Microcystin-LR-induced cytotoxicity and apoptosis in human embryonic kidney and human kidney adenocarcinoma cell lines

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Microcystin-LR (MC-LR) is a potent hepatotoxin, and increasing evidence suggests that it might also induce kidney injury. The aim of the present work was to evaluate the cytotoxicity and possible apoptotic effects of MC-LR on a human embryonic kidney cell line (HEK-293) and human kidney adenocarcinoma cell line (ACHN). Cells were exposed for 24 h to pure MC-LR (1 .0 -2 0 0 pM) and the cytotoxic effects were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) and sulphorhodamine B (SRB) cell viability assays. Cell viability in both cell lines was significantly decreased after treatment with M C-LR at 50 pM for 24 h (P < 0 .00 1 ). Moreover, MC-LR-treated ACHN and HEK -293 cells exhibited a marked dosedependent loss of confluence as judged by phase-contrast microscopy. Similarly, fluorescence microscopic observations following acridine orange-ethidium bromide (AO/EB) staining confirmed that both cell types were undergoing apoptosis after treatment with M C-LR for 24 h. Expression of three apoptosis-related genes, B ax, S u rvivin and p53, was analysed by quantitative reverse transcriptase PCR analysis. Both B a x and p 53 functioned as promoters of MC-LRmediated apoptosis in ACHN and HEK -293 cells. The S urvivin gene acted as a suppressor of apoptosis at lower M C-LR concentration (1 pM) and the gene was upregulated at higher MC-LR concentration (10 pM) (P < 0 .00 1 ). Significant increases of caspase 3 (P < 0 .0 0 0 1 ) and caspase 9 (P < 0 ;0 0 0 1 ) activity were detected in both cell lines after exposure to M C-LR for 24 h, indicating the M C-LR induces cytotoxicity and a marked apoptosis in both ACHN and H EK -293 kidney cell lines