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Acquired resistance to acetaminophen hepatotoxicity is associated with induction of multidrug resistance-associated protein 4 (Mrp4) in proliferating hepatocytes.

ABSTRACT: Treatment with hepatotoxicants such as acetaminophen (APAP) causes resistance to a second, higher dose of the same toxicant (autoprotection). APAP induces hepatic mRNA and protein levels of the multidrug resistance-associated proteins (Mrp) transporters in mice and humans. Basolateral efflux transporters Mrp3 and Mrp4 are the most significantly induced. We hypothesized that upregulation of Mrp3 and Mrp4 is one mechanism by which hepatocytes become resistant to a subsequent higher dose of APAP by limiting accumulation of xeno-, endobiotics, and byproducts of hepatocellular injury. The purpose of this study was to evaluate Mrp3 and Mrp4 expression in proliferating hepatocytes in a mouse model of APAP autoprotection. Plasma and livers were collected from male C57BL/6J mice treated with APAP 400 mg/kg for determination of hepatotoxicity and protein expression. Maximal Mrp3 and Mrp4 induction occurred 48 h after APAP. Mrp4 upregulation occurred selectively in proliferating hepatocytes. Additional groups of APAP-pretreated mice were challenged 48 h later with a second, higher dose of APAP. APAP-pretreated mice had reduced hepatotoxicity after APAP challenge compared to those pretreated with vehicle. A more rapid recovery of glutathione (GSH) in APAP-pretreated mice corresponded with increases in GSH synthetic enzymes. Interestingly, mice pretreated and challenged with APAP had dramatic increases in Mrp4 expression as well as enhanced hepatocyte proliferation. Inhibition of hepatocyte replication with colchicine not only restored sensitivity of APAP-pretreated mice to injury, but also blocked Mrp4 induction. Mrp4 overexpression may be one phenotypic property of proliferating hepatocytes that protects against subsequent hepatotoxicant exposure by mechanisms that are presently unknown.

SUBMITTER: Aleksunes LM

PROVIDER: S-EPMC2734298 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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Acquired resistance to acetaminophen hepatotoxicity is associated with induction of multidrug resistance-associated protein 4 (Mrp4) in proliferating hepatocytes.

Aleksunes Lauren M LM Campion Sarah N SN Goedken Michael J MJ Manautou José E JE

Toxicological sciences : an official journal of the Society of Toxicology 20080508 2

Treatment with hepatotoxicants such as acetaminophen (APAP) causes resistance to a second, higher dose of the same toxicant (autoprotection). APAP induces hepatic mRNA and protein levels of the multidrug resistance-associated proteins (Mrp) transporters in mice and humans. Basolateral efflux transporters Mrp3 and Mrp4 are the most significantly induced. We hypothesized that upregulation of Mrp3 and Mrp4 is one mechanism by which hepatocytes become resistant to a subsequent higher dose of APAP by ...[more]

PMID: 18468992

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Project description:Background & aimsAcetaminophen (APAP) overdose remains a frequent cause of acute liver failure, which is generally accompanied by increased levels of serum bile acids (BAs). However, the pathophysiological role of BAs remains elusive. Herein, we investigated the role of BAs in APAP-induced hepatotoxicity.MethodsWe performed intravital imaging to investigate BA transport in mice, quantified endogenous BA concentrations in the serum of mice and patients with APAP overdose, analyzed liver tissue and bile by mass spectrometry and MALDI-mass spectrometry imaging, assessed the integrity of the blood-bile barrier and the role of oxidative stress by immunostaining of tight junction proteins and intravital imaging of fluorescent markers, identified the intracellular cytotoxic concentrations of BAs, and performed interventions to block BA uptake from blood into hepatocytes.ResultsPrior to the onset of cell death, APAP overdose causes massive oxidative stress in the pericentral lobular zone, which coincided with a breach of the blood-bile barrier. Consequently, BAs leak from the bile canaliculi into the sinusoidal blood, which is then followed by their uptake into hepatocytes via the basolateral membrane, their secretion into canaliculi and repeated cycling. This, what we termed 'futile cycling' of BAs, led to increased intracellular BA concentrations that were high enough to cause hepatocyte death. Importantly, however, the interruption of BA re-uptake by pharmacological NTCP blockage using Myrcludex B and Oatp knockout strongly reduced APAP-induced hepatotoxicity.ConclusionsAPAP overdose induces a breach of the blood-bile barrier which leads to futile BA cycling that causes hepatocyte death. Prevention of BA cycling may represent a therapeutic option after APAP intoxication.Lay summaryOnly one drug, N-acetylcysteine, is approved for the treatment of acetaminophen overdose and it is only effective when given within ∼8 hours after ingestion. We identified a mechanism by which acetaminophen overdose causes an increase in bile acid concentrations (to above toxic thresholds) in hepatocytes. Blocking this mechanism prevented acetaminophen-induced hepatotoxicity in mice and evidence from patients suggests that this therapy may be effective for longer periods after ingestion compared to N-acetylcysteine.

Project description:Acetaminophen (APAP) is one of the most popular and safe pain medications worldwide. However, due to its wide availability, it is frequently implicated in intentional or unintentional overdoses where it can cause severe liver injury and even acute liver failure (ALF). In fact, APAP toxicity is responsible for 46% of all ALF cases in the United States. Early mechanistic studies in mice demonstrated the formation of a reactive metabolite, which is responsible for hepatic glutathione depletion and initiation of the toxicity. This insight led to the rapid introduction of N-acetylcysteine as a clinical antidote. However, more recently, substantial progress was made in further elucidating the detailed mechanisms of APAP-induced cell death. Mitochondrial protein adducts trigger a mitochondrial oxidant stress, which requires amplification through a MAPK cascade that ultimately results in activation of c-jun N-terminal kinase (JNK) in the cytosol and translocation of phospho-JNK to the mitochondria. The enhanced oxidant stress is responsible for the membrane permeability transition pore opening and the membrane potential breakdown. The ensuing matrix swelling causes the release of intermembrane proteins such as endonuclease G, which translocate to the nucleus and induce DNA fragmentation. These pathophysiological signaling mechanisms can be additionally modulated by removing damaged mitochondria by autophagy and replacing them by mitochondrial biogenesis. Importantly, most of the mechanisms have been confirmed in human hepatocytes and indirectly through biomarkers in plasma of APAP overdose patients. The extensive necrosis caused by APAP overdose leads to a sterile inflammatory response. Although recruitment of inflammatory cells is necessary for removal of cell debris in preparation for regeneration, these cells have the potential to aggravate the injury. This review touches on the newest insight into the intracellular mechanisms of APAP-induced cells death and the resulting inflammatory response. Furthermore, it discusses the translation of these findings to humans and the emergence of new therapeutic interventions.

Project description:Drug-induced liver injury (DILI) is a major clinical problem where natural compounds hold promise for its abrogation. Khaya grandifoliola (Meliaceae) is used in Cameroonian traditional medicine for the treatment of liver related diseases and has been studied for its hepatoprotective properties. Till date, reports showing the hepatoprotective molecular mechanism of the plant are lacking. The aim of this study was therefore to identify compounds from the plant bearing hepatoprotective activity and the related molecular mechanism by assessing their effects against acetaminophen (APAP)-induced hepatotoxicity in normal human liver L-02 cells line. The cells were exposed to APAP (10 mM) or co-treated with phytochemical compounds (40 ?M) over a period of 36 h and, biochemical and molecular parameters assessed. Three known limonoids namely 17-epi-methyl-6-hydroxylangolensate, 7-deacetoxy-7-oxogedunin and deacetoxy-7R-hydroxygedunin were identified. The results of cells viability and membrane integrity, reactive oxygen species generation and lipid membrane peroxidation assays, cellular glutathione content determination as well as expression of cytochrome P450 2E1 demonstrated the protective action of the limonoids. Immunoblotting analysis revealed that limonoids inhibited APAP-induced c-Jun N-terminal Kinase phosphorylation (p-JNK), mitochondrial translocation of p-JNK and Bcl2-associated X Protein, and the release of Apoptosis-inducing Factor into the cytosol. Interestingly, limonoids increased the expression of Mitogen-activated Protein Kinase Phosphatase (Mkp)-1, an endogenous inhibitor of JNK phosphorylation and, induced the nuclear translocation of Nuclear Factor Erythroid 2-related Factor-2 (Nrf2) and decreased the expression of Kelch-like ECH-associated Protein-1. The limonoids also reversed the APAP-induced decreased mRNA levels of Catalase, Superoxide Dismutase-1, Glutathione-S-Transferase and Methionine Adenosyltransferase-1A. The obtained results suggest that the isolated limonoids protect L-02 hepatocytes against APAP-induced hepatotoxicity mainly through increase expression of Mkp-1 and nuclear translocation of Nrf2. Thus, these compounds are in part responsible of the hepatoprotective activity of K. grandifoliola and further analysis including in vivo and toxicological studies are needed to select the most potent compound that may be useful as therapeutic agents against DILI.

Dataset Information

Acquired resistance to acetaminophen hepatotoxicity is associated with induction of multidrug resistance-associated protein 4 (Mrp4) in proliferating hepatocytes.

Publications

Acquired resistance to acetaminophen hepatotoxicity is associated with induction of multidrug resistance-associated protein 4 (Mrp4) in proliferating hepatocytes.

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