Genotoxicity and Carcinogenicity

DNA damage is a critical event preceding cellular apoptosis or necrosis. This study was carried out to investigate the effect of aristolochic acid I (AAI) on DNA damage and cell cycle in porcine proximal tubular epithelial cell lines (LLC-PK1 cells). LLC-PK1 cells were stimulated with AAI at the concentrations of 80, 320, and 1,280 ng/ml for 24 h. DNA damage was examined by comet assay and the cell cycle was assayed by flow cytometry (FCM), cellular apoptosis and lysis were examined simultaneously. Cellular nuclear changes were observed by electron microscopy and the expression of wild-type p53 protein and mRNA were measured by FCM and RT-PCR. We found that AAI-induced DNA damage prior to apoptosis and lysis in LLC-PK1 cells in a dose-dependent manner (P<0.01). The percentage of cells in the G2/M phase that were treated with AAI (320 and 1,280 ng/ml) for 24 h increased significantly (P<0.01). Electron micrographs showed the nuclear abnormalities in AAI-treated cells. The expression of p53 protein and mRNA did not change in the AAItreated cells. AAI may cause DNA damage and cell cycle arrest in LLC-PK1 cells through a wild-type p53independent pathway, prior to apoptosis or necrosis. This study on the molecular mechanism of AAI-induced toxicity may explain why tubular epithelial cells present limited proliferation and regeneration abilities in the clinical presentation of AAI-associated nephrotoxicity.