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 / 2 4h) and PM 2.5 (<= 11.6 mu g m-3 / 2 4h) with a maximum particle number mean of 4.4 x 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 decrease in cell viability and gene expression changes was observed. We propose this experimental set-up combining comprehensive aerosol characterization and longterm continuous ALI cell exposure for the identification of hazardous compounds or compound mixtures in ambient air.