N-Hydroxyacetaminophen: a postulated toxic metabolite of acetaminophen

Calder, I. C.; Hart, S. J.; Healey, K.; Ham, Kathryn N.

doi:10.1021/jm00140a014

Cited by 31 publications

(9 citation statements)

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“…However, even in the LS pathway, the energy barrier of 29.6 kcal/mol (PCM) for the FeO oxygen addition to the PAR anilide nitrogen is too large to contribute significantly to the P450-catalyzed NAPQI formation. The latter may also explain the lack of reports of N-hydroxy-PAR 4 (with a half-life of 15 min) 35 as a metabolic precursor of NAPQI. Accordingly, the OAR mechanism also does not offer a kinetically efficient way for the metabolic conversion of PAR to NAPQI.…”

Section: ■ Computational Methodologymentioning

confidence: 99%

Computational Biotransformation Profile of Paracetamol Catalyzed by Cytochrome P450

Schüürmann

2015

Chem. Res. Toxicol.

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The P450-catalyzed biotransformation of the analgesic drug paracetamol (PAR) is a long-debated topic, involving different mechanistic hypotheses as well as experimental evidence for the metabolites N-acetyl-p-benzoquinone imine (NAPQI), p-benzoquinone, acetamide, and 3-hydroxy-PAR. During the catalytic cycle of P450, a high-valent iron(IV)-oxo species known as Compound I (Cpd I) is formed as the ultimate oxidant, featuring two energetically close-lying ground states in the doublet (low-spin) and quartet (high-spin) spin states, respectively. In order to clarify the catalytic mechanism, a computational chemistry analysis has been undertaken for both the high- and low-spin routes, employing density functional theory (DFT) including PCM (polarized continuum-solvation model) that yields an approximate simulation of the bulk polarization exerted through the protein. The results demonstrate that hydrogen abstraction transfer (HAT) by the P450 oxidant Cpd I (FeO) is kinetically strongly preferred over the alternative pathways of an oxygen addition reaction (OAR) or two consecutive single-electron transfers (SET). Moreover, only the respective high-spin route yields N-acetyl-p-semiquinone imine (NAPSQI) as an intermediate that is converted to the electrophile N-acetyl-p-benzoquinone imine (NAPQI). By contrast, 3-hydroxy-PAR, acetamide, and p-benzoquinone as electrophilic and redox-active agent are formed predominantly in the low-spin state through reactions that do not involve NAPSQI. Thus, all experimentally observed PAR metabolites are in accord with an initial HAT from the phenolic oxygen, and NAPSQI should indeed be the precursor of NAPQI, both of which are generated only via the high-spin pathway.

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Section: ■ Computational Methodologymentioning

confidence: 99%

Computational Biotransformation Profile of Paracetamol Catalyzed by Cytochrome P450

Schüürmann

2015

Chem. Res. Toxicol.

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“…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.…”

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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

Dahlin¹,

Miwa²,

Lu³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

923

533

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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...

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“…This mechanism was subsequently modified following reports by Dr. Ian Calder's laboratory in Australia that N-acetyl-p-benzoquinone imine (NAPQI) was an electrophilic species (Calder et al 1973;Calder et al 1974;Calder et al 1981). They were able to synthesize it by oxidation with lead tetra-acetate, but it rapidly decomposed.…”

Section: Studies On the Mechanism Of Metabolic Activationmentioning

confidence: 99%

The development and hepatotoxicity of acetaminophen: reviewing over a century of progress

McGill

Hinson

2020

Drug Metabolism Reviews

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Acetaminophen (APAP) was first synthesized in the 1800s, and came on the market approximately 65 years ago. Since then, it has become one of the most used drugs in the world. However, it is also a major cause of acute liver failure. Early investigations of the mechanisms of toxicity revealed that cytochrome P450 enzymes catalyze formation of a reactive metabolite in the liver that depletes glutathione and covalently binds to proteins. That work led to the introduction of N acetylcysteine (NAC) as an antidote for APAP overdose. Subsequent studies identified the reactive metabolite N-acetyl-p-benzoquinone imine, specific P450 enzymes involved, the mechanism of P450-mediated oxidation, and major adducted proteins. The mechanisms downstream of metabolism remain poorly understood but several events appear critical. These events include development of an initial oxidative stress, reactive nitrogen formation, altered calcium flux, JNK activation and mitochondrial translocation, inhibition of mitochondrial respiration, the mitochondrial permeability transition, and nuclear DNA fragmentation. Additional research is necessary to fill remaining gaps in our knowledge of the toxicity, such as the source of the initial oxidative stress, and to improve our understanding liver regeneration after APAP overdose. A better understanding of these mechanisms may lead to additional treatment options. Even though NAC is an excellent antidote, its effectiveness is limited to the first 16 hours following overdose.

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N-Hydroxyacetaminophen: a postulated toxic metabolite of acetaminophen

Cited by 31 publications

References 0 publications

Computational Biotransformation Profile of Paracetamol Catalyzed by Cytochrome P450

Computational Biotransformation Profile of Paracetamol Catalyzed by Cytochrome P450

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

The development and hepatotoxicity of acetaminophen: reviewing over a century of progress

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