Cytochemical energy-filtering transmission electron microscopy of mitochondrial free radical formation in paraquat cytotoxicity

The generation of oxygen free radicals was investigated using cytochemistry and its energy-filtering transmission electron microscopy in reference to the toxic mediator for the herbicide paraquat. When isolated intact mitocho ndria from rat livers were incubated in a medium containing paraquat and NA DH, a mitochondrial NADH-quinone oxidoreductase activity generated superoxi de anions to cause the destruction of mitochondria which resulted in cell d eath. The superoxide anions were immediately converted into hydrogen peroxi de, which then formed cerium perhydroxide deposits in the presence of ceriu m ions and precipitated on the outer surface of the mitochondrial outer mem brane. This localization was also specifically identified by energy spectra l imaging and image-electron energy loss spectral analyses. Precipitation r eaction was scavenged by the addition of either cytochrome c or catalase an d inhibited by dicoumarol (an inhibitor of NAD(P)H-quinone oxidoreductases) . These cytochemical energy-filtering transmission electron microscopic res ults indicated that paraquat generated free radicals from the outer membran e of mitochondria.