The covalent binding of acetaminophen to protein. Evidence for cysteine residues as major sites of arylation in vitro

doi:10.1016/0009-2797(84)90145-5

Chemico-Biological Interactions

Volume 48, Issue 3, March 1984, Pages 349-366

https://doi.org/10.1016/0009-2797(84)90145-5 Get rights and content

Abstract

Covalent binding of the reactive metabolite of acetaminophen has been investigated in hepatic microsomal preparations from phenobarbital-pretreated mice. Low molecular weight thiols (cysteine and glutathione) were found to inhibit this binding, whereas several other amino acids which were tested did not. Bovine serum albumin (BSA), which contains a single free sulfhydryl group per molecule and which thus represents a macromolecular thiol compound, inhibited covalent binding of the reactive acetaminophen metabolite to microsomal protein in a concentration-dependent manner. The acetaminophen metabolite also became irreversibly bound to BSA in these experiments, although this binding was reduced by approx. 47% when the thiol function of BSA was selectively blocked prior to incubation. Covalent binding of the acetaminophen metabolite to bovine α_s1-casein, a soluble protein which does not contain any cysteine residues, was found to occur to an extent of 37% of that which became bound to native BSA. These results were taken to indicate that protein thiol groups are major sites of covalent binding of the reactive metabolite of acetaminophen in vitro. The covalent binding characteristics of synthetic N-acetyl-p-benzoquinoneimine (NAPQI), the putative electrophilic intermediate produced during oxidative metabolism of acetaminophen, paralleled closely those of the reactive species generated metabolically. These findings support the contention that NAPQI is indeed the reactive arylating metabolite of acetaminophen which binds irreversibly to protein.

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I read with interest the article “DNA-binding activities of compounds acting as enzyme inhibitors, ion channel blockers and receptor binders” recently published in Chemico-Biological Interactions. The authors suggested that acetaminophen, one of the most used drugs worldwide, alkylates DNA at therapeutic doses and is genotoxic. Given the implications of this statements for public health, it is important for the reader to hear a different perspective that is based on the entire literature on this subject. Everything considered, there is no credible evidence that acetaminophen is a genotoxic hazard or a carcinogen at therapeutic doses.
Amlexanox ameliorates acetaminophen-induced acute liver injury by reducing oxidative stress in mice
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Amlexanox, a clinically approved small-molecule therapeutic presently used to treat allergic rhinitis, ulcer, and asthma, is an inhibitor of the noncanonical IkB kinase-ε (IKKε) and TANK-binding kinase 1 (TBK1). This study was to investigate the protective mechanism of amlexanox in acetaminophen (APAP)-induced acute liver injury (ALI). Mice were intraperitoneally injected with APAP (300 mg/kg, 12 h) to induce ALI and were orally administrated with amlexanox (25, 50 and 100 mg/kg) one hour after APAP treatment. Inhibition of IKKε and TBK1 by treatment of amlexanox attenuated APAP-induced ALI as confirmed by decreased serum levels of aspartate aminotransferase and alanine aminotransferase. Furthermore, amlexanox significantly decreased hepatocellular apoptosis in injured livers of mice as evidenced by histopathologic observation. Consistently, reduced oxidative stress by amlexanox was observed by increased hepatic glutathione concomitant with decreased levels of malondialdehyde. Amlexanox also enhanced expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) target genes including heme oxygenase 1, NAD(P)H:quinone oxidoreductase 1, and glutamate-cysteine ligase in injured livers of mice. Mechanistic insights into the mode of action of amlexanox against APAP-induced hepatotoxicity were involved in increasing phosphorylation of AMP-activated protein kinase (AMPK) and nuclear translocation of Nrf2, both in vivo and in vitro. Furthermore, the protective effects of amlexanox on APAP-induced hepatotoxicity were abolished by compound C, an AMPK inhibitor. Taken together, our findings suggest that amlexanox exerts antioxidative activities against APAP-mediated hepatotoxicity via AMPK/Nrf2 pathway.
Mass isotopomer-guided decluttering of metabolomic data to visualize endogenous biomarkers of drug toxicity
2018, Biochemical Pharmacology
Metabolomics offers the opportunity to uncover endogenous biomarkers that can lead to metabolic pathways and networks and that underpin drug toxicity mechanisms. A novel protocol is presented and discussed that is applicable to drugs which generate urinary metabolites when administered to mice sensitive to its toxicity. The protocol would not apply to drugs that are not metabolized or eliminated by a different route. Separate stable isotope-labeled and unlabeled drug administration to mice is made together with collection of urines from control animals. Untargeted mass spectrometry-based metabolomic analysis of these three urine groups is conducted in addition to principal components analysis (PCA). In the case of unlabeled acetaminophen and [acetyl-²H₃]acetaminophen, each given at a hepatotoxic dose (400 mg/kg i.p.) to the sensitive mouse strain (wild-type 129), the PCA loadings plot showed a distribution of ions in the shape of a “fallen-Y” with the deuterated metabolites in one arm and the paired nondeuterated metabolites in the other arm of the fallen-Y. Ions corresponding to the endogenous toxicity biomarkers sat in the mouth of the fallen-Y. This protocol represents an innovative means to separate endogenous biomarkers from drug metabolites, thereby aiding the identification of biomarkers of drug toxicity. For acetaminophen, increased hepatic oxidative stress, mitochondrial damage, Ca²⁺ signaling, heme catabolism, and saturation of glucuronidation, together with decreased fatty acid β-oxidation and cellular energy dysregulation were all implied from the discovered biomarkers. The protocol can be applied to other drugs and may now be translated to clinical studies.
Experimental models of hepatotoxicity related to acute liver failure
2016, Toxicology and Applied Pharmacology
Citation Excerpt :
To effectively use the mouse model, a few issues need to be considered (Fig. 2). First, the mechanism of toxicity depends on the formation of a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), by the cytochrome P450 (CYP) enzyme system (Dahlin et al., 1984; Mitchell et al., 1973a; Streeter et al., 1984), especially CYP2E1 (Lee et al., 1996; McClain et al., 1980; Sato and Lieber, 1981; Sato et al., 1981). NAPQI is detoxified by GSH (Albano et al., 1985; Corcoran and Wong, 1986; Mitchell et al., 1973b; Rosen et al., 1984), but can equally bind to cysteine sulfhydryl groups of proteins (Hoffmann et al., 1985; Jollow et al., 1973; Streeter et al., 1984).
Acute liver failure can be the consequence of various etiologies, with most cases arising from drug-induced hepatotoxicity in Western countries. Despite advances in this field, the management of acute liver failure continues to be one of the most challenging problems in clinical medicine. The availability of adequate experimental models is of crucial importance to provide a better understanding of this condition and to allow identification of novel drug targets, testing the efficacy of new therapeutic interventions and acting as models for assessing mechanisms of toxicity. Experimental models of hepatotoxicity related to acute liver failure rely on surgical procedures, chemical exposure or viral infection. Each of these models has a number of strengths and weaknesses. This paper specifically reviews commonly used chemical in vivo and in vitro models of hepatotoxicity associated with acute liver failure.
Assessment of protein modifications in liver of rats under chronic treatment with paracetamol (acetaminophen) using two complementary mass spectrometry-based metabolomic approaches
2015, Journal of Proteomics
Liver protein can be altered under paracetamol (APAP) treatment. APAP-protein adducts and other protein modifications (oxidation/nitration, expression) play a role in hepatotoxicity induced by acute overdoses, but it is unknown whether liver protein modifications occur during long-term treatment with non-toxic doses of APAP. We quantified APAP-protein adducts and assessed other protein modifications in the liver from rats under chronic (17 days) treatment with two APAP doses (0.5% or 1% of APAP in the diet w/w). A targeted metabolomic method was validated and used to quantify APAP-protein adducts as APAP-cysteine adducts following proteolytic hydrolysis. The limit of detection was found to be 7 ng APAP–cysteine/mL hydrolysate i.e. an APAP–Cys to tyrosine ratio of 0.016‰. Other protein modifications were assessed on the same protein hydrolysate by untargeted metabolomics including a new strategy to process the data and identify discriminant molecules. These two complementary mass spectrometry (MS)-based metabolic approaches enabled the assessment of a wide range of protein modifications induced by chronic treatment with APAP.
APAP-protein adducts were detected even in the absence of glutathione depletion and hepatotoxicity, i.e. in the 0.5% APAP group, and increased by 218% in the 1% APAP group compared to the 0.5% APAP group. At the same time, the untargeted metabolomic method revealed a decrease in the binding of cysteine, cysteinyl–glycine and GSH to thiol groups of protein cysteine residues, an increase in the oxidation of tryptophan and proline residues and a modification in protein expression. This wide range of modifications in liver proteins occurred in rats under chronic treatment with APAP that did not induce hepatotoxicity.
Increased mitochondrial ROS formation by acetaminophen in human hepatic cells is associated with gene expression changes suggesting disruption of the mitochondrial electron transport chain
2015, Toxicology Letters
Acetaminophen (APAP) overdosage results in hepatotoxicity, but the underlying molecular mechanisms are still not completely understood. In the current study, we focused on mitochondrial-specific oxidative liver injury induced by APAP exposure. Owning to genetic polymorphisms in the CYP2E1 gene or varying inducibility by xenobiotics, the CYP2E1 mRNA level and protein activity vary extensively among individuals. As CYP2E1 is a known ROS generating enzyme, we chose HepG2 to minimize CYP2E1-induced ROS formation, which will help us better understand the APAP induced mitochondrial-specific hepatotoxicity in a subpopulation with low CYP2E1 activity. HepG2 cells were exposed to a low and toxic dose (0.5 and 10 mM) of APAP and analyzed at four time points for genome-wide gene expression. Mitochondria were isolated and electron spin resonance spectroscopy was performed to measure the formation of mitochondrial ROS. The yield of ATP was measured to confirm the impact of the toxic dose of APAP on cellular energy production. Our results indicate that 10 mM APAP significantly influences the expression of mitochondrial protein-encoding genes in association with an increase in mitochondrial ROS formation. Additionally, 10 mM APAP affects the expression of genes encoding the subunits of electron transport chain (ETC) complexes, which may alter normal mitochondrial functions by disrupting the assembly, stability, and structural integrity of ETC complexes, leading to a measurable depletion of ATP, and cell death. The expression of mitochondrium-specific antioxidant enzyme, SOD2, is reduced which may limit the ROS scavenging ability and cause imbalance of the mitochondrial ROS homeostasis. Overall, transcriptome analysis reveals the molecular processes involved in the observed APAP-induced increase of mitochondrial ROS formation and the associated APAP-induced oxidative stress.

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Chemico-Biological Interactions

Abstract

References (27)

Plasma paracetamol half-life and hepatic necrosis in patients with paracetamol overdosage

Lancet

Quantitative determination of the glutathione, cysteine, and N-acetyl cysteine conjugates of acetaminophen by high-pressure liquid chromatography

Anal. Biochem.

Drug residue formation from ronidazole, a 5-nitroimidazole. III. Studies on the mechanism of protein alkylation in vitro

Chem.-Biol. Interact.

Tissue sulfhydryl groups

Arch. Biochem. Biophys.

Protein measurement with the Folin phenol reagent

J. Biol. Chem.

Structural studies and organic ligand-binding properties of bovine plasma albumin

J. Biol. Chem.

On the sulfhydryl group of human plasma albumin

J. Biol. Chem.

Separation of multiple forms of protein-bound metabolites of the carcinogenic hydrocarbons 3-methylcholanthrene and 7,12-dimethylbenz[a]anthracene from several rodent species

Chem.-Biol. Interact.

Covalent modification of lysine during the suicide inactivation of rat liver cytochrome P-450 by chloramphenicol

Biochem. Pharmacol.

Mass spectrometric studies of a modified active-site tetrapeptide from Δ^s-3-ketosteroid isomerase of Pseudomonas testosteroni

J. Biol. Chem.

Liver necrosis from paracetamol

Br. J. Pharmacol. Chemother.

Toxic drug reactions

Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione

J. Pharmacol. Exp. Ther.

Cited by (139)

Comments on “DNA-binding activities of compounds acting as enzyme inhibitors, ion channel blockers and receptor binders.”

Amlexanox ameliorates acetaminophen-induced acute liver injury by reducing oxidative stress in mice

Mass isotopomer-guided decluttering of metabolomic data to visualize endogenous biomarkers of drug toxicity

Experimental models of hepatotoxicity related to acute liver failure

Assessment of protein modifications in liver of rats under chronic treatment with paracetamol (acetaminophen) using two complementary mass spectrometry-based metabolomic approaches

Increased mitochondrial ROS formation by acetaminophen in human hepatic cells is associated with gene expression changes suggesting disruption of the mitochondrial electron transport chain