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Organophosphorus compound-induced apoptosis in SH-SY5Y human neuroblastoma cells
K Carlson1, B S Jortner, M Ehrich
1Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, Virginia, 24061, USA.
Abstract:
Organophosphorus (OP) compounds have been shown to be cytotoxic to SH-SY5Y human neuroblastoma cell cultures. The mechanisms involved in OP compound-induced cell death (apoptosis versus necrosis) were assessed morphologically by looking at nuclear fragmentation and budding using the fluorescent stain Hoechst 33342 (10 microgram/ml). Hoechst staining revealed significant paraoxon (1 mM), parathion (1 mM), phenyl saligenin phosphate (PSP, 10 and 100 microM), tri-ortho-tolyl phosphate (TOTP, 100 microM and 1 mM), and triphenyl phosphite (TPPi, 1 mM) induced time-dependent increases in traditional apoptosis (p < 0.05). In many cells, PSP and TOTP (1 mM) also induced nuclear condensation with little fragmentation or budding. Pretreatment with cyclosporin A (500 nM, 30 h) decreased apoptosis following 1 mM parathion and TOTP exposures. Apoptotic nuclear changes were verified by DNA gel electrophoresis. Activation of caspase-3, a cysteine aspartate protease, was also monitored. OP compounds induced significant time-dependent increases in caspase-3 activation following paraoxon (1 mM), parathion (100 microM, 1 mM), PSP (10 microM, 100 microM, 1 mM), TOTP (100 microM, 1 mM), and TPPi (1 mM) exposure (p < 0.05). Pretreatment with cyclosporin A (500 nM, 30 h) significantly decreased caspase-3 activation during extended incubations with paraoxon, parathion, and TPPi (p < 0.05). In addition, pretreatment with the caspase-3 inhibitor Ac-DEVD-CHO and the caspase-8 inhibitor Ac-IETD-CHO (25 microM, 8 h) significantly decreased caspase-3 activation following exposure to 1 mM PSP and parathion (p < 0.05). Pretreatment with the serine protease inhibitor phenylmethyl sulfonyl fluoride (PMSF; 1 mM, 8 h) also significantly decreased caspase activation following 1 mM PSP and TOTP exposures (p < 0.05). Alteration of OP compound-induced nuclear fragmentation or caspase-3 activation by pretreatment with cyclosporin A, Ac-IETD-CHO, or PMSF suggested that OP compound-induced cytotoxicity may be modulated through multiple sites, including mitochondrial permeability pores, receptor-mediated caspase pathways, or serine proteases.
Insights
Organophosphorus compounds induce apoptosis and caspase-3 activation in neuroblastoma cells. Inhibitors suggest multiple pathways, including mitochondrial and serine protease involvement, modulate this cytotoxicity.
Area of Science:
- Neuroscience
- Toxicology
- Cell Biology
Background:
- Organophosphorus (OP) compounds are known neurotoxicants.
- Their cytotoxic mechanisms in neuronal cells require further elucidation.
Purpose of the Study:
- To investigate the mechanisms of cell death induced by OP compounds in SH-SY5Y human neuroblastoma cells.
- To determine if OP-induced cytotoxicity involves apoptosis and the activation of caspase-3.
Main Methods:
- SH-SY5Y cells were exposed to various OP compounds (paraoxon, parathion, PSP, TOTP, TPPi).
- Morphological assessment of apoptosis was performed using Hoechst 33342 staining and DNA gel electrophoresis.
- Caspase-3 activation was measured.
- Cells were pretreated with cyclosporin A, Ac-DEVD-CHO, Ac-IETD-CHO, or PMSF to assess the involvement of specific pathways.
Main Results:
- OP compounds induced significant time-dependent increases in apoptosis and caspase-3 activation.
- Cyclosporin A reduced apoptosis and caspase-3 activation in response to certain OP compounds.
- Inhibitors of caspase-3, caspase-8, and serine proteases partially blocked OP-induced caspase-3 activation and nuclear fragmentation.
Conclusions:
- OP compounds induce apoptosis and activate caspase-3 in SH-SY5Y cells.
- The cytotoxicity of OP compounds may be modulated through multiple cellular pathways, including mitochondrial permeability, receptor-mediated caspase activation, and serine proteases.