Li Lan, Du Yi, Ju Furong, Ma Shunxiang, Zhang Shengxiang
a Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University , Lanzhou , People's Republic of China.
Toxicol Mech Methods. 2016;26(3):211-20. doi: 10.3109/15376516.2016.1156796. Epub 2016 Mar 21.
Paraoxon (POX) is one of the most toxic organophosphorus pesticides, but its toxic mechanisms associated with apoptosis remain unclear. The aim of this study was to investigate calcium-associated mechanisms in POX-induced apoptosis in EL4 cells.
EL4 cells were exposed to POX for 0-16 h. EGTA was used to chelate Ca(2+ ) in extracellular medium, and heparin and procaine were used to inhibit Ca(2+ )efflux from the endoplasmic reticulum (ER). Z-ATAD-FMK was used to inhibit caspase-12 activity. The apoptotic rate assay, western blotting and immunocytochemistry (ICC) were used to reveal the mechanisms of POX-induced apoptosis.
POX significantly increased the expression and activation of caspase-12 and caspase-3, enhanced expression of calpain 1 and calpain 2, and induced the release of cyt c, but did not change the expression of Grp 78. Inhibiting caspase-12 activity alleviated POX-induced upregulation of calpain 1 and caspase-3, promoted POX-induced upregulation of calpain 2, and reduced POX-induced cyt c release, suggesting that there was a cross-talk between the ER-associated pathway and mitochondria-associated apoptotic signals. Attenuating intracellular calcium concentration with EGTA, heparin or procaine decreased POX-induced upregulation of calpain 1, calpain 2, caspase-12 and caspase-3, and reduced POX-induced cyt c release. After pretreatment with EGTA or procaine, POX significantly promoted expression of Grp 78.
Calcium played a key role in POX-induced apoptosis in EL4 cells by regulating both ER- and mitochondria-associated pathways. The cross-talk of ER- and mitochondria-associated pathways was accomplished through calcium signal.
对氧磷（POX）是毒性最强的有机磷农药之一，但其与细胞凋亡相关的毒性机制尚不清楚。本研究旨在探讨POX诱导EL4细胞凋亡过程中与钙相关的机制。
将EL4细胞暴露于POX中0 - 16小时。使用乙二醇双四乙酸（EGTA）螯合细胞外培养基中的Ca(2+)，使用肝素和普鲁卡因抑制内质网（ER）的Ca(2+)外流。使用Z-ATAD-FMK抑制半胱天冬酶-12（caspase-12）活性。采用凋亡率检测、蛋白质印迹法和免疫细胞化学（ICC）来揭示POX诱导凋亡的机制。
POX显著增加了caspase-12和caspase-3的表达与激活，增强了钙蛋白酶1和钙蛋白酶2的表达，并诱导了细胞色素c（cyt c）的释放，但未改变葡萄糖调节蛋白78（Grp 78）的表达。抑制caspase-12活性减轻了POX诱导的钙蛋白酶1和caspase-3的上调，促进了POX诱导的钙蛋白酶2的上调，并减少了POX诱导的cyt c释放，表明内质网相关途径与线粒体相关凋亡信号之间存在相互作用。用EGTA、肝素或普鲁卡因降低细胞内钙浓度可减少POX诱导的钙蛋白酶1、钙蛋白酶2、caspase-12和caspase-3的上调，并减少POX诱导的cyt c释放。用EGTA或普鲁卡因预处理后，POX显著促进了Grp 78的表达。
钙通过调节内质网和线粒体相关途径在POX诱导的EL4细胞凋亡中起关键作用。内质网和线粒体相关途径的相互作用是通过钙信号完成的。
