Extramitochondrial release of hydrogen peroxide from insect and mouse liver mitochondria using the respiratory inhibitors phosphine, myxothiazol, and antimycin and spectral analysis of inhibited cytochromes

Abstract The fumigant insecticide phosphine (PH 3 ) is known to inhibit cytochrome c oxidase in vitro . Inhibition of the respiratory chain at this site has been shown to stimulate the generation of superoxide radicals (O 2 − ), which dismutate to form hydrogen peroxide (H 2 O 2 ). This study was performed in order to investigate the production of H 2 O 2 by mitochondria isolated from granary weevil ( Sitophilus granarius ) and mouse liver on exposure to PH 3 . Other respiratory inhibitors, antimycin, myxothiazol, and rotenone were used with insect mitochondria. Hydrogen peroxide was measured spectrophotometrically using yeast cytochrome c peroxidase as an indicator. Insect and mouse liver mitochondria, utilizing endogenous substrate, both produced H 2 O 2 after inhibition by PH 3 . Insect organelles released threefold more H 2 O 2 than did mouse organelles, when exposed to PH 3 . Production of H 2 O 2 by PH 3 -treated insect mitochondria was increased significantly on addition of the substrate α-glycerophosphate. Succinate did not enhance H 2 O 2 production, however, indicating that the H 2 O 2 did not result from the autoxidation of ubiquinone. NAD+-linked substrates, malate and pyruvate also had no effect on H 2 O 2 production, suggesting that NADH-dehydrogenase was not the source of H 2 O 2 . Data obtained using antimycin and myxothiazol, both of which stimulated the release of H 2 O 2 from insect mitochondria, lead to the conclusion that glycerophosphate dehydrogenase is a source of H 2 O 2 . The effect of combining PH 3 , antimycin, and myxothiazol on cytochrome spectra in insect mitochondria was also recorded. It was observed that PH 3 reduces cytochrome c oxidase but none of the other cytochromes in the electron transport chain. There was no movement of electrons to cytochrome b when insect mitochondria are inhibited with PH 3 . The spectral data show that the inhibitors interact with the respiratory chain in a way that would allow the production of H 2 O 2 from the sites proposed previously.