Quinone-stimulated superoxide formation by subcellular fractions, isolated hepatocytes, and other cells

Quinones can undergo enzymatic one-electron reduction to the semiquinone radical which, in the presence of molecular oxygen, can transfer an electron and form the superoxide anion radical (O₂^-). Isolated hepatocytes do not liberate appreciable amounts of O₂^-. Simple quinones, such as 2,5-dimethyl-p-benzoquinone, stimulate the formation of O₂^- by hepatocytes up to 15 nmoles/min/10⁶ cells. Hepatocyte O₂^- formation stimulated by a variety of simple quinones and more complex antitumor quinones is maximal at a quinone one-electron reduction potential (E₇¹) of -70 mV and qualitatively similar to the pattern of O₂^- formation seen with mitochondrial NADH:ubiquinone oxidoreductase and microsomal NADH-cytochrome b₅ reductase. O₂^- production by microsomal NADPH-cytochrome P-450 reductase is maximal at a quinone E₇¹ of -200 mV. Phenobarbital induction, which increases NADPH-cytochrome P-450 reductase, has no effect on O₂^- formation by hepatocytes. It is concluded that NADPH-cytochrome P-450 reductase activity is not rate-limiting for quinone-stimulated O₂^- formation by hepatocytes. The sulfonated stilbenes, 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene have no marked effect on the formation of O₂^- by hepatocytes, suggesting that O₂^- is not transported through anion channels in the plasma membrane. Ethanol has no effect on hepatocyte O₂^- formation, which suggests that intracellular NADH is not rate-limiting. Treatment of hepatocytes with diethyldithiocarbamate, which inhibits cytosolic and mitochondrial superoxide dismutase, increases O₂^- formation by hepatocytes over 2-fold. Feeding rats a copper-deficient diet, which also decreases hepatic cytosolic and mitochondrial superoxide dismutase, has no effect on the quinone-dependent formation of O₂^- by hepatocytes.