A search for cellular and molecular cytotoxic mechanisms of Cochlodinium Polykricoides in isolated rat and fish hepatocytes
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AbstractIn this research, we investigated and compared the cytotoxic mechanisms of aqueous extract of C.polykricoides responsible for a severe and widespread HAB in the Persian Gulf and Gulf of Oman (2008-2009) in both isolated rat and trout liver hepatocytes. In addition, the role of oxidative stress and mitochondria in the induction of apoptosis were also investigated. Isolated hepatocytes were obtained by collagenase perfusion of the rat liver. To determine the hepatocyte “ROS” generation, dichlorofluorescin diacetate was used as the reagent. The uptake of the cationic fluorescent dye, rhodamine 123, has been used for the determination of hepatocytes mitochondrial membrane potential. Redistribution of lysosomotropic probe, acridine orange from lysosomes into cytosol was used for determination of lysosomal membrane damage. GSH and GSSG were determined using spectrofluorometric method. Caspase-3 activity and apoptosis phenotype were also determined using ‘‘Sigma’s caspase-3 assay kit and Sigma–Aldrich apoptosis detection kit, respectively. Incubation of algal extract with isolated rat hepatocytes caused hepatocyte membrane lysis, reactive oxygen species formation (ROS), glutathione depletion, collapse of mitochondrial membrane potential, ATP depletion and increase in ADP/ATP ratio, cytochrome c release in to the hepatocyte cytosol, activation of caspases cascade and appearance of apoptosis phenotype. antioxidants (α-tocopherol succinate and BHT), hydroxyl radical scavenger (mannitol and DMSO), Mitochondrial permeability transition (MPT) pore sealing agents (cyclosporine A, carnitine and trifluoperazine), NADPH P450 reductase inhibitor (Diphenyliodonium chloride), CYP2E1 inhibitors (Phenylimidazole and 4-Methylpyrazole) and ATP generators (L-glutamine, Fructose and Xylitol) inhibited the activation of caspase-3 and cell death. Our data showed, that algal extract activate apoptosis signaling via oxidative stress and mitochondrial pathway. ROS formation could directly be involved in mitochondrial MPT pore opening and activation of caspases cascade leading to toxic effect of C.polykricoides extract on both rat and trout hepatocytes. These findings contribute to a better understanding of C.polykricoides-toxic effects on mammalian and aquatic liver cells. Our findings revealed that trout hepatocytes are much more sensitive ( more than two hundred folds) to toxic effects of C.polykricoides extract than rat hepatocytes. On the other hand the algal extract induced lysosomal membrane damage only in trout but not rat hepatocytes.
Publisher or UniversityIranian Fisheries Science Research Institute
Series : Nr40839;