Organophosphorus compound-induced apoptosis in SH-SY5Y human neuroblastoma cells.
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.
Carlson
K
K
Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, Virginia, 24061, USA.
Jortner
B S
BS
Ehrich
M
M
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002800:Cholinesterase Inhibitors / Q000633:toxicity*; D053938:DNA Fragmentation; D004587:Electrophoresis, Agar Gel; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007306:Insecticides / Q000633:toxicity; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009336:Necrosis; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000473:pathology; D009943:Organophosphorus Compounds / Q000633:toxicity*; D014407:Tumor Cells, Cultured
D002800:Cholinesterase Inhibitors; D007306:Insecticides; D009943:Organophosphorus Compounds; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases