Abstract
The cytotoxic effects of 10 mM acetaminophen (APAP) in primary cultures of non-induced mouse hepatocytes are accompanied by depletion of intracellular glutathione (GSH), arylation of protein, and loss of protein sulfhydryl (PSH) groups. Investigation of the stoichiometry of the covalent binding and PSH loss after APAP exposure demonstrated a greater loss in PSH than could be accounted for by covalent binding to proteins and suggests that APAP exhibits both oxidative and arylative actions in cell culture. Subcellular fractionation revealed that the PSH oxidation induced by APAP was greatest in the microsomal fraction. Exposure of the hepatocytes to 10 mM 3,5-dimethyl-acetaminophen (3,5-DMA) or 2,6-dimethyl-acetaminophen (2,6-DMA) permitted dissociation of the oxidative and arylative properties of APAP. Even though treatment of cultured hepatocytes with 3,5-DMA did not result in covalent binding, there was a more rapid depletion of intracellular GSH, oxidation of PSH, and cytotoxicity compared to APAP. This investigation also provides the first evidence that the cytotoxic effects of both APAP and 3,5-DMA are accompanied by the formation of protein aggregates of high molecular weight that are not disulfide linked. The aggregates probably reflect the oxidative properties of these drugs and may be a mediator of their toxic effects. By contrast, 2,6-DMA, which did bind to cellular proteins and deplete GSH, did not lead to PSH loss, protein aggregation, or cytotoxicity. Since PSH oxidation and protein aggregation correlated well with cytotoxicity, these data suggest that the oxidative component of APAP and 3,5-DMA can play a significant role in eliciting cellular damage in cultured hepatocytes.