Hepatic Membrane Research Articles

Biosynthesis and trafficking of the bile salt export pump, BSEP: Therapeutic implications of BSEP mutations

The bile salt export pump (BSEP, ABCB11) is the primary transporter of bile acids from the hepatocyte to the biliary system. This rate-limiting step in bile formation is essential to the formation of bile salt dependent bile flow, the enterohepatic circulation of bile acids, and the digestion of dietary fats. Mutations in BSEP are associated with cholestatic diseases such as progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), drug-induced cholestasis, and intrahepatic cholestasis of pregnancy. Development of clinical therapies for these conditions necessitates a clear understanding of the cell biology of biosynthesis, trafficking, and transcriptional and translational regulation of BSEP. This chapter will focus on the molecular and cell biological aspects of this critical hepatic membrane transporter.

Abstract 883: Imatinib-induced modulation of gastrointestinal and hepatic membrane transporters in mice.

Abstract Drug-induced changes in the expression or activity of ADME genes may critically affect the pharmacokinetics (PK) and the efficacy of chemotherapy as well as the incidence and severity of unwanted side effects. Drug uptake and efflux transporters are likely to be involved in the intestinal absorption, distribution and hepatic excretion of imatinib, an oral tyrosine kinase inhibitor approved for the treatment of CML and GIST. Previously, we demonstrated that imatinib is a substrate drug of both ABCB1 and ABCG2. Furthermore, we showed that prolonged imatinib exposure in Caco-2 cell cultures leads to increased expression of these drug efflux transporters. Evidently, some GIST patients chronically treated with imatinib show a time-dependent decrease in imatinib plasma levels. Altered transporter activity in the intestine and/or liver affecting the PK of imatinib may (partially) account for the observed PK resistance. We hypothesized that acquired PK resistance by decreased absorption and/or increased clearance of imatinib may be attributable to induction of intestinal and hepatic transport proteins. So, we investigated in mice whether imatinib-induced modulation of drug transporters occurs over time upon prolonged daily use of imatinib. To mimic the clinical situation we chronically treated C57/Bl6 mice, either with imatinib or with a solute control (H2O) for 4, 8 or 12 weeks. Imatinib (100 mg/kg once daily) was administered to the mice via oral gavage (∼300 μl / mice). We collected all organs involved in the absorption and elimination of imatinib including the liver and the complete GI-tract (esophagus, stomach, small and large intestine and caecum) and subsequently isolated total RNA and protein from these tissues. We performed mRNA and miRNA profiling using mouse genome 430 PM (Affymetrix) and LNA-modified oligonucleotide arrays (Exiqon), respectively, and examined the expression of >80 transporter genes using the RT2 Profiler PCR array system (SABiosciences). Prolonged imatinib treatment resulted in the modulation of various intestinal and hepatic drug transporters and clearly changed the mRNA and miRNA profiles of ADME-involved organs. Apart from the imatinib-associated transporters Abcb1a/b, Abcg2, Slc22a1 and Slco1b2, indicating proof of concept, our search identified various other potential imatinib transporter which are currently tested in uptake experiments using transporter-transfected HEK293 cells. In summary, we showed that continuous exposure of mice to daily dosages of imatinib markedly affects the expression of drug transporters and changes the mRNA and miRNA expression profiles in organs involved in uptake, distribution and elimination of imatinib. These imatinib-induced changes may critically affect the PK, efficacy and adverse events of imatinib chemotherapy. Citation Format: Herman Burger, Alexander T. den Dekker, Antonius WM Boersma, Peter de Bruijn, Jaap Verweij, Ron HJ Mathijssen, Erik AC Wiemer. Imatinib-induced modulation of gastrointestinal and hepatic membrane transporters in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 883. doi:10.1158/1538-7445.AM2013-883

Proteomic and bioinformatic analysis of membrane proteome in type 2 diabetic mouse liver

Type 2 diabetes mellitus (T2DM) is the most prevalent and serious metabolic disease affecting people worldwide. T2DM results from insulin resistance of the liver, muscle, and adipose tissue. In this study, we used proteomic and bioinformatic methodologies to identify novel hepatic membrane proteins that are related to the development of hepatic insulin resistance, steatosis, and T2DM. Using FT-ICR MS, we identified 95 significantly differentially expressed proteins in the membrane fraction of normal and T2DM db/db mouse liver. These proteins are primarily involved in energy metabolism pathways, molecular transport, and cellular signaling, and many of them have not previously been reported in diabetic studies. Bioinformatic analysis revealed that 16 proteins may be related to the regulation of insulin signaling in the liver. In addition, six proteins are associated with energy stress-induced, nine proteins with inflammatory stress-induced, and 14 proteins with endoplasmic reticulum stress-induced hepatic insulin resistance. Moreover, we identified 19 proteins that may regulate hepatic insulin resistance in a c-Jun amino-terminal kinase-dependent manner. In addition, three proteins, 14-3-3 protein beta (YWHAB), Slc2a4 (GLUT4), and Dlg4 (PSD-95), are discovered by comprehensive bioinformatic analysis, which have correlations with several proteins identified by proteomics approach. The newly identified proteins in T2DM should provide additional insight into the development and pathophysiology of hepatic steatosis and insulin resistance, and they may serve as useful diagnostic markers and/or therapeutic targets for these diseases.

Analysis of differentially expressed genes and microRNAs in alcoholic liver disease

The purpose of this study was to screen differentially expressed genes and microRNAs in order to find a new target for the accurate diagnosis and effective therapy of alcoholic liver disease (ALD) at the gene and microRNA levels. The totalRNA of liver tissues was extracted from four groups of patients, ten subjects each. Microarrays were utilized to detect differentially expressed genes and microRNAs. According to gene values, significance levels and false discovery rate with a random variance model, gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, node genes and key microRNAs in networks were obtained and analyzed. A total of 878 differentially expressed genes and 26microRNAs were found. In co-expression genetic networks, node genes modulating the network were Acyl-coenzyme Asynthetase-3 (ACSF3), Frizzled-5 (FZD5), LOC727987 and C1orf222. In microRNA-gene networks, the key microRNAs were hsa-miR-570, hsa-miR-122, hsa-miR-34b, hsa-miR-29c, hsa-miR-922 and hsa-miR-185, which negatively regulated approximately 79 downstream target genes. In the course of ALD, we found 4 differentially expressed node genes and analyzed ACSF3 and FZD5. ACSF3 was significantly upregulated, and was involved in fatty acid and lipid metabolism and accelerated liver injury. These two genes were involved in fatty acids and lipid metabolism. FZD5 was downregulated and reduced the synthesis of membrane transport protein in the hepatic membrane and the membrane stability, and accelerated the liver cell apoptosis process. Six key microRNAs regulated numerous biological functions such as the immune response, the inflammatory response and glutathione metabolism. This finding provides valuable insight into the diagnosis and treatment of ALD.

Effect of the oral administration homeopathic Arnica montana on mitochondrial oxidative stress

To analyze the effect of homeopathic Arnica on mitochondrial oxidative stress induced by Ca(2+) plus inorganic phosphate and/or Fe(2+)-citrate-mediated lipid peroxidation through changes in oxygen consumption rates. Mitochondria were isolated by differential centrifugation from the livers of adult male Wistar rats which had been treated with Arnica montana 6cH, 12cH, 30cH or succussed 30% ethanol (control) for 21 days. In the presence of antimycin-A, electron transport chain inhibitor, as evidenced by antimycin-A insensitive O(2) consumption, Arnica inhibited lipid peroxidation of mitochondrial membranes. In oxidative stress conditions, in the presence of Ca(2+) and inorganic phosphate, animals receiving Arnica 30cH had a significant decrease in mitochondrial O(2) consumption compared to control animals. When administrated orally, Arnica 30cH protects against hepatic mitochondrial membrane permeabilization induced by Ca(2+) and/or Fe(2+)-citrate-mediated lipid peroxidation and fragmentation of proteins due to the attack by reactive oxygen species.

F-Actin Plaque Formation as a Transitional Membrane Microstructure Which Plays a Crucial Role in Cell-cell Reconnections of Rat Hepatic Cells After Isolation

Cell-cell reconnection after isolation or during healing process after injury is a cell membrane function. It is assumed that each tissue has its own architecture and accordingly, cells in different tissues could have different adaptability to resume their tissue-specific architectures. How membranes adapt themselves to make cell-cell reconnections has not been elucidated. This study investigated how rat hepatic cell membranes adapt themselves after isolation to establish cell-cell reconnections, which is essential to liver tissue healing process. Hepatic cells were obtained from rat liver by collagenase perfusion. The cells were maintained in suspension using a gyratory-mediated cell spheroid culture method. F-actin, claudin-2 and connexin-32 on cell membranes were stained and observed by confocal microscopy. Cells and spheroids were also observed by Scanning Electron Microscopy. It was found that hepatic cells underwent adaptive change after enzymatic isolation by diminishing membrane F-actin residues and forming F-actin plaques. Once an F-actin plaque is formed (within 1-4 h), it is ready to make initial contact with other cells through plaque-plaque overlapping in a manner similar to ‘Velcro-like’ binding. A cell could form one or more F-actin plaques and establish plaque-plaque contacts with one or more other cells. When the cells had established plaque-plaque contacts, their contacts would further develop to make full cell-cell bindings. Claudin-2, a tight-junction molecule, and connexin-32, a gap-junction molecule, gradually diminished and lost their original distribution patterns within 4 h after isolation. They did not show involvement in establishing initial cell-cell connections. It is concluded that F-actin plaque formation is a characteristic change that precedes cell-cell reconnection. F-actin plaques are transitional microstructures that serve as a special device to facilitate cell-cell contacts in the early stage after cell isolation. F-actin can be used as a biomarker to study membrane adaptability and the healing process of hepatic cells.

Mitochondrial electron transfer chain complexes inhibition by different organochalcogens

Mitochondrial dysfunction plays a pivotal role in the cell toxicology and death decision. The aim of the present study was to investigate the effect of three organocompounds (ebselen [Ebs], diphenyl diselenide [(PhSe)2] and diphenyl ditelluride [(PhTe)2]) on mitochondrial complexes (I, II, I–III, II–III and IV) activity from rat liver and kidney to determine their potential role as molecular targets of organochalcogens. All studied organochalcogens caused a statistically significant inhibition of the mitochondrial complex I activity. Ebs and (PhTe)2 caused a statistically significant inhibition of the mitochondrial complex II activity in both hepatic and renal membranes. Hepatic mitochondrial complex II activity was practically unchanged by (PhSe)2, whereas it significantly inhibited renal complex II activity. Mitochondrial complex IV activity was practically unchanged by the organochalcogens. Furthermore, organochalcogens inhibited the mitochondrial respiration supported by complex I or complex II substrates. The inhibitory effect of Ebs, (PhSe)2 and (PhTe)2 on mitochondrial complex I was prevented by NADH, but it was not prevented by catalase (CAT) and/or superoxide dismutase (SOD). Additionally, the organochalcogens-induced inhibition of complex I and II was completely reversed by reduced glutathione (GSH). In conclusion, Ebs, (PhSe)2 and (PhTe)2 were more effective inhibitors of renal and hepatic mitochondrial complex I than complex II, whereas complexes III and IV were little modified by these compounds. Taking into account the presented results, we suggest that organochalcogen-induced mitochondrial complexes I and II inhibition can be mediated by their thiol oxidation activity, i.e., Ebs, (PhSe)2 and (PhTe)2 can oxidize critical thiol groups from mitochondrial complexes I and II. So, mitochondrial dysfunction can be considered an important factor in the toxicity of Ebs, (PhSe)2 and (PhTe)2.

Taurine ameliorate alloxan induced oxidative stress and intrinsic apoptotic pathway in the hepatic tissue of diabetic rats

Oxidative stress is associated with various diabetic complications and taurine plays an important role in ameliorating those difficulties. In the present study we, therefore, investigated whether taurine plays any beneficial role against diabetes induced liver dysfunction and if it does, what cellular mechanism it follows during protective action. Induction of diabetes by alloxan (ALX) (at a dose of 120mg/kg body weight, i.p., once) reduced body weight and plasma insulin level, enhanced blood glucose and serum markers related to hepatic injury, accelerated ROS production, disturbed the intra-cellular antioxidant machineries and disintegrated hepatic cells near central vein. This pathophysiology leads to apoptotic cell death as evidenced from DNA fragmentation and TUNEL aasay. Studies on the mechanism of apoptosis showed that ALX accelerated the markers of mitochondrial dependent apoptotic pathway (enhanced cytochrome C release in cytosol from mitochondria, altered the expression of Bax, Bcl-2, Apaf-1, caspase-9, caspase-3). Treatment with taurine (1% w/v for three weeks) post-hyperglycemia, however, could restore all the alteration caused by ALX. Moreover, taurine activates hepatic PI3Kinase, Akt, hexokinase and augments the translocation of GLUT 2 to hepatic membrane in diabetic rats. Combining all, as a potential therapeutic, taurine may normalize the complications of diabetic liver injury.

Aberrantly elevated microRNA-34a in obesity attenuates hepatic responses to FGF19 by targeting a membrane coreceptor β-Klotho

MicroRNA-34a (miR-34a) is the most highly elevated hepatic miR in obese mice and is also substantially elevated in patients who have steatosis, but its role in obesity and metabolic dysfunction remains unclear. After a meal, FGF19 is secreted from the ileum; binds to a hepatic membrane receptor complex, FGF19 receptor 4 and coreceptor β-Klotho (βKL); and mediates postprandial responses under physiological conditions, but hepatic responses to FGF19 signaling were shown to be impaired in patients with steatosis. Here, we show an unexpected functional link between aberrantly elevated miR-34a and impaired βKL/FGF19 signaling in obesity. In vitro studies show that miR-34a down-regulates βKL by binding to the 3' UTR of βKL mRNA. Adenoviral-mediated overexpression of miR-34a in mice decreased hepatic βKL levels, impaired FGF19-activated ERK and glycogen synthase kinase signaling, and altered expression of FGF19 metabolic target genes. Consistent with these results, βKL levels were decreased and hepatic responses to FGF19 were severely impaired in dietary obese mice that have elevated miR-34a. Remarkably, in vivo antisense inhibition of miR-34a in obese mice partially restored βKL levels and improved FGF19 target gene expression and metabolic outcomes, including decreased liver fat. Further, anti-miR-34a treatment in primary hepatocytes of obese mice restored FGF19-activated ERK and glycogen synthase kinase signaling in a βKL-dependent manner. These results indicate that aberrantly elevated miR-34a in obesity attenuates hepatic FGF19 signaling by directly targeting βKL. The miR-34a/βKL/FGF19 axis may present unique therapeutic targets for FGF19-related human diseases, including metabolic disorders and cancer.

Hepatic Mitochondrial Energetics During Catch‐Up Fat With High‐Fat Diets Rich in Lard or Safflower Oil

We have investigated whether altered hepatic mitochondrial energetics could explain the differential effects of high-fat diets with low or high ω6 polyunsaturated fatty acid content (lard vs. safflower oil) on the efficiency of body fat recovery (catch-up fat) during refeeding after caloric restriction. After 2 weeks of caloric restriction, rats were isocalorically refed with a low-fat diet (LF) or high-fat diets made from either lard or safflower oil for 1 week, and energy balance and body composition changes were assessed. Hepatic mitochondrial energetics were determined from measurements of liver mitochondrial mass, respiratory capacities, and proton leak. Compared to rats refed the LF, the groups refed high-fat diets showed lower energy expenditure and increased efficiency of fat gain; these differences were less marked with high-safflower oil than with high-lard diet. The increase in efficiency of catch-up fat by the high-fat diets could not be attributed to differences in liver mitochondrial activity. By contrast, the lower fat gain with high-safflower oil than with high-lard diet is accompanied by higher mitochondrial proton leak and increased proportion of arachidonic acid in mitochondrial membranes. In conclusion, the higher efficiency for catch-up fat on high-lard diet than on LF cannot be explained by altered hepatic mitochondrial energetics. By contrast, the ability of the high-safflower oil diet to produce a less pronounced increase in the efficiency of catch-up fat may partly reside in increased incorporation of arachidonic acid in hepatic mitochondrial membranes, leading to enhanced proton leak and mitochondrial uncoupling.

Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability

Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H2O2 across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H2O2 release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p<0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H2O2 release, assessed by Amplex Red, was reduced by about 45% (p<0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+120%, p<0.05) and loss of mitochondrial membrane potential (−80%, p<0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H2O2 release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death.

Estrogenic compounds decrease growth hormone receptor abundance and alter osmoregulation in Atlantic salmon

Exposure of Atlantic salmon smolts to estrogenic compounds is shown to compromise several aspects of smolt development. We sought to determine the underlying endocrine mechanisms of estrogen impacts on the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. Smolts in freshwater (FW) were either injected 3 times over 10days with 2μgg−1 17β-estradiol (E2) or 150μgg−1 4-nonylphenol (NP). Seawater (SW)-acclimated fish received intraperitoneal implants of 30μgg−1 E2 over two weeks. Treatment with these estrogenic compounds increased hepatosomatic index and total plasma calcium. E2 and NP reduced maximum growth hormone binding by 30–60% in hepatic and branchial membranes in FW and SW, but did not alter the dissociation constant. E2 and NP treatment decreased plasma levels of IGF-I levels in both FW and SW. In FW E2 and NP decreased plasma GH whereas in SW plasma GH increased after E2 treatment. Compared to controls, plasma chloride concentrations of E2-treated fish were decreased 5.5mM in FW and increased 10.5mM in SW. There was no effect of NP or E2 on gill sodium–potassium adenosine triphosphatase (Na+/K+-ATPase) activity in FW smolts, whereas E2 treatment in SW reduced gill Na+/K+-ATPase activity and altered the number and size of ionocytes. Our data indicate that E2 downregulates the GH/IGF-I-axis and SW tolerance which may be part of its normal function for reproduction and movement into FW. We conclude that the mechanism of endocrine disruption of smolt development by NP is in part through alteration of the GH/IGF-I axis via reduced GH receptor abundance.

Pharmacological LXR activation reduces presence of SR-B1 in liver membranes contributing to LXR-mediated induction of HDL-cholesterol

ObjectivePharmacological LXR activation has anti-atherosclerotic actions in animal models. Part of these beneficial effects may be explained by accelerated reverse cholesterol transport since both plasma high density lipoprotein (HDL) cholesterol and fecal neutral sterol secretion are higher upon LXR activation. Mechanisms underlying these LXR-mediated effects have not been fully elucidated. MethodsWe investigated the roles of the isoforms LXRα and LXRβ and the HDL cholesterol uptake receptor SR-B1 in modulation of cholesterol metabolism upon treatment of mice with the LXR ligand T0901317. ResultsHDL cholesterol was maximally 60% increased in a time-dependent fashion due to appearance of more and larger HDL particles. Fecal neutral sterol secretion was maximally induced after 1week treatment. T0901317 treatment induced fecal neutral sterol secretion by ∼300% in wild-type but not in Lxrα deficient mice. Surprisingly, LXR activation reduced SR-B1 protein amount in hepatic membranes, suggesting that this might contribute to elevated HDL cholesterol. However, T0901317 still elevated plasma HDL cholesterol in Sr-b1 deficient mice, suggesting that SR-B1 is not the only step involved in LXR-mediated induction of plasma HDL cholesterol. In addition, SR-B1 is not essential for LXR-induced cholesterol removal from the body. ConclusionInduction of fecal neutral sterol secretion by T0901317 critically depends on LXRα but not on LXRβ. LXR activation reduces SR-B1 in hepatic membranes, probably partly contributing to elevated HDL cholesterol. SR-B1 is not required to enhance fecal neutral sterol secretion.

Analysis of Polarized Membrane Traffic in Hepatocytes and Hepatic Cell Lines

The protocols described in this unit were developed to monitor membrane traffic in cultured cell monolayers that display hepatic polarity. In general, the assays are designed to visualize and/or quantitate membrane trafficking by monitoring the fates of antibodies bound to specific membrane proteins. We first describe how to infect cells with recombinant adenovirus, the preferred method for introducing exogenous genes into hepatic cells. We next provide a morphological assay to monitor basolateral to apical transcytosis. In a supporting protocol, we describe how to visualize apical recycling and/or retention. In an additional supporting protocol, we provide a semi-quantitative method to measure the relative extents of apical delivery. Finally, we describe quantitative assays to measure basolateral internalization and recycling. The methods presented in this unit provide a relatively simple, yet powerful approach to examining hepatic membrane traffic.

Increased expression of chondroitin sulphate proteoglycans in rat hepatocellular carcinoma tissues

To investigate the expression of chondroitin sulphate proteoglycans (CSPGs) in rat liver tissues of hepatocellular carcinoma (HCC). Thirty male Sprague Dawley rats were randomly divided into two groups: control group (n = 10) and HCC model group (n = 20). Rats in the HCC model groups were intragastrically administrated with 0.2% (w/v) N-diethylnitrosamine (DEN) every 5 d for 16 wk, whereas 0.9% (w/v) normal saline was administered to rats in the control group. After 16 wk from the initiation of experiment, all rats were killed and livers were collected and fixed in 4% (w/v) paraformaldehyde. All tissues were embedded in paraffin and sectioned. Histological staining (hematoxylin and eosin and Toluidine blue) was performed to demonstrate the onset of HCC and the content of sulphated glycosaminoglycan (sGAG). Immunohistochemical staining was performed to investigate the expression of chondroitin sulphate (CS)/dermatan sulphate (DS)-GAG, heparan sulphate (HS)-GAG, keratan sulphate (KS)-GAG in liver tissues. Furthermore, expression and distribution of CSPG family members, including aggrecan, versican, biglycan and decorin in liver tissues, were also immunohistochemically determined. After 16 wk administration of DEN, malignant nodules were observed on the surface of livers from the HCC model group, and their hepatic lobule structures appeared largely disrupted under microscope. Toluidine blue staining demonstrated that there was an significant increase in sGAG content in HCC tissues when compared with that in the normal liver tissues from the control group [0.37 ± 0.05 integrated optical density per stained area (IOD/area) and 0.21 ± 0.01 IOD/area, P < 0.05]. Immunohistochemical studies demonstrated that this increased sGAG in HCC tissues was induced by an elevated expression of CS/DS (0.28 ± 0.02 IOD/area and 0.18 ± 0.02 IOD/area, P < 0.05) and HS (0.30 ± 0.03 IOD/area and 0.17 ± 0.02 IOD/area, P < 0.01) but not KS GAGs in HCC tissues. Further studies thereby were performed to investigate the expression and distribution of several CSPG components in HCC tissues, including aggrecan, versican, biglycan and decorin. Interestingly, there was a distinct distribution pattern for these CSPG components between HCC tissues and the normal tissues. Positive staining of aggrecan, biglycan and decorin was localized in hepatic membrane and/or pericellular matrix in normal liver tissues; however, their expression was mainly observed in the cytoplasm, cell membranes in hepatoma cells and/or pericellular matrix within HCC tissues. Semi-quantitative analysis indicated that there was a higher level of expression of aggrecan (0.43 ± 0.01 and 0.35 ± 0.03, P < 0.05), biglycan (0.32 ± 0.01 and 0.25 ± 0.01, P < 0.001) and decorin (0.29 ± 0.01 and 0.26 ± 0.01, P < 0.05) in HCC tissues compared with that in the normal liver tissues. Very weak versican positive staining was observed in hepatocytes near central vein in normal liver tissues; however there was an intensive versican distribution in fibrosis septa between the hepatoma nodules. Semi-quantitative analysis indicated that the positive rate of versican in hepatoma tissues from the HCC model group was much higher than that in the control group (33.61% and 21.28%, P < 0.05). There was no positive staining in lumican and keratocan, two major KSPGs, in either normal or HCC liver tissues. CSPGs play important roles in the onset and progression of HCC, and may provide potential therapeutic targets and clinical biomarkers for this prevalent tumor in humans.

Protective Effects of Tannic Acid against Methomyl-Induced-Oxidative Stress

Methomyl carbamate is a pesticide widely used in the control of insects. The present work aims at studying the effect of tannic acid on the antioxidant system of methomyl-treated-mice. Swiss albino mice were intraperitoneally administered a single dose of methomyl (7 mg/Kg b.wt). Mice of another group were injected with tannic acid (20 mg/ Kg b.wt.) 3 hrs before methomyl intoxication. After 24 hours, methomyl exposure resulted in significant increase in lactic dehydrogenase activity (LDH). The antioxidant capacity of hepatic cells in terms of the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione-S-transferase (GST) and glutathione (GSH) content was diminished. It appears that methomyl exerts its toxic effect via peroxidative damage to hepatic, renal and splenic cell membranes. Also, methomyl induced DNA damage in these organs as detected by alkaline filter elution technique. The distribution of methomyl in different organs of mice was detected by HPLC. Tannic acid administration prior to methomyl injection produced pronounced protective action against methomyl effects. It is observed that tannic acid enhances the endogenous antioxidant capacity of the cells by increasing the activities of SOD, CAT, GR and GST as well as increasing GSH content. The activity of LDH was decreased in liver and the damage of DNA was suppressed comparable to controls. In conclusion, this study concludes that tannic acid has a potential in mitigating most of the adverse effects induced by methomyl toxicity.

Serum Metabolite Profiles Are Reflective of Iron Overload in Thalassemia Major Patients on Chelation Therapy

Abstract 2112 Introduction:Iron overload (IO) either through blood transfusions or increased gastrointestinal absorption is associated with organ dysfunction and increased morbidity and mortality in patients with various hematological disorders including thalassemia. Despite improved methods of iron load detection and chelation therapy, patients still continue to be at risk for iron-associated toxicities. Methods for predicting IO include serum ferritin, liver iron content by MRI, and cardiac MRI T2*.Limited information is available regarding the effects of IO on the different metabolic systems in the liver and heart, which may change with chelation therapy. Different chelators, including deferoxamine (DFO), deferiprone (DFP) and deferasirox (DFX), chelate iron at different rates and effectiveness from the liver and heart. The liver is involved in maintenance of glucose and lipid homeostasis, and iron-triggered injury increases secondary metabolites including triacylglycerols and glucose. IO alters the stability of hepatic and myocardial lysosomal membranes releasing higher levels of lysosomal enzymes in liver compared to heart in animal models. These and other metabolic pathways have not been studied extensively in iron-overloaded patients. The new technology of metabolomics allows for the concurrent measurement and analysis of multiple metabolites and has the potential to provide more valuable information on the metabolic systems affected by iron overload that could have clinical relevance. Aim:To determine if novel metabolomics technologies can identify specific metabolites and pathways affected by iron overload including energy, carbohydrate, and lipid metabolism. Methods:In this pilot project, we included twelve iron overloaded patients (thalassemia major N=7, thalassemia intermedia N=2, hemoglobin H disease N=2, pure red cell aplasia N=1) (age > 18 years) and 12 sex and age-matched controls. Clinical parameters including age, sex, chelator therapy, pre-transfusion hemoglobin, serum ferritin, liver iron content, and cardiac MRI T2* were collected on patient samples. The metabolite profile on fasting serum samples was analyzed in triplicate using gas chromatography-mass spectrometry (GC-MS) along with MetaboliteDetector a software. Significant metabolites were identified using multivariate regression analysis by supervised projection methods (two-way orthogonal partial least squares discriminant analysis, O2PLS-DA) using Simca-P (Umetrics). Results:A total of 291 analytes were detected and quantified for each samples by GC-MS metabolomics. Clear differences were detected in serum metabolite profiles between patients with iron overload and control samples (p = 0.03) (Figure 1). Metabolite differences between the two groups consisted of amino acids, their breakdown products, and sugars. Multivariate regression analysis showed correlation between the different metabolite profiles and diagnosis of thalassemia major (p = 0.02). No significant differences were seen comparing age, sex, pre-transfusion hemoglobin, serum ferritin, LIC, and cardiac MRI T2*. There was a non-significant but detectable difference observed in the metabolic profile of the 3 patients on combination therapy with DFO and DFP (p<0.3) suggesting possible differences related to the presence DFP. Conclusion:We conclude that metabolomics is a valid and useful tool to detect differences in the iron-associated metabolic changes in iron overloaded patients, specifically, thalassemia major. Combination therapy with DFO and DFP has a possibly different metabolic profile compared to other chelators. Further work is needed to delineate the specific metabolic changes due to iron chelation, specifically, effects on oxidative damage, as chelation therapy is known to reduce the levels of non-transferrin bound iron and reactive oxygen species. A larger sample size may also be needed to further detect any metabolite differences in relation to organ iron load. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

측백나무 잎.열매 추출물의 이화학적 특성 및 항산화 효과

The contents of bioactive materials (e.g. polyphenolics compounds, flavonoids, minerals, and fatty acids) and antioxidative activities (DPPH (α,α'-diphenyl-β-picrylhydrazyl) free radical scavenging activity, peroxidation of linoleic acid and rat hepatocyte microsome, and Fe/Cu reducing power) were tested by in vitro experimental models using water, ethanol and methanol extracts of leaves (TOL) and fruits (TOF) from Thuja orientalis. Methanol extract from TOL showed the highest extraction yield (12.90%) as well as contents of polyphenolic compounds (16.02%) and flavonoids (0.25%). Major minerals were Ca, K, and Mg. Major fatty acids were palmitic and lauric acids in TOL and palmitic and decanoic acids in TOF. In oxidation of in vitro models using DPPH free radical scavenging activity, Fe/Cu reducing power, Fe²?/ascorbate-induced linolenic acid peroxidation by ferric thiocyanate and thiobarbituric acid (TBA) methods, and autooxidation of rat hepatic microsomes membrane, antioxidative activities were stronger in all extracts of TOL than in those of TOF in a dose-dependent manner. From these results, methanol extract of TOL was shown to have the most potent antioxidative properties and the highes content of antioxidative compounds such as polyphenolic compounds and flavonoids.

Preventive effect of a galactoglucomannan (GGM) from Dendrobium huoshanense on selenium-induced liver injury and fibrosis in rats

This study was carried out to investigate the preventive effects of galactoglucomannan (GGM), a homogeneous polysaccharide from Dendrobium huoshanense, on liver injury and fibrosis induced by sodium selenite. Sprague-Dawley rats injected subcutaneously with sodium selenite at the dosage of 3.28 mgkg(-1) b.wt. were set as the model groups. Rats treated with sodium selenite at the dosage of 3.28 mgkg(-1) b.wt. and GGM at 50-200 mgkg(-1) b.wt. were set as the prevention groups. Biochemical and histological analysis showed that GGM significantly ameliorated selenite-induced liver injury and fibrosis in rats. Oral administration of GGM effectively attenuated the toxicity of selenite to liver tissue, which was judged both by the decreased activities of serum hepatic enzymes, including alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH), and by liver histopathological examination. Meanwhile, GGM also reduced the levels of H(2)O(2) and malondialdehyde (MDA), elevated the levels of GSH, restored the fluidity of hepatic plasma membrane, and retained the activities of endogenous enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST). The prevention of selenite-induced liver injury and fibrosis by GGM was further supported by the reduced expression of transforming growth factor-β1 (TGF-β1) and type I collagen. These results suggested that GGM may be developed into a novel antifibrotic agent for the prevention of liver injury and fibrosis.

X-linked cholestasis in mouse due to mutations of the P4-ATPase ATP11C

Transporters at the hepatic canalicular membrane are essential for the formation of bile and the prevention of cholestatic liver disease. One such example is ATP8B1, a P4-type ATPase disrupted in three inherited forms of intrahepatic cholestasis. Mutation of the X-linked mouse gene Atp11c, which encodes a paralogous P4-type ATPase, precludes B-cell development in the adult bone marrow, but also causes hyperbilirubinemia. Here we explore this hyperbilirubinemia in two independent Atp11c mutant mouse lines, and find that it originates from an effect on nonhematopoietic cells. Liver function tests and histology revealed only minor pathology, although cholic acid was elevated in the serum of mutant mice, and became toxic to mutant mice when given as a dietary supplement. The majority of homozygous mutant females also died of dystocia in a maternal genotype-specific manner. ATP11C therefore represents a multifunctional transporter, essential for adult B-cell development, the prevention of intrahepatic cholestasis, and parturition, and is a new candidate for genetically undiagnosed cases of cholestasis and dystocia in humans.