Generation of Reactive Oxygen Species and Reduction of Ferric Chelates by Microsomes in the Presence of a Reconstituted System Containing Ethanol, NAD+ and Alcohol Dehydrogenase

Many of the toxic metabolic actions of ethanol on the liver have been ascribed to the enhanced cellular production of NADH, which arises as a consequence of the oxidation of ethanol by alcohol dehydrogenase (ADH). Experiments were conducted to evaluate whether NADH generated from a reconstituted system containing ethanol plus NAD⁺ plus ADH could interact with ferric chelates to promote microsomal lipid peroxidation and generation of a hydroxyl radical (OH)-like species. In the presence of the reconstituted system and iron, microsomes produced ? OH as assessed by the oxidation of ? OH scavenging agents. This oxidation was inhibited by catalase and competitive ? OH scavengers but not by superoxide dismutase. The ADH-dependent microsomal production of ? OH was effectively catalyzed by ferric-EDTA and -diethylenetriamine pentaacetic acid (-DTPA), but not by ferric-ATP or -citrate. However, all these ferric chelates were reduced by the microsomes in the presence of the reconstituted system. Hydrogen peroxide (H₂O₂) was produced in the presence of ADH and appeared to be a limiting factor for the production of ? OH. The reconstituted system also catalyzed microsomal lipid peroxidation, and the pattern of effectiveness of ferric chelates was opposite that of catalysis of ? OH production. There was little effect by catalase, superoxide dismutase or dimethyl sulfoxide (DMSO) on the ADH-dependent microsomal lipid peroxidation. The reconstituted system was characterized with respect to dependence on NAD⁺ and ADH; ethanol could be replaced by other alcohols, which are substrates for ADH. Pyrazole, a potent inhibitor of ADH, blocked the ability of the reconstituted system to interact with iron and microsomes to produce reactive oxygen species. The overall pattern of response of the ADH-dependent reactions with respect to rates of reactions, catalytic effectiveness of ferric chelates and sensitivity to radical scavengers is similar to that found with a NADPH-generating system. In view of the effectiveness of NADH derived from the oxidation of ethanol by ADH in reducing ferric chelates and promoting microsomal generation of reactive species, increased availability of cellular NADH as a consequence of ethanol oxidation could contribute to the development of oxidative stress and play a role in the toxic actions of ethanol to the liver.