Cytotoxic effects of N-acetyl-p-benzoquinone imine, a common arylating intermediate of paracetamol and N-hydroxyparacetamol

Abstract The cytotoxic effects of N -acetyl- p -benzoquinone imine (NAPQI), a postulated ultimate reactive metabolite of paracetamol (pHAA), was studied in suspensions of isolated rat hepatocytes. Incubation of cells for 10–300 min with 0.1–0.5 mM NAPQI led to concentration dependent cell damage. as determined by increased trypan blue exclusion, lactate dehydrogenase release and glutathione (GSH) depletion. NAPQI and N -hydroxyparacetamol (N-OH-pHAA), a postulated proximate metabolite of pHAA, caused cytotoxic effects in the same concentration range. In contrast, no toxic effects of pHAA (⩽ 20 mM) could be demonstrated. With the short half-life of NAPQI, less than 0.5% of the NAPQI added is expected to be left in the incubation medium after a 2 min incubated period. Nevertheless, 10–120 min (depending on the concentration of NAPQI) elapsed before the cells responded with increased membrane permeability. Clearly, the initial damage caused by NAPQI must be followed by subsequent cellular steps before toxicity becomes apparent. The addition of N-acetylcysteine, GSH or ascorbate during the NAPQI exposure period fully protected the hepatocytes from NAPQI damage. Lesser effects were demonstrated when these agents were added after the 5 min NAPQI exposure period. The results presented in this study further support the hypothesis that NAPQI is the ultimate reactive formed from pHAA.