Mitochondrial oxidoreductase of outer membrane is responsible for paraquat cytotoxicity

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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.