Gastroenterology

Gastroenterology

Volume 131, Issue 1, July 2006, Pages 165-178
Gastroenterology

Basic–liver, pancreas, and biliary tract
c-Jun N-Terminal Kinase Plays a Major Role in Murine Acetaminophen Hepatotoxicity

https://doi.org/10.1053/j.gastro.2006.03.045Get rights and content

Background & Aims: In searching for effects of acetaminophen (APAP) on hepatocytes downstream of its metabolism that may participate in hepatotoxicity, we examined the role of stress kinases. Methods: Mouse hepatocytes and C57BL/6 mice were administered a toxic dose of APAP with or without SP600125, a chemical c-jun N-terminal kinase (JNK) inhibitor. JNK activity as reflected in phospho-c-jun levels, serum alanine transaminase (ALT), and liver histology were assessed. Similar experiments were repeated in JNK1 and JNK2 knockout mice and by using antisense oligonucleotide (ASO) to knockdown JNK. Results: Sustained activation of JNK was observed in cultured mouse hepatocytes and in vivo in the liver after APAP treatment. The importance of this pathway was identified by the marked protective effect of SP600125 against APAP toxicity in vitro and in vivo. The specificity of this protective effect was confirmed in vivo by the knockdown of JNK1 and 2 using ASO pretreatment. JNK2 knockout mice and mice treated with JNK2 ASO exhibited partial protection against APAP. One potential target of JNK is Bax translocation, which was enhanced by APAP and blocked by the JNK inhibitor. Protection by the JNK inhibitor persisted in Kupffer cell-depleted mice, whereas there was no protection against CCl4 or concanavalin A toxicity. Conclusions: This work suggests that JNK acts downstream of APAP metabolism to promote hepatotoxicity. The results suggest that JNK2 plays a predominant role, although maximum protection was seen with decrease in both forms of JNK.

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Materials

JNK inhibitor SP600125, p38 inhibitor SB203580, and ERK inhibitor PD98059 were purchased from Calbiochem (San Diego, CA). Antisense oligonucleotides (ASO) targeting mouse JNK1 (Isis 104492), JNK2 (Isis 101759), and a chemical control oligonucleotide (Isis 141923) were synthesized as 20 nucleotide, uniform phosphorothioate chimeric oligonucleotides and purified as previously described.4 The oligonucleotides used in these studies were chimeric oligonucleotides containing 5 nuclease resistant

Effect of JNK Inhibitor, SP600125, on APAP-Induced Toxicity in Murine Hepatocytes

First, we confirmed the previous finding that 1-hour pretreatment with JNK inhibitor (20 μmol/L) provided significant protection (>50%) against 5 mmol/L APAP-induced necrosis (not shown). Equimolar concentrations of inhibitors of MAP kinases, p38 and Erk, did not provide significant protection against APAP-induced necrosis determined after 15 hours (not shown). Protection by the JNK inhibitor was not accompanied by a change in the extent of maximum GSH depletion Figure 1 (Figure 1A) or [14C]

Discussion

Conflicting evidence for a role of JNK in APAP toxicity has been published previously. In C6 glioma cells, APAP cytotoxicity was dependent on JNK activity,25 whereas, in Hep G2 cells, APAP treatment caused JNK activation, but inhibition of JNK did not afford protection against cytotoxicity.26 However, the findings in the present report unequivocally establish a role for JNK activation in APAP hepatotoxicity. In both cultured hepatocytes and in vivo livers, treatment with APAP induced a

References (56)

  • S. Eminel et al.

    JNK2 translocates to the mitochondria and mediates cytochrome c release in PC12 cells in response to 6-hydroxydopamine

    J Biol Chem

    (2004)
  • S. Kharbanda et al.

    Translocation of SAPK/JNK to mitochondria and interaction with Bcl-XL in response to DNA damage

    J Biol Chem

    (2000)
  • M. Fan et al.

    Vinblastine-induced phosphorylation of Bcl2 and Bcl-XL is mediated by JNK and occurs in parallel with inactivation of the Raf-1/MEK/ERK cascade

    J Biol Chem

    (2000)
  • H. El-Hassan et al.

    Involvement of mitochondria in acetaminophen-induced apoptosis and hepatic injuryRoles of cytochrome c, Bax, Bid, and caspases

    Toxicol Appl Pharmacol

    (2003)
  • M. Czaja et al.

    Prevention of carbon tetrachloride-induced rat liver injury by soluble tumor necrosis factor receptor

    Gastroenterology

    (1995)
  • C.R. Gardner et al.

    Exaggerated hepatotoxicity of acetaminophen in mice lacking tumor necrosis factor receptor-1Potential role of inflammatory mediators

    Toxicol Appl Pharmacol

    (2003)
  • T. Uehara et al.

    JNK mediates hepatis ischemia perfusion injury

    J Hepatol

    (2005)
  • S.D. Nelson et al.

    Mechanisms of acetaminophen-induced liver disease

  • J.C. Fernández-Checa et al.

    Impaired uptake of GSH by hepatic mitochondria from chronic ethanol-fed ratsTracer kinetic studies in vitro and in vivo and susceptibility to oxidant stress

    J Clin Invest

    (1991)
  • D.J. Jollow et al.

    Acetaminophen-induced hepatic necrosis. IIRole of covalent binding in vivo

    J Pharmacol Exp Ther

    (1973)
  • D. Han et al.

    Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space

    Biochem J

    (2001)
  • M. Wood et al.

    Halothane-induced hepatic necrosis in triiodothyronine-pretreated rats

    Anesthesiology

    (1980)
  • N. Van Rooijen et al.

    Liposome-mediated depletion of macrophagesmechanism of action, preparation of liposomes and applications

    J Immunol Methods

    (1994)
  • J.M. Austyn et al.

    F4/80, a monoclonal antibody directed specifically against the mouse macrophage

    Eur J Immunol

    (1981)
  • G.L. Su

    Lipopolysaccharides in liver injurymolecular mechanisms of Kupffer cell activation

    Am J Physiol Gastrointest Liver Physiol

    (2002)
  • C. Ji et al.

    Role of TNF-α in ethanol-induced hyperhomocysteinemia and murine alcoholic liver injury

    Hepatology

    (2004)
  • J. Bain et al.

    The specificities of protein kinase inhibitorsan update

    Biochem J

    (2003)
  • F. Tsuruta et al.

    JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins

    EMBO J

    (2004)
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    Supported by NIH grants DK30312 and DK48522; a postdoctoral training award from the Los Angeles Chapter of The American Liver Foundation (to B.K.G.); a postdoctoral training award from the Garrie-Budnick Foundation (to D.H.); and an NIH KO1 award (DK67149; to Z-X.L.).

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