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.

Paraquat（PQ）的急性毒性是一种在农业中广泛使用的除草剂，被认为是帕金森氏病的危险因素，是由活性氧（ROS）介导的，其环状氧化还原 - 还原氧化还原反应，并导致对肺部和其他多层型多型多层型的严重伤害。通常已经推测，在微粒体药物 - 代谢酶系统中，NADPH-胞染料P450还原酶形成ROS作为体外有毒机制。最近，我们证明了ROS是在细胞质NADH存在下从线粒体（MT）的外表面形成的，并受伤了MT。对于机制，我们发现NADH- Quinone氧化氧化胜地酶_M（NQO_M）的活性是在MT的隔离度上，并且在隔离的综合综合综合综合（NQO_M）的活性（NQO_M）。与2'，7'-二氯荧光素二乙酸酯一起孵育，H_2O_2的荧光探针，对照MT由于形成了2'，7'-二氯氟氟众素而显示出微弱的荧光。仅添加NADH或PQ并没有改变强度，但是NADH和PQ的共存提出了它。强度被苯喹酮抑制，苯喹酮是O_2^ - 的苯喹酮，并通过电压依赖性阴离子通道（VDAC）抑制剂和抗VDAC抗体降低。饥饿后，几乎所有MT似乎都是正统的和凝结的构象。当同时添加PQ和NADH时，MT肿胀和损坏的MT显着增加。抗VDAC抗体抑制了损伤。NQO_M活性是通过在外膜的洗涤剂提取中的氮气四唑（NBT）减少来观察到依赖性PQ的。它被抗VDAC抗体，DIDS和DCCD抑制。使用NBT减少，通过Zymography在500 kDa频带上的活性为正。该带包含由Western印迹确定的VDAC蛋白。这些结果表明VDAC蛋白可能引起PQ毒性。