Diphenyleneiodonium, an NAD(P)H Oxidase Inhibitor, also Potently Inhibits Mitochondrial Reactive Oxygen Species Production

Abstract

Diphenyleneiodonium (DPI) has frequently been used to inhibit reactive oxygen species (ROS) production mediated by flavoenzymes, particularly NAD(P)H oxidase. This study was undertaken to examine if DPI could also inhibit production of superoxide and H2O2 by mitochondria, the major source of cellular ROS. Detection of mitochondrial superoxide by lucigenin-derived chemiluminescence (CL) with unstimulated monocytes/macrophages showed that DPI at concentrations that inhibit NAD(P)H oxidase markedly diminished the production 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 DPI. DPI was as potent as rotenone in inhibiting the production of superoxide and H2O2 by mitochondrial respiration. With substrate-supported isolated mitochondria, DPI was shown to reduce mitochondrial superoxide production probably through inhibiting NADH-ubiquinone oxidoreductase (complex I).