Molecular Toxicology Of Microcystin-LR And Malachite Green In The Primary Hepatocytes Of Nile Tilapia (Oreochromis Niloticus)

In this study,in order to study mechanism of microcystin-LR(MC-LR) and malachite green (MG) toxicity,the isolated hepatocytes of Nile tilapia(Oreochromis niloticus) were exposed to MC-LR and MG.After hepatocytes were exposed to 10,50,150,500μg/L MC-LR for 24 h,the single cell gel electrophoresis assay(SCGE) was used to study the effects on DNA.Induction of DNA strand break in all MC-LR exposed groups was significantly higher than that of the control group.The different doses of MC-LR caused DNA damage in tilapia hepatocytes,and the time-and dose-dependent relationship between MC-LR and DNA migration were observed.Flow cytometry analysis indicates that the percentage of hepatocyte apoptosis increases in a time- and dose-dependent manner.The hepatocytes of Nile tilapia were incubated with MG(0.5,1.0,and 2.0 mg/L) and MC-LR(10μg/L) for 8 and 24 h,respectively.Using beta-actin as external control,RT-PCR within the exponential phase was conducted to determine the mRNA expression levels of cytochrome P4501 A1(CYP1A1),alpha and rho glutathione S-transferase (GSTA and GSTR),uncoupling protein 2(UCP2),glutathione peroxidase(GPX) and heat shock protein 70(HSP70).The significant increases of mRNA expression of these genes were detected at exposure of 0.5 mg/L MG(UCP2),and 1.0 mg/L MG(CYP1A1,GSTA,GSTR and UCP2). However,at exposure of 2.0 mg/L MG,no significant increase of any of these genes was observed.In contrast,the GSTA mRNA expression levels increase after 8 h MC-LR exposure whereas decreased after 24 h MC-LR exposure.The HSP70 mRNA expression level increased after 24 h MC-LR exposure.But no significant changes were found in CYP1A1 and GSTR after 8 h or 24 h MC-LR exposure.These results provided first experimental evidences on the mechanism of MG and MC-LR toxicity at cellular and molecular level,and indicated that the hepatocytes of Nile tilapia seemed to be more responsive to MG than MC-LR,and that CYP1A1, GSTA,GSTR and UCP2 might be involved in the detoxification of MG whereas GSTA might be important for the tolerance of MC-LR in Nile tilapia hepatocytes.