Elsevier

Toxicology Letters

Volume 144, Issue 3, 15 October 2003, Pages 279-288
Toxicology Letters

Short survey
The role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity

https://doi.org/10.1016/S0378-4274(03)00239-XGet rights and content

Abstract

Acetaminophen (AAP) overdose can cause severe hepatotoxicity and even liver failure in experimental animals and humans. Despite substantial efforts over the last 30 years, the mechanism of AAP-induced liver cell injury is still not completely understood. It is widely accepted that the injury process is initiated by the metabolism of AAP to a reactive metabolite, which first depletes glutathione and then binds to cellular proteins including a number of mitochondrial proteins. One consequence of this process may be the observed inhibition of mitochondrial respiration, ATP depletion and mitochondrial oxidant stress. In the presence of sufficient vitamin E, reactive oxygen formation does not induce severe lipid peroxidation but the superoxide reacts with nitric oxide to form peroxynitrite, a powerful oxidant and nitrating agent. Peroxynitrite can modify cellular macromolecules and may aggravate mitochondrial dysfunction and ATP depletion leading to cellular oncotic necrosis in hepatocytes and sinusoidal endothelial cells. Thus, we hypothesize that reactive metabolite formation and protein binding initiate the injury process, which may be then propagated and amplified by mitochondrial dysfunction and peroxynitrite formation. This concept also reconciles many of the controversial findings of the past and provides a viable hypothesis for the mechanism of hepatocellular injury after AAP overdose.

Introduction

Acetaminophen (AAP) is a safe and effective analgesic when used at therapeutic levels. However, an overdose can induce severe hepatotoxicity in experimental animals and in humans (Thomas, 1993). In fact, AAP overdose is the leading cause of drug-induced liver failure requiring transplantation in US (Ostapowicz et al., 2002). Early investigations into the mechanism identified the critical role of glutathione (GSH) in detoxifying the reactive metabolite of AAP (Mitchell et al., 1973a, Mitchell et al., 1973b). These studies led to the clinical use of N-acetylcysteine as standard treatment for patients with AAP overdose. However, despite this initial breakthrough, where the new insight into the mechanism of AAP-induced toxicity in animal studies resulted in the rapid translation of this knowledge into novel therapeutic strategies in the clinic, the mechanism of cell injury and liver failure is still not completely understood. In particular, the role of reactive oxygen species in the pathophysiology remains controversial despite three decades of research. In the present review, we will summarize this historic discussion and, based on more recent findings, we will attempt to reconcile these data and provide a unified hypothesis where protein binding of AAP is the initiating event, which is followed by mitochondrial dysfunction, oxidant stress and peroxynitrite formation, as critical events of an amplification phase of the cell injury mechanism.

Section snippets

Historical perspective: protein binding versus lipid peroxidation (LPO)

The first comprehensive mechanism for AAP hepatotoxicity was published in a series of landmark papers in 1973 (Mitchell et al., 1973a, Mitchell et al., 1973b, Potter et al., 1973, Jollow et al., 1973). These investigators demonstrated that the toxicity involved the activation of AAP to a reactive metabolite (Mitchell et al., 1973a, Jollow et al., 1973), which initially depletes GSH (Mitchell et al., 1973b) and subsequently covalently binds to cellular proteins (Jollow et al., 1973, Potter et

Oxidant stress during AAP-induced liver injury

Criticism of the protein binding theory led to the hypothesis that the overall protein binding of AAP was not as important as the covalent modification of specific vital protein targets in the cell. The groups of Cohen and Khairallah, and Hinson and Pumford developed antibodies against AAP adducts, which were used for immunohistochemistry to localize protein adducts in tissue and also for Western blot analyses (Roberts et al., 1987, Bartolone et al., 1988). Over the years, both groups could

Oxidant stress: cause or consequence of liver cell injury?

It is well-known that severe hepatocellular injury can lead to an intracellular, mitochondria-derived oxidant stress in other experimental models (Jaeschke and Mitchell, 1989). After AAP overdose, significant increases of the hepatic and mitochondrial GSSG levels, indicators of mitochondrial reactive oxygen formation, were only observed after 4–6 h, i.e. after the onset of cell injury (Jaeschke, 1990, Knight et al., 2001). Thus, it is feasible that the oxidant stress could be primarily a

Peroxynitrite formation and protein nitration

Enhanced generation of superoxide in the presence of equimolar concentrations of nitric oxide (NO) will lead to the formation of the potent oxidant and nitrating agent peroxynitrite (Squadrito and Pryor, 1998). The immunohistochemical detection of nitrotyrosine protein adducts in hepatocytes (Hinson et al., 1998) and in sinusoidal endothelial cells (Knight et al., 2001, Knight and Jaeschke, in press) suggested the formation of peroxynitrite after AAP overdose. The staining pattern for

Apoptosis versus oncotic necrosis

Although the mode of AAP-induced cell death was generally considered to be hemorrhagic necrosis (oncosis), several more recent reports suggest that apoptosis may play a significant role in the overall pathophysiology (Ray et al., 1996, Ferret et al., 2001). Oxidant stress and peroxynitrite can induce apoptosis under certain conditions (Hampton and Orrenius, 1998). A careful quantitative analysis of morphological changes in hepatocytes confirmed that the number of apoptotic cells increased

Summary and conclusions

AAP hepatotoxicity is initiated by the metabolic activation of AAP to a reactive metabolite, which first depletes cellular GSH pools (Fig. 1). When the liver GSH levels are exhausted, the reactive metabolite will covalently bind to cellular proteins including proteins of the plasma membrane and mitochondria. This may result in reduced Ca2+ ATPase activities and increased cytosolic Ca2+ levels. The direct effects of modified mitochondrial proteins or increased uptake of Ca2+ can both lead to

Acknowledgements

Work from the authors' laboratory was supported in part by National Institutes of Health grants ES06091 and AA12916.

References (89)

  • H. Jaeschke et al.

    The role of acrolein in allyl alcohol-induced lipid peroxidation and liver cell damage in mice

    Biochem. Pharmacol.

    (1987)
  • H. Jaeschke et al.

    NADH-dependent reductive stress and ferritin-bound iron in allyl alcohol-induced lipid peroxidation in vivo: the protective effect of vitamin E

    Chem. Biol. Interact.

    (1992)
  • T.R. Knight et al.

    Acetaminophen-induced inhibition of Fas receptor-mediated liver cell apoptosis: mitochondrial dysfunction versus glutathione depletion

    Toxicol. Appl. Pharmacol.

    (2002)
  • H. Kuthan et al.

    Generation of superoxide anion as a source of hydrogen peroxide in a reconstituted monooxygenase system

    FEBS Lett.

    (1978)
  • D.L. Laskin et al.

    Potential role of activated macrophages in acetaminophen hepatotoxicity. I. Isolation and characterization of activated macrophages from rat liver

    Toxicol. Appl. Pharmacol.

    (1986)
  • D.L. Laskin et al.

    Modulation of macrophages functioning abrogates the acute hepatotoxicity of acetaminophen

    Hepatology

    (1995)
  • J.A. Lawson et al.

    Inhibition of Fas receptor (CD95)-induced hepatic caspase activation and apoptosis by acetaminophen in mice

    Toxicol. Appl. Pharmacol.

    (1999)
  • L.L. Meyers et al.

    Acetaminophen-induced inhibition of mitochondrial respiration in mice

    Toxicol. Appl. Pharmacol.

    (1988)
  • S.L. Michael et al.

    Acetaminophen-induced hepatotoxicity in mice lacking inducible nitric oxide synthase

    Nitric Oxide

    (2001)
  • N.R. Pumford et al.

    Immunochemical quantitation of 3-(cystein-S-yl)acetaminophen protein adducts in subcellular liver fractions following a hepatotoxic dose of acetaminophen

    Biochem. Pharmacol.

    (1990)
  • Y. Qiu et al.

    Identification of the hepatic protein targets of reactive metabolites of acetaminophen in vivo in mice using two-dimensional gel electrophoresis and mass spectrometry

    J. Biol. Chem.

    (1998)
  • R.R. Ramsay et al.

    In vitro effects of acetaminophen metabolites and analogs on the respiration of mouse liver mitochondria

    Arch. Biochem. Biophys.

    (1989)
  • W. Shen et al.

    Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes: correlation of nuclear Ca2+ accumulation and early DNA fragmentation with cell death

    Toxicol. Appl. Pharmacol.

    (1991)
  • C.V. Smith et al.

    Effect of acetaminophen on hepatic content and biliary efflux of glutathione disulfide in mice

    Chem. Biol. Interact.

    (1989)
  • C.V. Smith et al.

    Acetaminophen hepatotoxicity in vivo is not accompanied by oxidant stress

    Biochem. Biophys. Res. Commun.

    (1985)
  • G.L. Squadrito et al.

    Oxidative chemistry of nitric oxide: the role of superoxide, peroxynitrite and carbon dioxide

    Free Radic. Biol. Med.

    (1998)
  • J.B. Tarloff et al.

    Sex- and age-dependent acetaminophen hepato- and nephrotoxicity in Sprague–Dawley rats: role of tissue accumulation, nonprotein sulfhydryl depletion, and covalent binding

    Fundam. Appl. Toxicol.

    (1996)
  • S.H.L. Thomas

    Paracetamol (acetaminophen) poisoning

    Pharmacol. Ther.

    (1993)
  • M.A. Tirmenstein et al.

    Subcellular binding and effects on calcium homeostasis produced by acetaminophen and a nonhepatotoxic regioisomer, 3′-hydroxyacetanilide, in mouse liver

    J. Biol. Chem.

    (1989)
  • M.A. Tirmenstein et al.

    Acetaminophen-induced oxidation of protein thiols. Contribution of impaired thiol-metabolizing enzymes and the breakdown of adenine nucleotides

    J. Biol. Chem.

    (1990)
  • J.O. Tsokos-Kuhn et al.

    Alkylation of the liver plasma membrane and inhibition of the Ca2+ ATPase by acetaminophen

    Biochem. Pharmacol.

    (1988)
  • S.E. Welty et al.

    Investigation of possible mechanisms of hepatic swelling and necrosis caused by acetaminophen in mice

    Biochem. Pharmacol.

    (1993)
  • A. Wendel et al.

    Drug-induced lipid peroxidation in mice. I. Modulation by monooxygenase activity, glutathione and selenium status

    Biochem. Pharmacol.

    (1981)
  • A. Wendel et al.

    Acute paracetamol intoxication of starved mice leads to lipid peroxidation in vivo

    Biochem. Pharmacol.

    (1979)
  • A. Wendel et al.

    Drug-induced lipid peroxidation in mice. II. Protection against paracetamol-induced liver necrosis by intravenous liposomally entrapped glutathione

    Biochem. Pharmacol.

    (1982)
  • M.L. Adams et al.

    Enhanced acetaminophen hepatotoxicity in transgenic mice overexpressing Bcl-2

    Mol. Pharmacol.

    (2001)
  • M.L. Bajt et al.

    Protection against Fas receptor-mediated apoptosis in hepatocytes and nonparenchymal cells by a caspase-8 inhibitor in vivo: evidence for postmitochondrial processing of caspase-8

    Toxicol. Sci.

    (2000)
  • M.L. Bajt et al.

    Scavenging peroxynitrite with glutathione enhances survival and promotes regeneration after acetaminophen overdose in mice: role of IL-6

    Toxicol. Sci.

    (2003)
  • A.P. Bautista et al.

    Superoxide anion generation in the liver during the early stage of endotoxemia in rats

    J. Leukoc. Biol.

    (1990)
  • S.D. Cohen et al.

    Selective protein arylation and acetaminophen-induced hepatotoxicity

    Drug Metab. Rev.

    (1997)
  • G.B. Corcoran et al.

    Effects of N-acetylcysteine on acetaminophen covalent binding and hepatic necrosis in mice

    J. Pharmacol. Exp. Ther.

    (1985)
  • D.C. Dahlin et al.

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

    Proc. Natl. Acad. Sci. USA

    (1984)
  • C.R. Gardner et al.

    Role of nitric oxide in acetaminophen-induced hepatotoxicity in the rat

    Hepatology

    (1998)
  • J.S. Gujral et al.

    Mode of cell death after acetaminophen overdose in mice: apoptosis or oncotic necrosis?

    Toxicol. Sci.

    (2002)
  • Cited by (393)

    View all citing articles on Scopus
    View full text