Effects of N-acetylcysteine and dithiothreitol on glutathione and protein thiol replenishment during acetaminophen-induced toxicity in isolated mouse hepatocytes

Isolated mouse hepatocytes were incubated with 1.0 mM acetaminophen (AA) for 1.5 h to initiate glutathione (GSH) and protein thiol (PSH) depletion and cell injury. Cells were subsequently washed to remove non-covalently bound AA and resuspended in medium containing N-acetylcysteine (NAC, 2.0 mM) or dithiothreitol (DTT, 1.5 mM). The effects of these agents on the replenishment of GSH and total PSH content were related to the development of cytotoxicity. When cells exposed to AA were resuspended in medium containing NAC or DTT, both agents replenished GSH and total PSH content to levels observed in untreated cells but only DTT was able to attenuate cytotoxicity. Addition of the GSH synthesis inhibitor, buthionine sulfoximine (BSO, 1.0 mM, 1.5 h), to cells in incubation medium containing AA, enhanced GSH and total PSH depletion and potentiated cytotoxicity. Resuspension of these cells in medium containing NAC did not alter the potentiating effects of BSO; GSH and PSH levels were not replenished and no cytoprotective effects were observed. However, when cells exposed to AA and BSO were resuspended in medium containing DTT, PSH content was replenished but GSH levels were not restored. In addition, DTT was able to delay the development of cytotoxicity. It appears that DTT, unlike NAC, has a GSH-independent mechanism of PSH content does not result in cytoprotection, the regeneration of critical PSH by DTT may play an important role in the maintenance of proper cell structure and/or function.