation included comprehensive toxicological assessments coupled to physical- and chemical-characterization of the aerosol. Exposure to ambient air resulted in increased significant cytotoxicity and a non-significant decrease in cell viability. Differential gene expressions were indicated for genes related to inflammation (IL1B, IL6) and to xenobiotic metabolism (CYP1A1, CYP1B1) with possible correlations to the PM_2.5 content. Common air pollutants were identified such as the carcinogenic benz[a]pyrene (≤ 3.4 ng m^-3 / 24h) and PM_2.5 (≤ 11.6 μg m^-3 / 24h) with a maximum particle number mean of 4.4 × 10^-3 m³/ 24h. For the first time, ALI human lung epithelial cells were exposed for 72 h to continuous airflow of ambient air. Despite direct exposure to ambient aerosols, only small decreased in cell viability and gene expression changes was observed. We propose this experimental set-up combining comprehensive aerosol characterization and long-term continuous ALI cell exposure for the identification of hazardous compounds or compound mixtures in ambient air.