Utilization of the CometChip assay for detecting PAH-induced DNA bulky adducts in a 3D primary human bronchial...

Polycyclic aromatic hydrocarbons (PAHs), which are formed during incomplete combustion of organic materials, may cause cancer through DNA damage mediated by formation of bulky DNA adducts from PAH reactive metabolites. The airway epithelium is a primary route of exposure for inhaled PAHs, and primary human bronchial epithelial cells (HBECs) in monolayer or organotypic cultures offer a more realistic testing scenario compared to traditional cell lines. However, lack of knowledge about their capacity to mediate DNA damage through generation of reactive chemical intermediates limits their use in quantitative studies for toxicity assessment or predictive modeling compared to in vivo studies. In this study, we explored the capacity of monolayer HBECs to generate DNA damage from metabolic activation of benzo[a]pyrene (BAP, 0.001 - 1 µg/mL, 24 h) using the high-throughput CometChip assay in comparison to HepG2 and MEF cells, as positive and negative metabolic controls, respectively. The CometChip assay was further adapted to evaluate DNA damage in HBECs cultured at the air-liquid interface (ALI) exposed to BAP (0.04-1.14 µg/cm², 24 h). Monolayer and ALI-HBECs displayed a statistically significant increase in DNA damage from BAP exposure with repair trapping agents in a dose-dependent manner similar to the response from HepG2 cells. Monolayer HBECs also showed a greater sensitivity to DNA damage compared to ALI-HBECs, which correlated with induction of CYP1A1 activity at similar exposure conditions. Results from the CometChip assay were also observed at lower BAP concentrations compared to CYP1A1 activity, cytotoxicity, or barrier integrity disruption demonstrating the sensitivity of the CometChip assay.