Formation of glutathione adducts of carbon tetrachloride metabolites in a rat liver microsomal incubation system.

Metabolism of CCl4 by rat liver microsomes causes damage to the membrane. GSH diminishes that damage. One mechanism of GSH protection has been characterized. It involves formation of diglutathione carbonate from two molecules of GSH and one molecule of phosgene, an oxygenated metabolite of CCl4. The present studies were done to seek other GSH adducts of CCl4 metabolites and to examine the effect of oxygen tension on their formation. Incubations were carried out in sealed flasks under oxygen concentrations ranging from 0.14 to 21% at 37 degrees. The CCl4 concentration was 72 microM. 14CCl4 and 35S-GSH were used to label metabolites. High pressure liquid chromatographic analysis of the aqueous phase demonstrated two GSH adducts of CCl4 metabolites. One adduct was oxygen-dependent and was identified as diglutathione carbonate by its co-elution with a diglutathione carbonate standard. Its formation showed no evidence of saturation when GSH concentrations as high as 10 mM were used, indicating that the overall process was nonenzymatic. Formation of the other adduct was greatest under the lowest oxygen concentration studied and none occurred at oxygen tensions of 5% or greater. Based on experiments with radiolabeled CCl4 and GSH, this metabolite appeared to be a product of one molecule each of CCl4 and GSH. Formation of this adduct had enzymatic characteristics. It was saturable with respect to GSH with an apparent Km of 70 microM, and other thiol compounds that were tested could not substitute for GSH. The adduct was unstable during isolation attempts and was not characterized further. Formation of these two GSH adducts could account for some of the protection by GSH against CCl4 injury.