Diphenyleneiodonium, an NAD(P)H oxidase inhibitor, also potently inhibits mitochondrial reactive oxygen species production

Diphenyleneiodonium (DPI) has frequently been used to inhibit reactive oxyg en species (ROS) production mediated by flavoenzymes, particularly NAD(P)H oxidase. This study was undertaken to examine if DPI could also inhibit pro duction of superoxide and H2O2 by mitochondria, the major source of cellula r ROS. Detection of mitochondrial superoxide by lucigenin-derived chemilumi nescence (CL) with unstimulated monocytes/macrophages showed that DPI at co ncentrations that inhibit NAD(P)H oxidase markedly diminished the productio n of superoxide by mitochondrial respiration. Similarly, the extracellular H2O2 derived from mitochondrial respiration as detected by luminol-derived CL in the presence of horseradish peroxidase was also greatly reduced by DP I. DPI was as potent as rotenone in inhibiting the production of superoxide and H2O2 by mitochondrial respiration. With substrate-supported isolated m itochondria, DPI was shown to reduce mitochondrial superoxide production pr obably through inhibiting NADH-ubiquinone oxidoreductase (complex I). (C) 1 998 Academic Press.