Comparison of cellular and molecular cytotoxic mechanisms of Cochlodinium polykrikoides in isolated trout and rat hepatocytes

Abstract

Harmful algal blooms produced by the marine ichthyotoxic dinoflagellate Cochlodinium polykrikoides are responsible for mass mortalities of wild and farmed fish globally. This study compared the cytotoxic mechanisms of C. polykrikoides total extract on both trout and rat liver hepatocytes. Trout hepatocytes were more sensitive than rat hepatocytes against C. polykrikoides extract. The effective concentration 50 after 3 hour incubation (EC50_3hr) concentrations found for C. polykrikoides extract in trout and rat hepatocytes (i.e., 50% membrane lysis in 3 hr) were Eq. 1 cell/ml and Eq. 240 cell/ml, respectively. C. polykrikoides extract exposure in both isolated trout and rat hepatocytes resulted in membrane lysis, reactive oxygen species formation, glutathione depletion, collapse of mitochondrial membrane potential, ATP depletion, increase in adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio, cytochrome c release into the hepatocyte cytosol, and activation of caspases cascade. Trout hepatocyte toxicity was also associated with lysosomal membrane injury. Mitochondrial permeability transition in both trout and rat hepatocytes produced cytochrome c release from the mitochondrial intramembrane space into the cytosol. Thus, the cytochrome c release triggered activation of caspase-3 and apoptosis. Finally, data demonstrated that C. polykrikoides extract may induce more apoptotic phenotype in rat than trout hepatocytes, which in the latter favored predominantly necrotic mode of cell death.

Keywords:

• Cochlodinium polykrikoides
• rat hepatocytes
• trout hepatocytes
• oxidative stress
• apoptosis
• lysosomes

Acknowledgments

The results presented in this article were partly extracted from the thesis of Dr Jafar Shahraki (PhD graduate of Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences) and Dr Iman Barekati (Pharm.D. graduate of International Branch, Shahid Beheshti University of Medical Sciences) who performed their theses under supervision of Prof. Jalal Pourahmad. The investigation was performed in Prof. J. Pourahmad's laboratory in the Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Abbreviations

HAB:	=	harmful algal blooms
MPT pore:	=	mitochondrial permeability transition pore
GSH:	=	glutathione, reduced glutathione
GSSG:	=	glutathione disulfide, oxidized glutathione
ROS:	=	reactive oxygen species
MMP:	=	mitochondrial membrane potential
DCFH-DA:	=	dichlorofluorescein diacetate
DCF:	=	dichlorofluorescein
Caspases:	=	cysteineeaspartic proteases
pNA:	=	p-nitroaniline
AnnCy3:	=	Annexin V-Cy3.18
ELISA:	=	enzyme-linked immunosorbent assay
Apaf-1:	=	apoptotic protease activating factor 1

Additional information

Funding

The work was financially supported by research grant [grant number 1392-1-153-1173] from the Shahid Beheshti University of Medical Sciences, Deputy of Research.