Cochemé Helena M, Murphy Michael P
Institute of Healthy Ageing, University College London, London, United Kingdom.
Methods Enzymol. 2009;456:395-417. doi: 10.1016/S0076-6879(08)04422-4.
Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride) is widely used as a redox cycler to stimulate superoxide production in organisms, cells, and mitochondria. Paraquat is also used to induce symptoms of Parkinson's disease in experimental models of this neurodegenerative disorder. Paraquat causes extensive mitochondrial oxidative damage, and in mammalian systems, complex I of the respiratory chain has been identified as the major site of superoxide production by paraquat. Although much progress has been made at explaining how paraquat interacts with mitochondria, several aspects remain to be clarified-most notably the pathway of paraquat uptake into mitochondria. This chapter describes methods for further investigating the interaction of paraquat with mitochondria and also provides practical information for the general use of paraquat as a superoxide generator and agent of oxidative stress. The techniques covered include the detection and quantitation of the paraquat dication and the paraquat monocation radical (by electron paramagnetic resonance, spectrophotometry, and with an ion-selective electrode); assays for measuring paraquat-induced superoxide production by intact mitochondria or mitochondrial membranes (including aconitase inactivation, and coelenterazine chemiluminescence); methods for assessing paraquat uptake by mitochondria; and screens for identifying paraquat sensitivity or resistance in yeast mutants.
百草枯（1,1'-二甲基-4,4'-联吡啶二氯化物）作为一种氧化还原循环剂被广泛用于刺激生物体、细胞和线粒体中超氧化物的产生。百草枯还被用于在这种神经退行性疾病的实验模型中诱发帕金森病症状。百草枯会导致广泛的线粒体氧化损伤，在哺乳动物系统中，呼吸链复合体I已被确定为百草枯产生超氧化物的主要部位。尽管在解释百草枯如何与线粒体相互作用方面已经取得了很大进展，但仍有几个方面有待阐明——最显著的是百草枯进入线粒体的途径。本章描述了进一步研究百草枯与线粒体相互作用的方法，并为将百草枯普遍用作超氧化物生成剂和氧化应激剂提供了实用信息。所涵盖的技术包括百草枯二价阳离子和百草枯一价阳离子自由基的检测和定量（通过电子顺磁共振、分光光度法以及离子选择性电极）；测量完整线粒体或线粒体膜中百草枯诱导的超氧化物产生的测定方法（包括乌头酸酶失活和腔肠素化学发光）；评估线粒体对百草枯摄取的方法；以及在酵母突变体中鉴定百草枯敏感性或抗性的筛选方法。
