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

Abstract

Attached

Microcystin-LR (M C -L R ) 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 M C -L R on a human embryonic kidney cell line (H E K -2 9 3 ) and human kidney adenocarcinoma cell line (A C H N ). Cells were exposed for 24 h to pure M C -L R (1 .0 -2 0 0 pM ) and the cytotoxic effects were evaluated by 3-(4,5-dim ethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (M T T ) and sulphorhodamine B (S R B ) cell viability assays. Cell viability in both cell lines was significantly decreased after treatment with M C -L R at 50 pM for 24 h (P c O .0 0 1 ). Moreover, M C-LR-treated A C H N and H E K -2 9 3 cells exhibited a marked dosedependent loss of confluence as judged by phase-contrast microscopy. Similarly, fluorescence microscopic observations following acridine orange-rethidium bromide (A O / E B ) staining confirmed that both cell types were undergoing apoptosis after treatment with M C -L R for 24 H. Expression of three apoptosis-related genes, Bax, Survivinand p53, was analysed by quantitative reverse transcriptase P C R analysis. Both Bax and p 5 3 functioned as promoters of M C -L R - mediated apoptosis in A C H N and H E K -2 9 3 cells. The Survivin gene acted as a suppressor of apoptosis at lower M C -L R concentration (1 jiM ) and the gene was upregulated at higher M C -L R concentration (1 0 pM ) (P < 0 .0 0 1 ). Significant increases of caspase 3 (P < 0 .0 0 0 1 ) and caspase 9 ( P < 0.0001) activity were detected in both call lines after exposure to M C -L R for 24 h