Differential cytotoxic effects of denitroaristolochic acid II and aristolochic acids on renal epithelial cells

Aristolochic acids (AAs), naturally occurring nephrotoxins and rodent carcinogens, are commonly found in medicinal plants such as Radix aristolochiae. The toxicity of AAs is believed to be associated with the formation of promutagenic AA-DNA adducts, and it has also been suggested that the nitro group in AAs might be important in the process. In order to investigate the role of the nitro group in AA-mediated cytotoxicity, the effects of denitroaristolochic acid II (dN-AAII), aristolochic acid II (AAII) and aristolochic acid I (AAI) on renal tubular epithelial Madin–Darby canine kidney (MDCK) cells were examined and compared. The cytotoxicity of AAI, AAII and dN-AAII was found to be time- and concentration-dependent. As determined by MTT assay, AAI was found to be most toxic in MDCK cells upon exposure for 24, 48 and 72 h, followed by AAII, and dN-AAII showed the least cytotoxicity. The effect of AAI and AAII on the integrity of cell membrane was found to be similar and appeared to be more prominent than that of dN-AAII. Based on the results obtained from the flow cytometric analysis, significant apoptosis in MDCK cells was observed with AAI and AAII at as low as 25 μmol/L following exposure for 24 h, whereas significant apoptosis was induced by dN-AAII at a much higher concentration, 300 μmol/L, suggesting that both AAI and AAII were significantly more cytotoxic than dN-AAII. In addition, the levels of reactive oxygen species (ROS) were increased following treatment with AAI, AAII and dN-AAII at concentrations of 5, 25 and 25 μmol/L, respectively, for 4 h. The results suggest that the nitro group plays an important role in AA-mediated cytotoxicity in MDCK cells and increased intracellular ROS levels may be associated, at least in part, with the cell injury observed in MDCK cells.