Dataset Information

Screening for drug-induced hepatotoxicity in primary mouse hepatocytes using acetaminophen, amiodarone, and cyclosporin a as model compounds: an omics-guided approach.

ABSTRACT: Drug-induced hepatotoxicity is a leading cause of attrition for candidate pharmaceuticals in development. New preclinical screening methods are crucial to predict drug toxicity prior to human studies. Of all in vitro hepatotoxicity models, primary human hepatocytes are considered as 'the gold standard.' However, their use is hindered by limited availability and inter-individual variation. These barriers may be overcome by using primary mouse hepatocytes. We used differential in gel electrophoresis (DIGE) to study large-scale protein expression of primary mouse hepatocytes. These hepatocytes were exposed to three well-defined hepatotoxicants: acetaminophen, amiodarone, and cyclosporin A. Each hepatotoxicant induces a different hepatotoxic phenotype. Based on the DIGE results, the mRNA expression levels of deregulated proteins from cyclosporin A-treated cells were also analyzed. We were able to distinguish cyclosporin A from controls, as well as acetaminophen and amiodarone-treated samples. Cyclosporin A induced endoplasmic reticulum (ER) stress and altered the ER-Golgi transport. Moreover, liver carboxylesterase and bile salt sulfotransferase were differentially expressed. These proteins were associated with a protective adaptive response against cyclosporin A-induced cholestasis. The results of this study are comparable with effects in HepG2 cells. Therefore, we suggest both models can be used to analyze the cholestatic properties of cyclosporin A. Furthermore, this study showed a conserved response between primary mouse hepatocytes and HepG2 cells. These findings collectively lend support for use of omics strategies in preclinical toxicology, and might inform future efforts to better link preclinical and clinical research in rational drug development.

SUBMITTER: Van Summeren A

PROVIDER: S-EPMC3567623 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Screening for drug-induced hepatotoxicity in primary mouse hepatocytes using acetaminophen, amiodarone, and cyclosporin a as model compounds: an omics-guided approach.

Van Summeren Anke A Renes Johan J Lizarraga Daneida D Bouwman Freek G FG Noben Jean-Paul JP van Delft Joost H M JH Kleinjans Jos C S JC Mariman Edwin C M EC

Omics : a journal of integrative biology 20130111 2

Drug-induced hepatotoxicity is a leading cause of attrition for candidate pharmaceuticals in development. New preclinical screening methods are crucial to predict drug toxicity prior to human studies. Of all in vitro hepatotoxicity models, primary human hepatocytes are considered as 'the gold standard.' However, their use is hindered by limited availability and inter-individual variation. These barriers may be overcome by using primary mouse hepatocytes. We used differential in gel electrophores ...[more]

PMID: 23308384

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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.

Dataset Information

Screening for drug-induced hepatotoxicity in primary mouse hepatocytes using acetaminophen, amiodarone, and cyclosporin a as model compounds: an omics-guided approach.

Publications

Screening for drug-induced hepatotoxicity in primary mouse hepatocytes using acetaminophen, amiodarone, and cyclosporin a as model compounds: an omics-guided approach.

Similar Datasets

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