Molecular mechanism for prevention of N-acetyl-p-benzoquinoneimine cytotoxicity by the permeable thiol drugs diethyldithiocarbamate and dithiothreitol

Abstract

The present study was carried out to elucidate the mechanism by which the permeable thiol drug diethyldithiocarbamate (DEDC) exhibited an antidotal effect against acetaminophen-induced hepatotoxicity in vivo. DEDC was found to act as an antidote against acetaminophen-induced cytotoxicity in hepatocytes isolated from a pyrazole-pretreated rat without affecting cytochrome P-450 levels. The mechanism of protection exhibited against reactive intermediate N-acetyl-p-benzoquinoneimine (NAPQI)-induced cytotoxicity by DEDC was then investigated and compared with that exhibited by the permeable thiol-reductant dithiothreitol (DTT). Cytotoxicity induced by the dimethylated analogue 2,6-dimethyl-N-acetyl-p-benzoquinoneimine (2,6-diMeNAPQI) was prevented if the hepatocytes were preincubated with DEDC for 5 min and removed before addition of 2,6-diMeNAPQI. Both DEDC and DTT were also found to act as antidotes against NAPQI- and 2,6-diMeNAPQI-induced cytotoxicity in isolated rat hepatocytes if added within 2 min of the addition of the quinoneimines. However, the addition of DEDC or DTT 10 min after either quinoneimine did not prevent subsequent cytotoxicity or restore GSH levels, indicating that the alkylation of GSH and of protein thiols was irreversible at that time. Fast atom bombardment mass spectrometry was used to show that DEDC formed conjugates with both NAPQI and 2,6-diMeNAPQI. Furthermore, these conjugates were found to be nontoxic. This suggests that DEDC acts as a trap for the toxic quinoneimines, thus preventing alkylation of essential macromolecules. In contrast, DTT reduced the quinoneimines to their respective nontoxic parent compounds and presumably also reduced mixed-protein disulfides and GSSG, thereby regenerating protein thiols and GSH. Therefore, this study suggests that DEDC and DTT act as antidotes by two different mechanisms.