PROTECTIVE STRATEGIES AGAINST ACETAMINOPHEN INDUCED HEPATOTOXICITY
University of Kansas
Pharmacology, Toxicology & Therapeutics
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Acetaminophen (APAP) is a widely used analgesic, which is safe at therapeutic levels. APAP is mainly conjugated with glucuronic acid and sulfate to form water-soluble, nontoxic metabolites. Only a small portion of APAP is metabolized by P-450 isoenzymes, thereby forming the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). NAPQI can react with sulfhydryl groups such as GSH. After APAP overdose, hepatic GSH is dramatically reduced, and NAPQI is able to bind to cellular proteins including mitochondrial proteins. This protein binding results in mitochondrial dysfunction and reactive oxygen species formation. This chain of events eventually leads to necrotic cell death of hepatocytes. My goal was to investigate the mechanisms and signaling pathways of APAP-induced cell necrosis in the liver, and to identify therapeutic approaches to prevent liver failure. Three protective strategies were investigated in detail: 1) Glutathione (GSH) and N-acetylcysteine (NAC) 2) Metallothionein (MT) 3) C-jun N-terminal kinase (JNK) inhibitor 1) Both in humans and in experimental animals, NAC is used as an antidote against APAP-induced liver injury. The doses of NAC that are being used clinically and experimentally are higher than needed for re-synthesis of hepatic GSH levels. In fact, our laboratory demonstrated that lower doses of GSH are highly effective in protecting against APAP toxicity. Therefore, I investigated whether there is a difference between the efficacy of NAC and GSH in protecting against APAP hepatotoxicity. Our data indicate that the amino acids supplied with the delayed treatment of the same dose of GSH or NAC are used for the re-synthesis of hepatic glutathione at similar levels, which protect against APAP-induced reactive oxygen species and peroxynitrite in the mitochondria. However, excess amino acids derived from GSH also serve as energy substrates for the Krebs cycle, which results in better protection against APAP hepatotoxicity than NAC treatment. Thus, the optimal protection by delayed GSH or NAC treatment involves the combination of two mechanisms, which are the accelerated recovery of mitochondrial GSH levels and the support of the mitochondrial bioenergetics. 2) Metallothionein (MT) expression attenuates APAP-induced liver injury; however, the mechanism of this protection remains incompletely understood. To address this issue, mice were treated with ZnCl2 for three days to induce MT. Twenty-four hours after the last dose of zinc, the animals received 300 mg/kg APAP. We found that the protective effect of MT in vivo was not due to the direct scavenging of reactive oxygen species and peroxynitrite. In addition, zinc treatment had no effect on the early GSH depletion kinetics after APAP administration, which is an indicator of the metabolic activation of APAP to its reactive metabolite NAPQI. MT was able to effectively trap NAPQI by covalent binding. We conclude that MT scavenges some of the excess NAPQI after GSH depletion and prevents covalent binding to cellular proteins,which is the trigger for the propagation of the cell injury mechanisms through mitochondrial dysfunction and nuclear DNA damage. 3) C-jun N-terminal kinase (JNK) has been suggested to contribute to APAP-induced liver injury. The postulated mechanism of JNK involvement was the promotion of mitochondrial Bax translocation, which triggers mitochondrial outer membrane pore formation and results in the release of intermembrane proteins such as apoptosis inducing factor (AIF) and endonuclease G (EndoG). However, it was reported that Bax-deficient mice were only temporally protected against APAP-induced liver injury (Bajt et al., 2008). In contrast, the protective effect of a JNK inhibitor was observed consistently up to 24 h. Therefore, additional mechanisms of injury involving JNK activation need to be considered. To address this issue, I treated mice with the JNK inhibitor, SP600125 1h before APAP (600 mg/kg). SP600125 reduced peroxynitrite formation; however, it did not have any significant effect on the level of nitrate and nitrite in plasma. Moreover, L-N-(1-iminoethyl)lysine (L-Nil), a specific iNOS inhibitor, attenuated neither plasma nitrate and nitrite levels nor hepatic injury after APAP injection. Taken together, SP600125 reduced peroxynitrite formation by decreasing superoxide formation. In summary, my investigation demonstrated that JNK is a critical factor for Bax translocation, which causes mitochondria outer membrane pore formation. In addition, JNK accelerates peroxynitrite generation via induction of superoxide formation. In conclusion, I demonstrated the efficacy of three protective strategies against APAP-induced hepatotoxicity: Mechanism of protection A: Preventing NAPQI binding to proteins (e.g., induction of MT gene expression); Mechanism of protection B: Scavenging (GSH, NAC) or reducing (JNK inhibition) the formation of reactive oxygen species; and Mechanism of protection C: Supplying mitochondrial energy substrates (GSH, NAC).
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