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Acute Cytotoxic Mechanisms and Detoxic Mechanisms of Paraquat Poisoning

百草枯中毒的急性细胞毒性机制和解毒机制

基本信息

批准号：
14571474
负责人：
HIRAI Keiichi
金额：
$ 1.79万
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：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

The acute cytotoxicity of paraquat, which is a widely used potent herbicide in agriculture and became recently accepted 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 was classically misunderstood that NADPH-CYP reductase-CYP in the microsomal phase I drug- metabolizing enzyme systems formed ROS as the in vitro toxic mechanisms. However, we discovered that paraquat was first metabolized in the postmicrosomal cytosomal fractions into paraquat monopyridone, which was subsequently hydroxylated by the NADPH-CYP reductase-CYP, including CYP3A, CYP2B and CYP2C, as detoxification systems.The ROS were formed from the outer surface of the mitochondria in the presence of cytoplasmic NADH and injured the mitochondria. As for the mechanisms, we have found an NADH-quinone oxidoreductase activity located on the outer membrane of mitochondria and propose the participation of voltage dependent anion channel (VDAC).When isolated rat mitochondria were incubated wit coexistence of NADH and paraquat, ROS production was raised and resulted into the structural injury. The intensity was suppressed by benzoquinone, a scavenger of O_2^- and decreased by voltage dependent anion channel (VDAC) inhibitors, such as DIDS and DCCD, and anti-VDAC antibody. 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 at 31 kDa.

百草枯是一种在农业中广泛使用的强效除草剂，最近被认为是帕金森病的危险因素，其急性细胞毒性是由其循环氧化还原反应产生的活性氧介导的，并对哺乳动物的肺和其他多器官造成严重损伤。经典的体外毒性机制被误解为微粒体I相药物代谢酶系统中的NADPH还原酶-γ形成ROS。然而，我们发现百草枯首先在微粒体后的细胞质组分中代谢为百草枯单吡啶酮，然后被作为解毒系统的NADPH还原酶CYP3A、CYP2B和CYP2C羟基化，在细胞质NADH存在下，从线粒体外表面产生ROS，损伤线粒体。在其作用机制上，我们发现线粒体外膜上存在一个NADH-醌氧化还原酶活性，并推测电压依赖性阴离子通道（VDAC）参与了这一过程。电压依赖性阴离子通道（VDAC）抑制剂DIDS、DCCD及抗VDAC抗体均能降低O_2 ^-清除剂苯醌的抑制作用。通过使用NBT还原的非变性PAGE上的酶谱分析，在500 kDa条带处活性为阳性。该条带含有通过蛋白质印迹法测定为31 kDa的VDAC蛋白。