Quinone-Stimulated Superoxide Formation by Subcellular Fractions, Isolated Hepatocytes, and Other Cells

It is concluded that NADPH-cytochrome P-450 reductase activity is not rate-limiting for quinone-stimulated O2[unknown] formation by hepatocytes, suggesting that intracellular NADH is not rates-limited.

Abstract

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[unknown]). Isolated hepatocytes do not liberate appreciable amounts of O2[unknown]. Simple quinones, such as 2,5-dimethyl- p -benzoquinone, stimulate the formation of O2[unknown] by hepatocytes up to 15 nmoles/min/106 cells. Hepatocyte O2[unknown] formation stimulated by a variety of simple quinones and more complex antitumor quinones is maximal at a quinone one-electron reduction potential ( E 71) of -70 mV and qualitatively similar to the pattern of O2[unknown] formation seen with mitochondrial NADH:ubiquinone oxidoreductase and microsomal NADH-cytochrome b 5 reductase. O2[unknown] production by microsomal NADPH-cytochrome P-450 reductase is maximal at a quinone E 71 of -200 mV. Phenobarbital induction, which increases NADPH-cytochrome P-450 reductase, has no effect on O2[unknown]formation by hepatocytes. It is concluded that NADPH-cytochrome P-450 reductase activity is not rate-limiting for quinone-stimulated O2[unknown] 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[unknown]by hepatocytes, suggesting that O2[unknown] is not transported through anion channels in the plasma membrane. Ethanol has no effect on hepatocyte O2[unknown] formation, which suggests that intracellular NADH is not rate-limiting. Treatment of hepatocytes with diethyldithiocarbamate, which inhibits cytosolic and mitochondrial superoxide dismutase, increases O2[unknown] 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[unknown] by hepatocytes.