Jiangsu Center for Drug Screening, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China.
Toxicol In Vitro. 2010 Jun;24(4):1092-7. doi: 10.1016/j.tiv.2010.03.012. Epub 2010 Mar 23.
Aristolochic acid nephropathy, a progressive tubulointerstitial renal disease, is primarily caused by aristolochic acid I (AA-I) intoxication. Aristololactam I (AL-I), the main metabolite of AA-I, may also participate in the processes that lead to renal damage. To investigate the role and mechanism of the AL-I-mediated cytotoxicity, we determined and compared the cytotoxic effects of AA-I and AL-I on cells of the human proximal tubular epithelial (HK-2) cell line. To this end, we treated HK-2 cells with AA-I and AL-I and assessed the cytotoxicity of these agents by using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, flow cytometry, and an assay to determine the activity of caspase 3. The proliferation of HK-2 cells was inhibited in a concentration- and time-dependent manner. Cell-cycle analysis revealed that the cells were arrested in the S-phase. Apoptosis was evidenced by the results of the annexin V/propidium iodide (PI) assay and the occurrence of a sub-G1 peak. In addition, AA-I and AL-I increased caspase 3-like activity in a concentration-dependent manner. These results also suggested that the cytotoxic potency of AL-I is higher than that of AA-I and that the cytotoxic effects of these molecules are mediated through the induction of apoptosis in a caspase 3-dependent pathway.
马兜铃酸肾病是一种进行性的肾小管间质性肾疾病，主要由马兜铃酸 I（AA-I）中毒引起。马兜铃内酰胺 I（AL-I）是 AA-I 的主要代谢物，也可能参与导致肾损伤的过程。为了研究 AL-I 介导的细胞毒性的作用和机制，我们测定并比较了 AA-I 和 AL-I 对人近端肾小管上皮（HK-2）细胞系细胞的细胞毒性。为此，我们用 AA-I 和 AL-I 处理 HK-2 细胞，并通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定法、流式细胞术和测定 caspase 3 活性的测定法评估这些药物的细胞毒性。HK-2 细胞的增殖呈浓度和时间依赖性抑制。细胞周期分析显示细胞在 S 期被阻滞。通过 Annexin V/碘化丙啶（PI）测定和出现亚 G1 峰，证实了细胞凋亡。此外，AA-I 和 AL-I 以浓度依赖的方式增加 caspase 3 样活性。这些结果还表明，AL-I 的细胞毒性效力高于 AA-I，这些分子的细胞毒性作用是通过 caspase 3 依赖性途径诱导细胞凋亡介导的。
