Liver Failure In Humans Research Articles

Protective effect of kombucha tea against acetaminophen-induced hepatotoxicity in mice: a biochemical and histopathological study

Acetaminophen overdose causes severe hepatotoxicity leading to liver failure in experimental animals and humans. This study was undertaken to evaluate the protective effect of kombucha tea (KT) against acetaminophen-induced hepatotoxicity. Forty male Balb/c mice were divided into four equal groups: (1) the control group, (2) KT-treated group, (3) acetaminophen-treated group, and (4) KT/acetaminophen-treated group. All mice in group 4 were given KT orally for 7 days before a single hepatotoxic dose of acetaminophen (1,000 mg/kg orally). Activities of liver marker enzymes in serum; aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP); and total protein (TP), albumin (ALB), and direct and total bilirubin levels were determined. Acetaminophen challenge caused significant increases in the levels of bilirubin and liver enzymes (AST, ALT, ALP, and LDH), while TP and ALB levels were reduced significantly. Histopathologic assessments showed that severe glycogen storage in hepatocytes, hepatocellular degeneration and necrosis, mononuclear cell infiltration in portal area, dilation of central veins, and capillarization also reduced in KT/acetaminophen group compared to acetaminophen-treated mice. In conclusion, these findings suggest that KT has protective effect on acetaminophen-induced hepatotoxicity.

Interferon-gamma negatively regulates Th17-mediated immunopathology during mouse hepatitis virus infection

Fulminant hepatitis can cause acute liver failure and death in both humans and mice. However, the cellular and molecular mechanisms underlying the acute disease are still not well understood. Here, we examine the role of Th17 response in the development of the acute hepatitis following infection with mouse hepatitis virus (MHV). We show that IL-17 levels in serum are rapidly elevated and positively correlated to liver damage and death of the mice. In IFN-γR−/− mice, Th17 response is enhanced and the elevated IL-17 production contributes to severe liver damage as well as detrimental inflammation because neutralization of IL-17 effectively suppresses inflammation and protects mice from liver injury. We further show that IFN-γ facilitates antigen-induced apoptosis of Th17 cells and adoptive transferred IFN-γR−/−, but not IFN-γR+/+; CD4+ T cells promotes an enhanced liver damage in wild-type mice. The results demonstrate an essential role of Th17 cells in MHV-induced immunopathology and the importance of IFN-γ in maintaining immune balance between Th1 and Th17 responses during acute viral infection.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-010-0711-5) contains supplementary material, which is available to authorized users.

Cyclophilin D deficiency protects against acetaminophen-induced oxidant stress and liver injury

Acetaminophen (APAP) hepatotoxicity is the main cause of acute liver failure in humans. Although mitochondrial oxidant stress and induction of the mitochondrial permeability transition (MPT) have been implicated in APAP-induced hepatotoxicity, the link between these events is unclear. To investigate this, this study evaluated APAP hepatotoxicity in mice deficient of cyclophilin D, a protein component of the MPT. Treatment of wild type mice with APAP resulted in focal centrilobular necrosis, nuclear DNA fragmentation and formation of reactive oxygen (elevated glutathione disulphide levels) and peroxynitrite (nitrotyrosine immunostaining) in the liver. CypD-deficient (Ppif−/−) mice were completely protected against APAP-induced liver injury and DNA fragmentation. Oxidant stress and peroxynitrite formation were blunted but not eliminated in CypD-deficient mice. Thus, mitochondrial oxidative stress and induction of the MPT are critical events in APAP hepatotoxicity in vivo and at least part of the APAP-induced oxidant stress and peroxynitrite formation occurs downstream of the MPT.

Effects of Sesame Oil Against After the Onset of Acetaminophen‐Induced Acute Hepatic Injury in Rats

Acetaminophen (APAP) is a safe and effective analgesic and antipyretic when used at therapeutic levels. However, an acute or cumulative overdose can cause severe liver injury with the potential to progress to liver failure in humans and experimental animals. Much attention has been paid to the development of an antioxidant that protects against APAP-induced acute hepatic injury. Hence, we aimed to investigate the effect of sesame oil against after the onset of acute hepatic injury in APAP-overdosed rats. Male Wistar rats were first given 2 oral doses (1,000 mg/kg each) of APAP (at 0 and 24 hours) and then 1 oral dose of sesame oil (8 mL/kg at 24 hours). After 48 hours, APAP increased aspartate and alanine aminotransferase levels in the rats' serum and centrilobular necrosis in liver tissue. In addition, APAP significantly decreased the rats' glutathione levels and mitochondrial aconitase activity, but increased superoxide anion, hydroxyl radical, and lipid peroxidation levels. Oral sesame oil (8 mL/kg, given at 24 hours) reversed all APAP-altered parameters and protected the rats against APAP-induced acute liver injury. We hypothesize that sesame oil acts as a useful agent that maintains intracellular glutathione levels and inhibits reactive oxygen species, thereby protecting rats against after the onset of APAP-induced acute oxidative liver injury.

825 COAGULATION PROTEINS IN ACUTE LIVER INJURY: A ROLE IN INITIATION AND PROPAGATION

Background: Recent evidence suggests liver injury is in part activated by the coagulation cascade. With the exception of thrombin the function and expression of individual coagulation proteins in the pathogenesis of liver injury is poorly understood. Paracetamol toxicity is a leading cause of liver failure in humans, but the mechanisms of initiation and progression are not clear. Aims: To determine if tissue factor (TF) and fibrin are upregulated in an animal model of acute liver injury and to quantify and qualify the expression of these proteins. Methods: Acute liver injury was induced in mice (C57BL/6 background) by a single intraperitoneal injection of 300mg/kg paracetamol. Control mice received intraperitoneal saline injection. Mice were culled by 24 hours and whole livers extracted and fixed in formalin. Each liver injury was graded based on extent and distribution of necrosis (Grade 0: No injury; Grade 1: perivenular necrosis, no bridging; Grade 2: focal bridging necrosis; Grade 3: extensive bridging necrosis; Grade 4: panlobular necrosis). An indirect immunohistochemical detection technique was employed to qualify and quantify expression of TF and fibrin. Results: Fibrin was expressed in all grades of acute liver injury. Grade 3 and 4 injuries demonstrated confluent fibrin expression with a centrilobular distribution. Less widespread expression was noted in milder injuries. A very strong correlation was demonstrated between fibrin expression and grade of liver injury on visual analogue scale (r = 0.956, p 0.05). Centrilobular TF expression strongly correlated with both grade of injury (r = 0.77, p < 0.001) and fibrin expression (r = 0.731, p < 0.001). Conclusions: TF is expressed normally in a sinusoidal pattern and upregulated with fibrin in a centrilobular pattern of expression in paracetamol induced acute liver injury. These findings suggest a possible role for these proteins in the initiation and propagation of necrosis and the therapeutic potential of their targeted inhibition should be considered in this type of injury.

Polymorphisms in RIG-I like helicases and their influence on the course of Hepatitis C Virus infection

Hepatitis C is a major cause of liver cirrhosis, liver failure and hepatocellular carcinoma in humans. Hepatitis C Virus (HCV) infects about 3% of the human population. Interestingly the course of infection varies widely among patients. Approximately 15–20% of the infected individuals clear the infection spontaneously, whereas in 80% the infection presides in a chronic state. Virus-related factors and clinical risk factors of the host such as sex, alcohol consumption and age at infection do not allow to predict the development of a chronic status. It is therefore suspected that the susceptibility to chronic HCV infection is determined by genetic polymorphisms in the host's immune system. The RIG-I like helicase (RLH) RIG-I was recently shown to be responsible for triggering the innate immune response after infection with HCV in cell culture systems. This led us to examine single nucleotide polymorphisms (SNPs) in RIG-I and other components of the RIG-I-like helicase pathway and correlate them with the outcome after HCV-infection. We focused our analysis on non-synonymous SNPs in coding regions and found in a european population that the SNP rs3747517 in Mda-5 (leading to mutation H843R) has a significantly higher prevalence in individuals that spontaneously resolved HCV infection as compared to chronically infected patients. In addition we made expression constructs of RLHs and analysed in complementary in vitro assays the functional consequences of the mutations caused by the analysed SNPs. Together these results suggest an important role of RLHs for the clinical outcome of an HCV infection.

Toward Second Generation Hepatitis C Virus NS3 Serine Protease Inhibitors: Discovery of Novel P4 Modified Analogues with Improved Potency and Pharmacokinetic Profile

Hepatitis C virus (HCV) infection is a global health crisis leading to liver cirrhosis, hepatocellular carcinoma, and liver failure in humans. Recently, we disclosed the discovery of Boceprevir, SCH 503034 (1), a novel, potent, selective, orally bioavailable NS3 protease inhibitor that is currently undergoing phase III clinical trials. Our efforts toward a second generation HCV NS3 serine protease inhibitor were directed at improving the overall profile of the inhibitor. This article will elaborate on our studies leading to the discovery of new P4 modified inhibitors with enhanced potency and improved oral bioavailability. Thus, introduction of ether and carbamate-derived P4 moieties resulted in improving the replicon potency significantly. Incorporation of the P' secondary amide residue afforded significant improvement in pharmacokinetic properties. Combining the preferred moieties, identified from comprehensive SAR studies, resulted in inhibitors that displayed superior potency and very good oral as well as target organ exposure in rats.

Hepatic stem/progenitor cells and stem-cell transplantation for the treatment of liver disease

Allogeneic liver transplantation is still the only effective treatment available to patients with liver failure. However, because there is a serious shortage of liver donors, an alternative therapeutic approach is needed. Transplantation of mature hepatocytes has been evaluated in clinical trials, but the long-term efficacy remains unclear and the paucity of donor cells limits this strategy. Stem-cell transplantation is a more promising alternative approach. Several studies have provided information about the mechanism underlying the proliferation and differentiation of hepatic stem/progenitor cells. Moreover, in experimental models of liver disease, transplantation of hepatic stem/progenitor cells or hepatocyte-like cells derived from multipotent stem cells led to donor cell-mediated repopulation of the liver and improved survival rates. However, before stem-cell transplantation can be applied in the clinic to treat liver failure in humans, it will be necessary to overcome several difficulties associated with the technique.

Curcumin reduces the toxic effects of iron loading in rat liver epithelial cells

Iron overload can cause liver toxicity and increase the risk of liver failure or hepatocellular carcinoma in humans. Curcumin (diferuloylmethane), a component of the food spice turmeric, has antioxidant, iron binding and hepatoprotective properties. The aim of this study was to quantify its effects on iron overload and the resulting downstream toxic effects in cultured T51B rat liver epithelial cells. T51B cells were loaded with ferric ammonium citrate (FAC) with or without the iron delivery agent 8-hydroxyquinoline. Cytotoxicity was measured by methylthiazolyldiphenyl-tetrazolium bromide assay. Iron uptake and iron bioavailability were documented by chemical assay, quench of calcein fluorescence and ferritin induction. Reactive oxygen species (ROS) were measured by a fluorescence assay using 2',7'-dichlorodihydrofluorescein diacetate. Oxidative stress signalling to jnk, c-jun and p38 was measured by a Western blot with phospho-specific antibodies. Curcumin bound iron, but did not block iron uptake or bioavailability in T51B cells given FAC. However, it reduced cytotoxicity, blocked the generation of ROS and eliminated signalling to cellular stress pathways caused by iron. Inhibition was observed over a wide range of FAC concentrations (50-500 microM), with an apparent IC(50) in all cases between 5 and 10 microM curcumin. In contrast, desferoxamine blocked both iron uptake and toxic effects of iron at concentrations that depended on the FAC concentration. The effects of curcumin also differed from those of alpha-tocopherol, which did not bind iron and was less effective at blocking iron-stimulated ROS generation. Curcumin reduced iron-dependent oxidative stress and iron toxicity in T51B cells without blocking iron uptake.

Improved Xenogenic Hepatocyte Implantation into Nude Mouse Liver Parenchyma with Acute Liver Failure when Followed by Repeated Anti-Fas Antibody (Jo2) Treatment

Hepatocyte transplantation is a promising therapy for acute liver failure in humans. Recently, we succeeded in inducing various acute and chronic liver failures in nude mice. Engraftment of transplanted xenogeneic rat hepatocytes, visualized in the host liver by anti-MHC class I immunohistochemistry, revealed that liver repopulation was limited, and equivalent in nude mice with and without acute liver failure. In the present study, acute liver failure was induced in nude mice by a single injection of sublethal anti-Fas antibody Jo2, followed 24 h later by rat hepatocyte transplantation and than by a weekly repeated injection of Jo2. Rat hepatocyte engraftment into the recipient liver parenchyma 3 weeks following hepatocyte transplantation was about sevenfold increased when nude mice were subsequently subjected to weekly repeated Jo2 injection. Genomic analysis of these mice showed an overall transcriptome profile of upregulation of cellular cycle blocking transcripts, activation of liver injury inducing IFN-gamma/STAT1 pathway, and circadian transcript signature of antiproliferative cell status compared to mice submitted to hepatocyte transplantation only. The findings of the present study suggest that the induction of cell proliferation blockade in recipient livers could promote sufficient engraftment of transplanted hepatocytes to allow transient or definitive treatment of liver failure in humans.

Disruption of NaS1 sulfate transport function in mice leads to enhanced acetaminophen‐induced hepatotoxicity†

Sulfate is required for detoxification of xenobiotics such as acetaminophen (APAP), a leading cause of liver failure in humans. The NaS1 sulfate transporter maintains blood sulfate levels sufficiently high for sulfonation reactions to work effectively for drug detoxification. In the present study, we identified two loss-of-function polymorphisms in the human NaS1 gene and showed the Nas1-null mouse to be hypersensitive to APAP hepatotoxicity. APAP treatment led to increased liver damage and decreased hepatic glutathione levels in the hyposulfatemic Nas1-null mice compared with that in normosulfatemic wild-type mice. Analysis of urinary APAP metabolites revealed a significantly lower ratio of APAP-sulfate to APAP-glucuronide in the Nas1-null mice. These results suggest hyposulfatemia increases sensitivity to APAP-induced hepatotoxicity by decreasing the sulfonation capacity to metabolize APAP. In conclusion, the results of this study highlight the importance of plasma sulfate level as a key modulator of acetaminophen metabolism and suggest that individuals with reduced NaS1 sulfate transporter function would be more sensitive to hepatotoxic agents.

Essential Elements of the Capsid Protein for Self-Assembly into Empty Virus-Like Particles of Hepatitis E Virus

Hepatitis E virus (HEV) is a noncultivable virus that causes acute liver failure in humans. The virus's major capsid protein is encoded by an open reading frame 2 (ORF2) gene. When the recombinant protein consisting of amino acid (aa) residues 112 to 660 of ORF2 is expressed with a recombinant baculovirus, the protein self-assembles into virus-like particles (VLPs) (T.-C. Li, Y. Yamakawa, K. Suzuki, M. Tatsumi, M. A. Razak, T. Uchida, N. Takeda, and T. Miyamura, J. Virol. 71:7207-7213, 1997). VLPs can be found in the culture medium of infected Tn5 cells but not in that of Sf9 cells, and the major VLPs have lost the C-terminal 52 aa. To investigate the protein requirement for HEV VLP formation, we prepared 14 baculovirus recombinants to express the capsid proteins truncated at the N terminus, the C terminus, or both. The capsid protein consisting of aa residues 112 to 608 formed VLPs in Sf9 cells, suggesting that particle formation is dependent on the modification process of the ORF2 protein. In the present study, electron cryomicroscopy and image processing of VLPs produced in Sf9 and Tn5 cells indicated that they possess the same configurations and structures. Empty VLPs were found in both Tn5 and Sf9 cells infected with the recombinant containing an N-terminal truncation up to aa residue 125 and C-terminal to aa residue 601, demonstrating that the aa residues 126 to 601 are the essential elements required for the initiation of VLP assembly. The recombinant HEV VLPs are potential mucosal vaccine carrier vehicles for the presentation of foreign antigenic epitopes and may also serve as vectors for the delivery of genes to mucosal tissue for DNA vaccination and gene therapy. The results of the present study provide useful information for constructing recombinant HEV VLPs having novel functions.

Hepatocyte transplantation

Hepatocyte transplantation

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

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.

Protective Role of Hydroxysteroid Sulfotransferase in Lithocholic Acid-induced Liver Toxicity

Supplement of 1% lithocholic acid (LCA) in the diet for 5-9 days resulted in elevated levels of the marker for liver damage aspartate aminotransferase and alkaline phosphatase activities in both farnesoid X receptor (FXR)-null and wild-type female mice. The levels were clearly higher in wild-type mice than in FXR-null mice, despite the diminished expression of a bile salt export pump in the latter. Consistent with liver toxicity marker activities, serum and liver levels of bile acids, particularly LCA and taurolithocholic acid, were clearly higher in wild-type mice than in FXR-null mice after 1% LCA supplement. Marked increases in hepatic sulfating activity for LCA (5.5-fold) and hydroxysteroid sulfotransferase (St) 2a (5.8-fold) were detected in liver of FXR-null mice. A 7.4-fold higher 3alpha-sulfated bile acid concentration was observed in bile of FXR-null mice fed an LCA diet compared with that of wild-type mice. Liver St2a content was inversely correlated with levels of alkaline phosphatase. In contrast, microsomal LCA 6beta-hydroxylation was not increased and was in fact lower in FXR-null mice compared in wild-type mice. Clear decreases in mRNA encoding sodium taurocholate cotransporting polypeptide, organic anion transporting polypeptide 1, and liver-specific organic anion transporter-1 function in bile acid import were detected in LCA-fed mice. These transporter levels are higher in FXR-null mice than wild-type mice after 1% LCA supplement. No obvious changes were detected in the Mrp2, Mrp3, and Mrp4 mRNAs. These results indicate hydroxysteroid sulfotransferase-mediated LCA sulfation as a major pathway for protection against LCA-induced liver damage. Furthermore, Northern blot analysis using FXR-null, pregnane X receptor-null, and FXR-pregnane X receptor double-null mice suggests a repressive role of these nuclear receptors on basal St2a expression.

Region selective alterations of soluble guanylate cyclase content and modulation in brain of cirrhotic patients

Modulation of soluble guanylate cyclase (sGC) by nitric oxide (NO) is altered in brain from experimental animals with hyperammonemia with or without liver failure. The aim of this work was to assess the content and modulation of sGC in brain in chronic liver failure in humans. Expression of the α-1, α-2, and β-1 subunits of sGC was measured by immunoblotting in autopsied frontal cortex and cerebellum from cirrhotic patients and controls. The contents of α-1 and α-2 subunits of guanylate cyclase was increased both in cortex and cerebellum, whereas the β-1 subunit was not affected. Addition of the NO-generating agent S-nitroso- N-acetyl-penicillamine (SNAP) to homogenates of frontal cortex from controls increased the activity of sGC 87-fold, whereas, in homogenates from cirrhotic patients, the increase was significantly higher (183-fold). In contrast, in cerebellum, activation of guanylate cyclase by NO was significantly lower in patients (156-fold) than in controls (248-fold). A similar regional difference was found in rats with portacaval anastomosis. In conclusion, these findings show that the NO-guanylate cyclase signal transduction pathway is strongly altered in brain in patients with chronic liver failure and that the effects are different in different brain areas. Given that activation of sGC by NO in brain is involved in the modulation of important cerebral processes such as intercellular communication, learning and memory, and the sleep-wake cycle, these changes could be implicated in the pathogenesis of hepatic encephalopathy in these patients. (H EPATOLOGY 2002;36:1155-1162.)

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

Acetaminophen (AAP) overdose can cause severe liver injury and liver failure in experimental animals and humans. Recently, several authors proposed that apoptosis might be a major mechanism of cell death after AAP treatment. To address this controversial issue, we evaluated a detailed time course of liver injury after AAP (300 mg/kg) in fasted C3Heb/FeJ mice. Apoptotic hepatocytes were quantified in H&E-stained liver sections using morphologic criteria (cell shrinkage, chromatin condensation and margination, and apoptotic bodies). The number of apoptotic hepatocytes remained at baseline (0.2 +/- 0.1 cells/10 high-power fields [HPF]) up to 2 h after AAP administration. However, between 3 and 24 h, apoptotic cell death increased significantly, e.g., 6.3 +/- 0.8 cells/10 HPF at 6 h. Despite the increase in the number of hepatocytes meeting the morphological criteria of apoptosis, this cell fraction remained well below 1% of all parenchymal cells. No evidence for caspase-3 processing or increase in enzyme activity was detected at any time. These results were compared to the overall percent of necrotic cells in liver sections. Confluent areas of centrilobular necrosis were estimated to involve 40-60% of all hepatocytes between 3 and 24 h after AAP administration. These numbers correlated with the increase in plasma alanine aminotransferase activities, which reached a peak level of 5900 +/- 1350 U/l at 24 h. A similar result was obtained with higher doses of AAP and with the use of fed animals. Thus, oncotic necrosis and not apoptosis is the principal mechanism of liver-cell death after AAP overdose in vivo.

Evidence for a serotonin transporter deficit in experimental acute liver failure

It has been suggested that alterations of serotonin transport may be implicated in the pathogenesis of the neuropsychiatric symptoms encountered in acute liver failure. In order to address this issue, microdialysate concentrations of serotonin, its precursor L-tryptophan and metabolite 5-hydroxyindoleacetic acid (5-HIAA) as well as brain regional distribution of serotonin transporter ([ 3H]-citalopram) sites were measured in rats with acute liver failure resulting from hepatic devascularization. A significant loss of [ 3H]-citalopram sites was observed in dorsal Raphe nucleus, in frontal and frontoparietal cortices as well as in substantia nigra of rats with severe encephalopathy resulting from acute liver failure. In frontal cortex, this loss of transporter binding sites was accompanied by significant increases of L-tryptophan, serotonin and 5-HIAA concentrations in extracellular fluid. Pharmacological manipulation of the brain serotonin system could afford a novel therapeutic approach to the prevention of the neuropsychiatric symptoms characteristic of acute liver failure in humans.

Replacement of Liver Function in Rats by Transplantation of Microcarrier-Attached Hepatocytes

Isolated hepatocytes, harvested from normal rat livers by portal vein collagenase perfusion, can be attached to collagen-coated dextran microcarriers and transplanted by intraperitoneal injection into rats. Survival and function of the transplanted hepatocytes have been demonstrated in mutant rats lacking bilirubin-uridine diphosphate glucuronosyltransferase activity (Gunn strain) and rats with inherited lack of plasma albumin (Nagase analbuminemia rat strain). This simple technique promises to be useful in the treatment of acute liver failure in humans.