Metabolism of acetaminophen by cultured rat hepatocytes. Depletion of protein thiol groups without any loss of viability

Over the course of 4 hr, the metabolism of acetaminophen (APAP) by cultured rat hepatocytes resulted in a depletion of protein thiols and an accumulation of oxidized glutathione (GSSG) in the medium. With 20 mM APAP, arylation and the formation of glutathione mixed disulfides accounted for a loss of 22% of the total protein thiols in the absence of any loss of viability. With 20 mM APAP and an inhibition of glutathione reductase by 1.3-(2-chloroethyl)-1-nitrosourea (BCNU), protein thiols were depleted by 40% by arylation and the formation of glutathione mixed disulfides, again without a loss of viability. With 20 mM APAP and BCNU in the presence of 20 mM deferoxamine, there was still little or no cell killing after 8 hr despite a loss now of almost 60% of the total protein thiols. These data do not support the hypothesis that a depletion of protein thiols is related to the toxicity of APAP. One millimolar APAP and BCNU killed 60% of the hepatocytes within 4 hr. In this circumstance, the loss of protein thiols was not attributable to either arylation by APAP metabolites or the formation of glutathione mixed disulfides. The antioxidant N,N'-diphenyl-phenylenediamine prevented the cell killing and the loss of protein thiols, a result implicating a role for lipid peroxidation in the depletion of protein-bound thiols. However, protein thiol depletion under these circumstances is not necessarily related to the lethal cell injury and most likely represents an epiphenomenon of the peroxidation of cellular lipids.