N-acetyl-p-benzoquinone imine: a cytochrome P-450-mediated oxidation product of acetaminophen.

Abstract

N-acetyl-p-benzoquinone imine (NAPQI) has been proposed as the toxic metabolite of acetaminophen for the past 10 years, although it has never been detected as an enzymatic oxidation product of acetaminophen. We report (i) direct detection of NAPQI formed as an oxidation product of acetaminophen by cytochrome P-450 and cumene hydroperoxide and (it) indirect evidence that is compelling for NAPQI formation from acetaminophen by cytochrome P-450, NADPH, and NADPH-cytochrome P-450 reductase. Evidence is provided for the rapid reduction of NAPQI back to acetaminophen by NADPH and NADPH-cytochrome P-450 reductase such that steady-state levels of NAPQI were below our detection limits of 6.7 X 10-8 M. In mouse liver microsomal incubations, radiolabeled analogs of both NAPQI and acetaminophen bound covalently to microsomal protein with the loss of -20% of the acetyl group as acetamide. The binding in each case was decreased by glutathione with concomitant formation of 3-Sglutathionylacetaminophen. The binding also was decreased by L-ascorbic acid, NADPH, and NADH with reduction of NAPQI to acetaminophen. Results of partitioning experiments indicate that NAPQI is a major metabolite of acetaminophen, a considerable fraction of which is rapidly reduced back to acetaminophen.The widely used analgesic-antipyretic drug acetaminophen is known to cause serious liver necrosis at high doses in man and experimental animals (1, 2). An electrophilic product of cytochrome P-450 oxidation that depletes cellular glutathione (GSH) and covalently binds to tissue macromolecules has been implicated in this toxic reaction (3). Initially, the formation of the arylating metabolite was believed to involve N-oxidation of acetaminophen to N-hydroxyacetaminophen followed by dehydration to N-acetyl-p-benzoquinone imine (NAPQI) (4). However, subsequent kinetic studies and carrier pool trapping experiments in vitro (5, 6) and metabolism studies of N-hydroxyacetaminophen in vivo (7) have shown that if N-hydroxyacetaminophen is an intermediate, it must decompose at the enzymatic site of hydroxylation.Although it appears that N-hydroxyacetaminophen is not significantly involved in acetaminophen toxicity, evidence still favors NAPQI as an ultimate toxin. We recently synthesized NAPQI in pure crystalline form, and studies have shown this reactive quinoneimine to be highly toxic in mice and to isolated hepatocytes (8). Other investigators, using aqueous solutions of NAPQI generated either electrochemically (9) or by dehydration of N-hydroxyacetaminophen (7, 10, 11), have shown NAPQI to be an electrophile and an oxidant. Similar properties were exhibited by stable benzene solutions of chemically synthesized NAPQI (12).We present in this report direct evidence for the formation of NAPQI from acetaminophen in incubations of acetaminophen with cumene hydroperoxide (CHP) and hepatic cytochrome P-450 purified from phenobarbital-pretreated rats. Similar attempts to directly detect NAPQI in incubations of acetaminophen with either purified P-450, NADPH, and...

Use of horseradish peroxidase for gene-directed enzyme prodrug therapy with paracetamol

“…Therefore, it would not require clinical evaluation as a prodrug alone. In cases of overdose, however, the normal elimination pathways become saturated, often due to glutathione (GSH) depletion, and liver damage develops due to the production of N-acetyl-p-benzoquinoneimine (NAPQI) by cytochrome P450 (CYP) enzymes (Dahlin et al, 1984). Alterations in cell cycle progression have also been observed.…”

Neutrophil depletion protects against murine acetaminophen hepatotoxicity

“…The toxic response to APAP is thought to be initiated by a highly electrophilic intermediate, N-acetyl-p-benzoquinone-imine (NAPQI), generated by hepatic CYP2E1. [2][3][4] Excessive NAPQI depletes hepatic glutathione (GSH) and covalently binds to cellular macromolecules, resulting in oxidative stress reactions, dysfunction of mitochondria, and DNA damage. 4 These initiation events ultimately may result in direct damage to hepatocytes, leading to cell death of hepatocytes.…”

Evaluation of Acetaminophen Effect on Oxidative Stressed Mice by Peroxide Hydrogen

“…Results obtained in this study can be explained by the fact that APAP (N-acetyl-p-aminophenol) by CYP (cytochrome P450) leads to the formation of N-acetylpbenzoquinoneimine (NAPQI), a highly reactive intermediate metabolite [30], which is normally detoxified by conjugation with reduced glutathione (GSH). This may explain the highest activity of GR observed in mice treated with paracetamol + H 2 O 2 ( Figure 2).…”