Biochemical effects of organophosphorous compounds on cultured rat and human cardiomyocyte-like cells
At present, little is known about the effect(s) of organophosphorous compounds (OPs) on cardiomyocytes. The present study aimed to investigate the effects of phenyl saligenin phosphate (PSP), two organophosphorothioate insecticides (diazinon and chlorpyrifos), and their acutely toxic metabolites (diazoxon and chlorpyrifos oxon) on rat H9c2 and human cardiomyocyte-like cells. The rat embryonic H9c2 myoblast cell line, which has the ability to differentiate into a cardiac muscle phenotype, can be instrumental in understanding OP cytotoxicity at different differentiation stages. Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) were used for the validation of selected OP effects in a more human relevant system. The differentiation of both H9c2 and human cardiomyocytes resulted in increased expression of differentiated muscle markers such as troponin 1, tropomyosin and α-actin. OP-induced cytotoxicity was assessed by monitoring MTT reduction, LDH release, and caspase-3 activity. Cell death was not observed in mitotic or differentiated H9c2 cells with diazinon, diazoxon, or chlorpyrifos oxon (48 h exposure; 200 μM). Chlorpyrifos-induced cell death was only evident at concentrations >100 μM. In marked contrast, PSP displayed pronounced cytotoxicity towards both mitotic and differentiated H9c2 cells. PSP triggered the activation of JNK1/2, suggesting a role for this pro-apoptotic protein kinase in PSP-induced cell death, which was attenuated by the JNK1/2 inhibitor SP 600125, confirming the role of JNK1/2 activation in PSP-induced cytotoxicity. Dansylated PSP was used to identify novel PSP binding proteins. 2D-gel electrophoresis profiles of cells treated with dansylated PSP (25 μM) were used to identify proteins fluorescently labeled with dansylated PSP. Proteomic analysis identified tropomyosin, heat shock protein β-1 and nucleolar protein 58 as novel protein targets for PSP. The present study also examined the effect of sublethal concentrations of OP on differentiating H9c2 cells. This was assessed by monitoring morphological changes, levels of cardiac cytoskeleton protein expression and AChE activity in cells induced to differentiate in the presence and absence of OPs. Results showed that exposure to diazinon and chlorpyrifos induced morphological changes, AChE inhibition and decreases in troponin 1 expression. Morphological changes were observed with PSP treated cells concomitant with altered expression of cardiac cytoskeleton proteins, troponin 1, tropomyosin, α-actin and other novel proteins. When hiPSC-CMs were employed to validate differences in cardiac toxicity induced by OPs, a similar cardiotoxic pattern when compared to differentiated H9c2 cells. In summary, PSP induced cytotoxicity was associated with JNK activation and apoptrosis whereas little cytotoxicity was observed with the other OPs. However PSP, chlorpyrifos and diazinon induced sub-lethal effects in cultured H9c2 and hiPSC-CMs were associated with decrease levels of cardiac cytoskeleton protein expression.
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