Spheroid formation and functional restoration of human fetal hepatocytes on poly-amino acid-coated dishes after serial proliferation

Primary human fetal hepatocytes proliferated in monolayer culture up to the 9th passage. During proliferation, the cells changed their morphology from a fibroblast-like shape after inoculation to an epithelia-like polygonal shape after they reached confluence. The proliferation was associated with the loss of ammonia detoxification capacity, which is essential for the function of bioartificial liver. The cells formed spheroids on a poly-glutamic acid- or poly-aspartic acid-coated polystyrene dish that had a negatively charged surface at neutral pH. However, the cells did not form spheroids on a poly-lysine- or poly-arginine-coated dish that had a positively charged surface, which is reportedly suitable to form spheroids for adult hepatocytes. The activity of cytochrome P450 (CYP 1A1, CYP1A2) of the cells in spheroid culture was about twice as high as that of the cells in monolayer culture. The ammonia detoxification activity of the cells was restored in spheroid culture by treatment with 2% dimethylsulfoxide. These results suggest that the conditions for human fetal hepatocytes to form spheroids are different from that for adult hepatocytes, and the use of poly-glutamic acid or poly-aspartic acid coating may improve spheroid culture of proliferative human fetal hepatocytes. This revised version was published online in July 2006 with corrections to the Cover Date.     

The functional role of microRNAs in alcoholic liver injury

The function of microRNAs (miRNAs) during alcoholic liver disease (ALD) has recently become of great interest in biological research. Studies have shown that ALD associated miRNAs play a crucial role in the regulation of liver‐inflammatory agents such as tumour necrosis factor‐alpha (TNF‐α), one of the key inflammatory agents responsible for liver fibrosis (liver scarring) and the critical contributor of alcoholic liver disease. Lipopolysaccharide (LPS), a component of the cell wall of gram‐negative bacteria, is responsible for TNF‐α release by Kupffer cells. miRNAs are the critical mediators of LPS signalling in Kupffer cells, hepatocytes and hepatic stellate cells. Certain miRNAs, in particular miR‐155 and miR‐21, show a positive correlation in up‐regulation of LPS signalling when they are exposed to ethanol. ALD is related to enhanced gut permeability that allows the levels of LPS to increase, leads to increased secretion of TNF‐α by the Kupffer cells and subsequently promotes alcoholic liver injury through specific miRNAs. Meanwhile, two of the most frequently dysregulated miRNAs in steatohepatitis, miR‐122 and miR‐34a are the critical mediators in ethanol/LPS activated survival signalling during ALD. In this review, we summarize recent findings regarding the experimental and clinical aspects of functions of specific microRNAs, focusing mainly on inflammation and cell survival after ethanol/LPS treatment, and advances on the role of circulating miRNAs in human alcoholic disorders.     

A combined proteomics and computational approach provides a better understanding of HCV-induced liver disease

Evaluation of: Diamond DL, Krasnoselsky AL, Burnum KE et al. Proteome and computational analyses reveal new insights into the mechanisms of hepatitis C virus-mediated liver disease posttransplantation. Hepatology 56(1), 28–38 (2012).

HCV is a major cause of chronic liver disease worldwide and is a formidable therapeutic challenge. Recently, Diamond et al. analyzed the proteomic profiles of liver samples from HCV-positive liver transplant recipients, supplemented with an independent metabolite analysis. They used a computational approach, which highlighted the enriched functional themes and topological attributes associated with the protein association network based on their clinical data and suggested a crucial role of oxidative stress in fibrosis progression in HCV infection. Their findings provide new insights into the mechanisms that regulate the progression of HCV-associated liver fibrosis, which may be useful for identification of suitable biomarkers to evaluate the onset and severity of hepatic fibrosis and the development of new therapeutic and anti-HCV strategies.     

Evidence for cytochrome P450 2B1/2B2 isoenzymes in freshly prepared peripheral blood lymphocytes

Cytochrome P450 2B1 and 2B2, the major hepatic drug metabolizing enzymes belonging to CYP2 family and associated constitutive androstane receptor (CAR) were found to be expressed in peripheral blood lymphocytes (PBL) isolated from rats. As observed in liver, pretreatment of phenobarbital (PB) or phenytoin were found to increase the expression of CYP2B1, CYP2B2 and associated enzyme activity in PBL. Like in liver, blood lymphocyte CYP2B1/2B2 catalyzed the activity of 7-pentoxyresorufin O-dealkylase (PROD). The present data, demonstrating similarities in the regulation of blood lymphocyte CYP2B-isoenzymes with the liver enzymes, suggests that blood lymphocyte CYP2B-isoenzymes could be used as a biomarker to monitor tissue levels.     

Schisandrin B elicits a glutathione antioxidant response and protects against apoptosis via the redox-sensitive ERK/Nrf2 pathway in AML12 hepatocytes

Abstract

This study examined the effects of (?)schisandrin B [(?)Sch B] on MAPK and Nrf2 activation and the subsequent induction of glutathione antioxidant response and cytoprotection against apoptosis in AML12 hepatocytes. Pharmacological tools, such as cytochrome P-450 (CYP) inhibitor, antioxidant, MAPK inhibitors and Nrf2 RNAi, were used to delineate the signalling pathway. (?)Sch B caused a time-dependent activation of MAPK in AML12 cells, particularly the ERK1/2. The MAPK activation was followed by an enhancement in Nrf2 nuclear translocation and the eliciting of a glutathione antioxidant response. Reactive oxygen species arising from a CYP-catalysed reaction with (?)Sch B seemed to be causally related to the activation of MAPK and Nrf2. ERK inhibition by U0126 or Nrf2 suppression by Nrf2 RNAi transfection almost completely abrogated the cytoprotection against menadione-induced apoptosis in (?)Sch B-pre-treated cells. (?)Sch B pre-treatment potentiated the menadione-induced ERK activation, whereas both p38 and JNK activations were suppressed. Under the condition of ERK inhibition, Sch B treatment did not protect against carbon tetrachloride-hepatotoxicity in an in vivo mouse model. In conclusion, (?)Sch B triggers a redox-sensitive ERK/Nrf2 signalling, which then elicits a cellular glutathione antioxidant response and protects against oxidant-induced apoptosis in AML12 cells.     

Relations Between Tocopherol Depletion and Coenzyme Q During Lipid Peroxidation in rat Liver Mitochondria

In order to evaluate different mitochondrial antioxidant systems, the depletion of alpha-tocopherol and the levels of the reduced and oxidized forms of CoQ were measured in rat liver mitochondria during Fe⁺⁺/ascorbate and NADPH/ADP/Fe⁺⁺ induced lipid peroxidation. During the induction phase of malondialdehyde formation, alpha-tocopherol declined moderately to about 80% of initial contents, whereas the total CoQ pool remained nearly unchanged, but reduced CoQ9 continuously declined. At the start of massive malondialdehyde formation, CoQ9 reaches its fully oxidized state. At the same time alpha-tocopherol starts to decline steeply, but never becomes fully exhausted in both experimental systems. Evidently the oxidation of the CoQ9 pool constitutes a prerequisite for the onset of massive lipid peroxidation in mitochondria and for the subsequent depletion of alpha-tocopherol. Trapping of the GSH by addition of dinitrochlorbenzene (a substrate of the GSH transferase), results in a moderate acceleration of lipid peroxidation, but alpha-tocopherol and ubiquinol levels remained unchanged when compared with the controls. Addition of succinate to GSH depleted mitochondria effectively suppressed MDA formation as well as alpha-tocopherol and ubiquinol depletion. The data support the assumption that the protective effect of respiratory substrates against lipid peroxidation in the absence of mitochondrial GSH is mediated by the regeneration of the lipid soluble antioxidants CoQ and alpha-tocopherol.