Actin Filaments-related Toxicity Mechanism Of Microcystin

Objective: The outbreak of cyanobacerial blooms due to eutrophication, it reduces theutilization value of the water and threats to human health by virtue of their ability to produce thecyclic heptapeptides, microcystins. The most toxic and widely distributed MC variants are MC-LRand RR. It has been suggested that the liver is the prime target organ affected using tritium-labledMC, which can induce acute liver bleeding, liver necrosis, and primary liver cancer. So far, theexact mechanism of MC-induced potent tumor promoting activity and hepatotoxicity is not veryclear, especially the role of the cytoskeleton involved. Therefore, this study selected normal humanliver cell line HL7702based on actin filaments to suggest toxic mechanism of MC-LR.Methods:1、In the present study, cell proliferation was detected by MTT on exposure of cellsto different concentrations of MC-LR, and the subsequent exposure concentration was determinedby the results of MTT.2、We further examined the effects of MC-LR on the structure and distribution of actinfilaments using FITC-conjugated phalloidin.3、We examined the mRNA, protein and phosphorylation levels of actin filament proteins andthe effect on the reorganization of actin filaments by real-time PCR and Western blotting.Results:1、After treatment with MC-LR (0,0.001,0.01,0.1,1,10μM) for24h cellproliferation decreased in a concentration-dependent manner. When exposed to1μM or higherconcentration of MC-LR, cell proliferation significantly decreased (P <0.05).2、Treatment with MC-LR did not result in any alterations of cell shape or size. However,more actin filaments accumulated around the nucleus, dense bundles were observed, and a loss oftheir filamentous distribution, which implies that MC-LR induced the reorganization of actinfilaments.3、After treatment with MC-LR for24h the mRNA and protein levels of actin filament familymembers did not change.4、After treatment with MC-LR for24h the levels of p-VASP(Ser239), p-VASP(Ser157) andp-Ezrin(Thr567) significantly increased(P <0.05), but the p-Cofilin remained unchanged.5、Total ERK1/2, JNK and P38remained unchanged, however, phosphorylated ERK1/2, JNKand P38were markedly upregulated by MC-LR treatment in a concentration-dependent manner(P<0.05).6、Pretreatment with P38or ERK1/2inhibitor, hyperphosphorylation of VASP and Ezrin werediminished to normal level and compared to the treatment group it significantly decreased (P <0.05). However, JNK inhibitor has no significant effect. Conclusions:1、Cell proliferation decreased when exposed to MC-LR, which can cause thedisruption and reorganization of the actin filaments.2、MC-LR induced hyperphosphorylation of VASP and Ezrin.3、MC-LR can activate MAPK pathway, and ERK1/2and P38involved in MC-LR-inducedhyperphosphorylation of actin filament proteins.4、MC-LR exerts its potential hepatotoxicity through MAPK pathway activation, which causehyperphosphorylation of actin filament proteins and result in cytoskeletal architecture remodeling.