Carlson K, Jortner B S, Ehrich M
Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, Virginia, 24061, USA.
Toxicol Appl Pharmacol. 2000 Oct 15;168(2):102-13. doi: 10.1006/taap.2000.8997.
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.
有机磷（OP）化合物已被证明对SH-SY5Y人神经母细胞瘤细胞培养物具有细胞毒性。通过使用荧光染料Hoechst 33342（10微克/毫升）观察核碎裂和出芽情况，从形态学角度评估了OP化合物诱导细胞死亡（凋亡与坏死）的机制。Hoechst染色显示，对氧磷（1毫摩尔）、对硫磷（1毫摩尔）、苯基水杨苷磷酸酯（PSP，10和100微摩尔）、磷酸三邻甲苯酯（TOTP，100微摩尔和1毫摩尔）以及亚磷酸三苯酯（TPPi，1毫摩尔）均能显著诱导传统凋亡随时间增加（p < 0.05）。在许多细胞中，PSP和TOTP（1毫摩尔）还诱导了核浓缩，几乎没有碎裂或出芽现象。用环孢菌素A（500纳摩尔，30小时）预处理可降低1毫摩尔对硫磷和TOTP暴露后的凋亡率。通过DNA凝胶电泳验证了凋亡性核变化。还监测了半胱天冬酶-3（一种半胱氨酸天冬氨酸蛋白酶）的激活情况。对氧磷（1毫摩尔）、对硫磷（100微摩尔、1毫摩尔）、PSP（10微摩尔、100微摩尔、1毫摩尔）、TOTP（100微摩尔、1毫摩尔）以及TPPi（1毫摩尔）暴露后，OP化合物显著诱导半胱天冬酶-3激活随时间增加（p < 0.05）。用环孢菌素A（500纳摩尔，30小时）预处理可在与对氧磷、对硫磷和TPPi长时间孵育期间显著降低半胱天冬酶-3激活（p < 0.05）。此外，用半胱天冬酶-3抑制剂Ac-DEVD-CHO和半胱天冬酶-8抑制剂Ac-IETD-CHO（25微摩尔，8小时）预处理可在暴露于1毫摩尔PSP和对硫磷后显著降低半胱天冬酶-3激活（p < 0.05）。用丝氨酸蛋白酶抑制剂苯甲基磺酰氟（PMSF；1毫摩尔，8小时）预处理也可在暴露于1毫摩尔PSP和TOTP后显著降低半胱天冬酶激活（p < 0.05）。用环孢菌素A、Ac-IETD-CHO或PMSF预处理改变了OP化合物诱导的核碎裂或半胱天冬酶-3激活，这表明OP化合物诱导的细胞毒性可能通过多个位点进行调节，包括线粒体通透性孔、受体介导的半胱天冬酶途径或丝氨酸蛋白酶。
