INCREASED PRODUCTION OF REACTIVE OXYGEN SPECIES BY RAT-LIVER MITOCHONDRIA AFTER CHRONIC ETHANOL TREATMENT

Rat liver microsomes and, to a lesser extent, nuclei were previously s hown to produce reactive oxygen species at elevated rates after chroni c ethanol treatment. The ability of intact rat liver mitochondria to i nteract with iron and either NADH or NADPH, and the effects of ethanol treatment, on production of reactive oxygen intermediates was determi ned. In the presence of ferric-ATP, NADH or NADPH catalyzed mitochondr ial lipid peroxidation. Rates were elevated two- to threefold with mit ochondria from ethanol-fed rats with both reductants. Mitochondrial li pid peroxidation was insensitive to superoxide dismutase, catalase, or hydroxyl radical scavengers but was sensitive to GSH and anti-oxidant s such as trolox. Mitochondrial generation of hydroxyl radical-like sp ecies (assayed by oxidation of chemical scavengers) was increased afte r chronic ethanol treatment, as was H2O2 production. Modifiers of mito chondrial metabolism such as rotenone, cyanide, or an uncoupling agent , had no effect on mitochondrial production of reactive oxygen interme diates. The membrane-impermeable thiol reagent, p-chloromercuribenzoat e, was completely inhibitory with both mitochondrial preparations. The activity of the rotenone-insensitive NADH-cytochrome c reductase, an enzyme of the outer mitochondrial membrane, was increased 40 to 60% by the ethanol treatment. These results suggest that NADH acting via the outer membrane NADH reductase can catalyze an iron-dependent producti on of oxygen radicals by rat liver mitochondria. The outer mitochondri al membrane fraction, prepared by digitonin fractionation, displayed i ncreased rotenone-insensitive NADH-cytochrome c reductase activity aft er ethanol treatment and was more reactive in catalyzing scission of p BR322 DNA from the supercoiled form to the open circular forms. Rates of oxygen radical production by mitochondria and the extent of increas e produced by chronic ethanol treatment are similar to those previousl y found with microsomes when NADH is the cofactor. Oxidation of ethano l by alcohol dehydrogenase generates NADH, and NADH-dependent producti on of reactive oxygen species by various organelles is increased after chronic ethanol treatment. These acute metabolic interactions coupled to induction by chronic ethanol treatment may play an important role in the development of a state of oxidative stress in the liver by etha nol. (C) 1994 Academic Press, Inc.