Mitochondrial oxidoreductase of outer membrane is responsible for paraquat cytotoxicity

外膜线粒体氧化还原酶负责百草枯的细胞毒性

基本信息

批准号：
15591664
负责人：
SHIMADA Hiroki
金额：
$ 1.66万
依托单位：
KANAZAWA MEDICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

The acute toxicity of paraquat (PQ), which is a widely used herbicide in agriculture and believed to be a risk factor of Parkinson's disease, is mediated by reactive oxygen species (ROS) produced by their cyclic oxidation-reduction reaction and causes severe injury to the lungs and other multiorgans in mammals. It has generally been speculated that NADPH-cytochrome P450 reductase in the microsomal drug-metabolizing enzyme systems formed ROS as the in vitro toxic mechanisms. Recently, we demonstrated that the ROS were formed from the outer surface of the mitochondria (Mt) in the presence of cytoplasmic NADH and injured the Mt. As for the mechanisms, we have found an NADH-quinone oxidoreductase_m (NQO_m) activity located on the outer membrane of Mt and propose the participation of voltage dependent anion channel (VDAC).When isolated rat Mt were incubated with 2', 7'-dichlorofluorescin-diacetate, a fluorescent probe of H_2O_2 production, control Mt showed a faint fluorescence due to the formation of 2', 7'-dichlorofluorescein. An addition of NADH or PQ alone did not change the intensity, but coexistence of NADH and PQ raised it. The intensity was suppressed by benzoquinone, a scavenger of O_2^- and decreased by voltage dependent anion channel (VDAC) inhibitors and anti-VDAC antibody.After the starvation, almost all of the Mt appeared to be orthodox and condensed conformation. When PQ and NADH were added simultaneously, swollen and broken Mt were markedly increased. Anti-VDAC antibody inhibited the damage.NQO_m activity as NADH dependent PQ reduction was observed by the reduction of nitroblue tetrazolium (NBT) in the detergent-extract of the outer membrane. It was suppressed by anti-VDAC antibody, DIDS and DCCD. The activity was positive at the 500 kDa band by zymography on native-PAGE using NBT reduction. This band contained VDAC protein determined by Western blot.These results indicate that VDAC protein may cause PQ toxicity.

百草枯 (PQ) 是一种在农业中广泛使用的除草剂，被认为是帕金森病的危险因素，其急性毒性是由其循环氧化还原反应产生的活性氧 (ROS) 介导的，会对人体造成严重伤害。哺乳动物的肺和其他多器官。一般推测微粒体药物代谢酶系统中的NADPH-细胞色素P450还原酶形成ROS是其体外毒性机制。最近，我们证明了ROS是在细胞质NADH存在的情况下从线粒体（Mt）外表面形成的，并损伤了Mt。至于机制，我们发现了位于线粒体外表面的NADH-醌氧化还原酶_m（NQO_m）活性。 Mt 的外膜，并提出电压依赖性阴离子通道 (VDAC) 的参与。当分离的大鼠 Mt 与 2', 7'-二氯荧光素二乙酸盐一起孵育时，作为H_2O_2产生的荧光探针，对照Mt由于2',7'-二氯荧光素的形成而显示出微弱的荧光。单独添加 NADH 或 PQ 不会改变强度，但 NADH 和 PQ 共存会提高强度。其强度被O_2^-清除剂苯醌抑制，并被电压依赖性阴离子通道(VDAC)抑制剂和抗VDAC抗体减弱。饥饿后，几乎所有的Mt都呈现正统和浓缩构象。当同时添加PQ和NADH时，肿胀和破裂的Mt显着增加。抗VDAC抗体抑制损伤。通过外膜洗涤剂提取物中硝基蓝四唑(NBT)的还原观察到NADH依赖性PQ还原的NQO_m活性。它被抗VDAC抗体、DIDS和DCCD抑制。使用 NBT 还原在非变性 PAGE 上进行酶谱分析，发现 500 kDa 条带的活性呈阳性。通过Western blot测定该条带含有VDAC蛋白。这些结果表明VDAC蛋白可能引起PQ毒性。