Evidence for the involvement of N-ACETYL-p-quinoneimine in acetaminophen metabolism

doi:10.1016/0006-2952(79)90123-0

Biochemical Pharmacology

Volume 28, Issue 22, 15 November 1979, Pages 3285-3290

https://doi.org/10.1016/0006-2952(79)90123-0 Get rights and content

Abstract

Evidence for the presence of N-acetyl-p-quinoneimine (NAPQI), a postulated toxic intermediate of acetaminophen metabolism, in mouse liver microsomal incubations is reported. The intermediate was tentatively identified by comparison with synthetic NAPQI generated electrochemically from acetaminophen in a coulometric flow reactor. All but one of the reaction products of NAPQI with a number of nucleophiles were found in vitro as well, and in similar relative amounts. The NAPQI intermediate is moderately stable at physiological pH and temperature with a lifetime which is dependent on the components of the medium.

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Cited by (221)

The role of alcohol consumption on acetaminophen induced liver injury: Implications from a mathematical model
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Acetaminophen (APAP) overdose is one of the predominant causes of drug induced acute liver injury in the U.S and U.K. Clinical studies show that ingestion of alcohol may increase the risk of APAP induced liver injury. Chronic alcoholism may potentiate APAP hepatotoxicity and this increased risk of APAP toxicity is observed when APAP is ingested even shortly after alcohol is cleared from the body. However, clinical reports also suggest that acute alcohol consumption may have a protective effect against hepatotoxicity by inhibiting microsomal acetaminophen oxidation and thereby reducing N-acetyl-p-benzoquinone imine (NAPQI) production. The aim of this study is to model this dual role of alcohol to determine how the timing of alcohol ingestion affects APAP metabolism and resulting liver injury and identify mechanisms of APAP induced liver injury. The mathematical model is developed to capture condition of a patient of single time APAP overdose who may be an acute or chronic alcohol user. The analysis suggests that the risk of APAP-induced hepatotoxicity is increased if APAP is ingested shortly after alcohol is cleared from the body in chronic alcohol users. A protective effect of acute consumption of alcohol is also observed in patients with APAP overdose. For example, simultaneous ingestion of alcohol and APAP overdose or alcohol intake after or before few hours of APAP overdose may result in less APAP-induced hepatotoxicity when compared to a single time APAP overdose. The rate of hepatocyte damage in APAP overdose patients depends on trade-off between induction and inhibition of CYP enzyme.
Human superoxide dismutase 1 attenuates quinoneimine metabolite formation from mefenamic acid
2021, Toxicology
Citation Excerpt :
In addition to the detoxification reaction, some drugs are metabolized to reactive metabolites, which cause various toxicities such as liver injury and skin reactions. For example, it has been suggested that acetaminophen (APAP) and diclofenac (DIC) are metabolized by cytochrome P450 s (P450 or CYP) to produce quinoneimine, then bind to DNA or proteins as the cause of liver injury (Miner and Kissinger, 1979; Poon et al., 2001). Mefenamic acid (MFA), one of the nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibits cyclooxygenase to suppress production of prostaglandin (Byron and Mark, 1998), is used for headaches, arthritis, and dysmenorrhea (Mcguak et al., 1996; Grillo et al., 2012).
Mefenamic acid (MFA), one of the nonsteroidal anti-inflammatory drugs (NSAIDs), sometimes causes liver injury. Quinoneimines formed by cytochrome P450 (CYP)-mediated oxidation of MFA are considered to be causal metabolites of the toxicity and are detoxified by glutathione conjugation. A previous study reported that NAD(P)H:quinone oxidoreductase 1 (NQO1) can reduce the quinoneimines, but NQO1 is scarcely expressed in the human liver. The purpose is to identify enzyme(s) responsible for the decrease in MFA-quinoneimine formation in the human liver. The formation of MFA-quinoneimine by recombinant CYP1A2 and CYP2C9 was significantly decreased by the addition of human liver cytosol, and the extent of the decrease in the metabolite formed by CYP1A2 was larger than that by CYP2C9. By column chromatography, superoxide dismutase 1 (SOD1) was identified from the human liver cytosol as an enzyme decreasing MFA-quinoneimine formation. Addition of recombinant SOD1 into the reaction mixture decreased the formation of MFA-quinoneimine from MFA by recombinant CYP1A2. By a structure-activity relationship study, we found that SOD1 decreased the formation of quinoneimines from flufenamic acid and tolfenamic acid, but did not affect those produced from acetaminophen, amodiaquine, diclofenac, and lapatinib. Thus, SOD1 may selectively decrease the quinoneimine formation from fenamate-class NSAIDs. To examine whether SOD1 can attenuate cytotoxicity caused by MFA, siRNA for SOD1 was transfected into CYP1A2-overexpressed HepG2 cells. The leakage of lactate dehydrogenase caused by MFA treatment was significantly increased by knockdown of SOD1. In conclusion, we found that SOD1 can serve as a detoxification enzyme for quinoneimines to protect from drug-induced toxicity.
Identification of an isoform catalyzing the CoA conjugation of nonsteroidal anti-inflammatory drugs and the evaluation of the expression levels of acyl-CoA synthetases in the human liver
2021, Biochemical Pharmacology
Nonsteroidal anti-inflammatory drugs (NSAIDs) containing carboxylic acid are conjugated with coenzyme A (CoA) or glucuronic acid in the body. It has been suggested that these conjugates are associated with toxicities, such as liver injury and anaphylaxis, through their binding via trans-acylation to cellular proteins. Although studies on glucuronidation have progressed, studies on CoA conjugation of drugs catalyzed by acyl-CoA synthetase (ACS) enzymes are still in the early stages. This study aimed to clarify the human ACS isoforms responsible for CoA-conjugation of NSAIDs through consideration of the hepatic expression levels of ACS isoforms. We found that among 10 types of NSAIDs, propionic acid-class NSAIDs, namely, alminoprofen, flurbiprofen, ibuprofen, ketoprofen, and loxoprofen, were conjugated with CoA in the human liver, whereas NSAIDs in the other classes, including diclofenac and mefenamic acid, were not. qRT-PCR revealed that among the 26 ACS isoforms, ACSL1 was the most highly expressed in the human liver, followed by ACSM2B. The propionic acid-class NSAIDs were conjugated with CoA by recombinant human ACSL1. The protein binding abilities of the CoA conjugates and the glucuronide forms of propionic acid-class NSAIDs were compared as an index of toxicity. The CoA conjugates had stronger adduct formation with liver microsomal proteins than glucuronides for all 5 propionic acid-class NSAIDs. In conclusion, we found that propionic acid-class NSAIDs could be conjugated to CoA by ACSL1 in the human liver to form CoA conjugates, which likely cause toxicity by protein adduct formation.
Electrochemical oxidation of acetaminophen in the presence of diclofenac and piroxicam - Synthesis of new derivatives and kinetic investigation of toxic quinone imine/drugs interactions
2018, Journal of Electroanalytical Chemistry
Citation Excerpt :
This general use of these drugs together, on the one hand, and the importance of their interactions pharmaceutically and therapeutically, on the other hand, stimulates us to assess the possible acetaminophen/diclofenac acetaminophen/piroxicam interactions. It has been shown that N-acetyl-p-benzoquinone-imine (NAPQI) is the main in-vivo and in-vitro oxidation product of acetaminophen [18–20]. Previously reported studies show that the electrochemically generated NAPQI is a toxic and reactive intermediate and it participates in different types of reactions [20–25].
In this research, electrochemical oxidation of acetaminophen was investigated in the presence of steroidal anti-inflammatory drugs diclofenac and piroxicam by cyclic voltammetry and controlled-potential coulometry techniques. The results indicated that N-acetyl-p-benzoquinone-imine (NAPQI), produced by electrooxidation of acetaminophen, reacts with diclofenac and piroxicam, via the Michael addition reaction. Corresponding products were electrochemically synthesized in aqueous solutions using a carbon electrode in an undivided cell. These products were identified by spectroscopic methods (FT-IR, ¹HNMR, ¹³CNMR and MS). The homogeneous rate constants (k_obs), based on the suggested electrode mechanism (i.e. EC), were estimated by comparing the experimental cyclic voltammetric responses with those digital simulated results. A comparison of the estimated k_obs for the reaction of electrochemically generated N-acetyl-p-benzoquinone-imine (NAPQI) with steroidal anti-inflammatory drugs revealed that k_{obs (piroxicam)} > k_{obs(diclofenac)} at biological pH.
Timescale analysis of a mathematical model of acetaminophen metabolism and toxicity
2015, Journal of Theoretical Biology
Acetaminophen is a widespread and commonly used painkiller all over the world. However, it can cause liver damage when taken in large doses or at repeated chronic doses. Current models of acetaminophen metabolism are complex, and limited to numerical investigation though provide results that represent clinical investigation well. We derive a mathematical model based on mass action laws aimed at capturing the main dynamics of acetaminophen metabolism, in particular the contrast between normal and overdose cases, whilst remaining simple enough for detailed mathematical analysis that can identify key parameters and quantify their role in liver toxicity. We use singular perturbation analysis to separate the different timescales describing the sequence of events in acetaminophen metabolism, systematically identifying which parameters dominate during each of the successive stages. Using this approach we determined, in terms of the model parameters, the critical dose between safe and overdose cases, timescales for exhaustion and regeneration of important cofactors for acetaminophen metabolism and total toxin accumulation as a fraction of initial dose.
Mini-dialysis tubes as tools to prepare drug-protein adducts of P450-dependent reactive drug metabolites
2015, Journal of Pharmaceutical and Biomedical Analysis
Citation Excerpt :
Madsen and co-workers formed the reactive intermediate of APAP (N-acetyl-p-benzoquinone imine) by electrochemical oxidation and reported a half-life of 47 min in 0.1 M phosphate buffer (pH 7.4) in the absence of biological material [34]. However, N-acetyl-p-benzoquinone imine was found to have a half-life of less than 10 s in presence of microsomes [35], which may explain the absence of hGST P1-1 adducts of APAP in the current study. Similarly, the previously observed hGST P1-1 adducts of the CLZ nitrenium ion were not detected upon CLZ bioactivation in the present work [11].
The modification of critical cellular proteins by reactive metabolites (RMs) resulting from P450-dependent drug bioactivation is considered essential to the onset of many idiosyncratic drug reactions. In this study, we report a novel method that can be used to prepare and study drug-protein adducts. Drug bioactivation by P450s was performed in a small container containing a mini-dialysis tube with the model target protein human glutathione-S-transferase P1-1 (hGST P1-1), allowing RMs to translocate from P450 to hGST P1-1 via a semi-permeable membrane (6–8 kDa). GST P1-1 modification was evaluated by LC–MS analysis of intact protein adducts and following digestion of protein with trypsin. As proof of principle, the described methodology was first applied to the direct electrophile monochlorobimane. A highly active P450 BM3 mutant (CYP102A1M11H) was subsequently used for bioactivation of acetaminophen, clozapine, diclofenac (DF) and mefenamic acid (MFA), but hGST P1-1 adducts were only observed for the latter two drugs. CYP2C9 and CYP3A4, which metabolize DF to p-benzoquinone imines, were tested to investigate the applicability of human P450s. Finally, it was evaluated whether bioactivation of MFA by human and rat liver microsomes resulted in modification of hGST P1-1. The results show that our adduct preparation method can also be used in combination with membrane-bound P450 bioactivation systems, as long as formed RMs have sufficient life-time to reach hGST P1-1 inside the dialysis tube.

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Evidence for the involvement of N-ACETYL-p-quinoneimine in acetaminophen metabolism

Abstract

J. electroanal. Chem.

Electrochim. Acta

J. pharm. Sci.

J. electroanal. Chem.

Analyt. Biochem.

Biochem. Pharmac.

Pharmacology

J. med. Chem.