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Primary Hepatocytes (primary + hepatocyte)
Selected AbstractsLiver endothelial cells promote LDL-R expression and the uptake of HCV-like particles in primary rat and human hepatocytes,HEPATOLOGY, Issue 2 2006Yaakov Nahmias Low-density lipoprotein (LDL) is an important carrier of plasma cholesterol and triglycerides whose concentration is regulated by the liver parenchymal cells. Abnormal LDL regulation is thought to cause atherosclerosis, while viral binding to LDL has been suggested to facilitate hepatitis C infection. Primary hepatocytes quickly lose the ability to clear LDL during in vitro culture. Here we show that the coculture of hepatocytes with liver sinusoidal endothelial cells (LSEC) significantly increases the ability of hepatocytes to uptake LDL in vitro. LDL uptake does not increase when hepatocytes are cocultured with other cell types such as fibroblasts or umbilical vein endothelial cells. We find that LSECs induce the hepatic expression of the LDL receptor and the epidermal growth factor receptor. In addition, while hepatocytes in single culture did not take up hepatitis C virus (HCV)-like particles, the hepatocytes cocultured with LSECs showed a high level of HCV-like particle uptake. We suggest that coculture with LSECs induces the emergence of a sinusoidal surface in primary hepatocytes conducive to the uptake of HCV-like particles. In conclusion, our findings describe a novel model of polarized hepatocytes in vitro that can be used for the study of LDL metabolism and hepatitis C infection. (HEPATOLOGY 2006;43:257,265.) [source] Growth factor attenuation of IFN,-mediated hepatocyte apoptosis requires p21waf,1INTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2006Christian T. McCullough Summary Interferon gamma (IFN,) is an important mediator of inflammatory liver damage as part of a complex cytokine network. In vitro, IFN, induces hepatocyte apoptosis. We hypothesized that the hepatocyte response to IFN signalling is context-dependent, and that specific growth factors, via phosphatidylinositol 3 kinase (PI(3)K) and protein kinase B/Akt signalling pathways, confer a cytoprotective effect. We established an in vitro model of IFN,-mediated primary hepatocyte injury. We show that epidermal growth factor (EGF) and hepatocyte growth factor (HGF) attenuate the IFN,-induced hepatocyte apoptosis. IRF-1, but not p53, is required for IFN,-mediated apoptosis. The loss of p21waf,1 not only sensitizes the hepatocyte to IFN,-mediated injury but is required for survival factor mediated cytoprotection. We show that the PI(3)K inhibitor, LY294002, partially inhibits the apoptotic response of the hepatocyte to IFN,. In summary, we present evidence that a component of pro-apoptotic IFN, signalling in the primary hepatocyte occurs via the PI(3)K pathway. We show that the hepatocyte response to IFN, is modulated by external survival factors and that this survival signalling requires p21waf,1. [source] Induction of cytochrome P4501A in African brown house snake (Lamprophis fuliginosus) primary hepatocytesENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 2 2006Markus Hecker Abstract Ahough there have been numerous sudies involving fish, birds, and mammals, little is known about the response of the cytochrome P4501A system of snakes to halogenated aromatic hydrocarbons (HAHs). The present study describes the induction of ethoxyresorufin- O -deethylase (EROD) in primary hepatocytes of the African brown house snake (Lamprophis fuliginosus). Hepatocytes were exposed in multiwell plates to 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) and four different non- ortho -substi-tuted coplanar polychlorinated biphenyls (PCBs 77, 81, 126, and 169). Exposure to TCDD and PCB 126 resulted in a dose-dependent increase in EROD activity, with maximum inducible EROD activities of 177 ± 56 (mean ± SEM) and 101.1 ± 55 pmol/min/mg protein for TCDD and PCB 126, respectively. None of the other PCBs caused a measurable induction of EROD, which suggests reduced inducibility of snake hepatocytes compared to some vertebrate taxa. Median effective concentrations (EC50s) were 0.16 ± 0.03 nM for TCDD and 8.25 ± 4.14 nM for PCB 126. The relative potency (REP20,80) range for PCB 126 was 0.044 to 0.046. Compared to results from in vitro systems using other vertebrate species, both the maximum inducibility and the REPs estimated for L. fuliginosus were within the same range as those reported for mammals and the more sensitive bird species but were greater than the values reported for most fish species. In conclusion, induction of EROD activity in primary hepatocytes appears to be a useful approach for evaluating the dioxin-like potencies of aryl hydrocarbon,receptor agonists in snakes. The test system offers a method for rapid screening of reptilian responsiveness to these compounds using smaller numbers of organisms than with in vivo studies, an important consideration for many declining reptile species. [source] Polycyclic aromatic hydrocarbons as inducers of cytochrome P4501A enzyme activity in the rainbow trout liver cell line, RTL-W1, and in primary cultures of rainbow trout hepatocytesENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2001Anja Behrens Abstract In order to investigate cell-specific differences in the response of in vitro models to environmental toxicants, we compared the capacity of nine polycyclic aromatic hydrocarbons (PAHs) to induce cytochrome P4501A (CYP1A) in primary rainbow trout (Oncorhynchus mykiss) hepatocytes and a rainbow trout liver cell line, RTL-W1. Induction of CYP1A was estimated from the catalytic activity of 7-ethoxyresorufin- O -deethylase (EROD) and compared by median effective concentration (EC50) values, induction spans, and benzo[a]pyrene induction equivalency factors for inducing PAHs. The influence of culture conditions was investigated with respect to the presence or absence of serum and varying exposure times. Both in vitro systems lead to an identical classification of the PAHs in noninducing (anthracene, fluoranthene, phenanthrene, and pyrene) and inducing compounds with a similar ranking of inducing PAHs. Mean EC50 values in RTL-W1 cells were, respectively, 343 and 266 nM for benzo[a]anthracene, 57 and 92 nM for BaP, 134 and 283 nM for benzo[b]fluoranthene, 455 and 270 nM for chrysene, and 98 and 116 nM for 3-methylcholanthrene. Compared to primary hepatocytes, the RTL-W1 cell line was more sensitive in its EROD response to the presence or absence of serum and to the increase in exposure time, which led to higher EC50 values. [source] The PPAR, agonist GW501516 suppresses interleukin-6-mediated hepatocyte acute phase reaction via STAT3 inhibitionEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 5 2007T. Kino Abstract Background, Interleukin-6 and downstream liver effectors acute phase reactants are implicated in the systemic inflammatory reaction. Peroxisome proliferator-activated receptor , (PPAR,), which binds to and is activated by a variety of fatty acids, was recently shown to have anti-inflammatory actions. Materials and methods, We examined the ability of the synthetic PPAR, agonist GW501516 to suppress interleukin-6-induced expression of acute phase proteins in human hepatoma HepG2 cells and rat primary hepatocytes. Results, GW501516 dose-dependently suppressed interleukin-6-induced mRNA expression of the acute phase protein ,1-antichymotrypsin in HepG2 cells. The compound also suppressed interleukin-6-induced mRNA expression of ,2-acid glycoprotein, ,-fibrinogen and ,2-macroglobulin in and the secretion of C-reactive protein by rat primary hepatocytes. Depletion of the PPAR, receptor, but not of PPAR, or ,, attenuated the suppressive effect of GW501516 on interleukin-6-induced ,1-antichymotrypsin mRNA expression, indicating that PPAR, specifically mediated this effect. Since interleukin-6 stimulates the transcriptional activity of the ,1-antichymotrypsin promoter by activating the signal transducer and activator of transcription (STAT) 3, we examined functional interaction of this transcription factor and PPAR, on this promoter. Overexpression of PPAR, enhanced the suppressive effect of GW501516 on STAT3-activated transcriptional activity of the ,1-antichymotrypsin promoter, while GW501516 suppressed interleukin-6-induced binding of this transcription factor to this promoter. Conclusions, These findings indicate that agonist-activated PPAR, interferes with interleukin-6-induced acute phase reaction in the liver by inhibiting the transcriptional activity of STAT3. PPAR, agonists might be useful for the suppression of systemic inflammatory reactions in which IL-6 plays a central role. [source] Primary hepatocyte culture supports hepatitis C virus replication: A model for infection-associated hepatocarcinogenesis,HEPATOLOGY, Issue 6 2010Krishna Banaudha Analysis of progressive changes in hepatic gene expression that underlie hepatocarcinogenesis following hepatitis C virus (HCV) infection require examination of long-term cultures of normally differentiating primary human hepatocytes. We report a culture system of primary hepatocytes that support productive replication of infectious HCV. Hepatic functions were analyzed by reverse-transcription polymerase chain reaction amplification of total cell RNA from cultures maintained in serum-free defined medium for up to 190 days. Sustained hepatic function was assessed by expression of albumin, alpha-fetoprotein, cytochrome P4502E1, cytokeratin-18, type-1 collagen, transforming growth factor-beta 1, matrix metalloproteinase-2 (MMP-2), MMP-13, and interferon alpha-receptors 1 and 2. Normally differentiated human primary hepatocytes supported productive replication of infectious clones of HCV genotypes 1a, 1b, and 2a; virus infection was inhibited by antibodies against CD81 virus entry factor. Virus released into the culture media of HCV-infected primary hepatocytes repeatedly passage to naďve hepatocytes. Replication of the three HCV genotypes shows interferon sensitivity observed in natural infections. Conclusion: Sustained cultures of physiologic host cells for the propagation of infectious HCV strains should accelerate studies of host response to HCV infection and progressive liver disease. Hepatology 2010;51:1922,1932 [source] Generation of endoderm-derived human induced pluripotent stem cells from primary hepatocytes,HEPATOLOGY, Issue 5 2010Hua Liu Recent advances in induced pluripotent stem (iPS) cell research have significantly changed our perspective on regenerative medicine. Patient-specific iPS cells have been derived not only for disease modeling but also as sources for cell replacement therapy. However, there have been insufficient data to prove that iPS cells are functionally equivalent to human embryonic stem (hES) cells or are safer than hES cells. There are several important issues that need to be addressed, and foremost are the safety and efficacy of human iPS cells of different origins. Human iPS cells have been derived mostly from cells originating from mesoderm and in a few cases from ectoderm. So far, there has been no report of endoderm,derived human iPS cells, and this has prevented comprehensive comparative investigations of the quality of human iPS cells of different origins. Here we show for the first time reprogramming of human endoderm-derived cells (i.e., primary hepatocytes) to pluripotency. Hepatocyte-derived iPS cells appear indistinguishable from hES cells with respect to colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, and differentiation potential in embryoid body formation and teratoma assays. In addition, these cells are able to directly differentiate into definitive endoderm, hepatic progenitors, and mature hepatocytes. Conclusion: The technology to develop endoderm,derived human iPS cell lines, together with other established cell lines, will provide a foundation for elucidating the mechanisms of cellular reprogramming and for studying the safety and efficacy of differentially originated human iPS cells for cell therapy. For the study of liver disease pathogenesis, this technology also provides a potentially more amenable system for generating liver disease-specific iPS cells. (HEPATOLOGY 2010;51:1810,1819) [source] Specific role for acyl CoA:Diacylglycerol acyltransferase 1 (Dgat1) in hepatic steatosis due to exogenous fatty acids,HEPATOLOGY, Issue 2 2009Claudio J. Villanueva Nonalcoholic fatty liver disease, characterized by the accumulation of triacylglycerols (TGs) and other lipids in the liver, often accompanies obesity and is a risk factor for nonalcoholic steatohepatitis and fibrosis. To treat or prevent fatty liver, a thorough understanding of hepatic fatty acid and TG metabolism is crucial. To investigate the role of acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), a key enzyme of TG synthesis, in fatty liver development, we studied mice with global and liver-specific knockout of Dgat1. DGAT1 was required for hepatic steatosis induced by a high-fat diet and prolonged fasting, which are both characterized by delivery of exogenous fatty acids to the liver. Studies in primary hepatocytes showed that DGAT1 deficiency protected against hepatic steatosis by reducing synthesis and increasing the oxidation of fatty acids. In contrast, lipodystrophy (aP2-SREBP-1c436) and liver X receptor activation (T0901317), which increase de novo fatty acid synthesis in liver, caused steatosis independently of DGAT1. Pharmacologic inhibition of Dgat1 with antisense oligonucleotides protected against fatty liver induced by a high-fat diet. Conclusion: Our findings identify a specific role for hepatic DGAT1 in esterification of exogenous fatty acids and indicate that DGAT1 contributes to hepatic steatosis induced by this mechanism. (HEPATOLOGY 2009.) [source] Rat hepatocyte spheroids formed by rocked technique maintain differentiated hepatocyte gene expression and function,HEPATOLOGY, Issue 2 2009Colleen M. Brophy The culture of primary hepatocytes as spheroids creates an efficient three-dimensional tissue construct for hepatic studies in vitro. Spheroids possess structural polarity and functional bile canaliculi with normal differentiated function. Thus, hepatocyte spheroids have been proposed as the cell source in a variety of diagnostic, discovery, and therapeutic applications, such as a bioartificial liver. Using a novel rocking technique to induce spheroid formation, kinetics of spheroid formation, cell-cell adhesion, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. Evidence was provided that the formation of spheroids occurred faster and with fewer nonadherent hepatocytes in rocked suspension culture compared to a traditional rotational system. Hepatocyte spheroids in rocked culture showed stable expression of more than 80% of 242 liver-related genes including those of albumin synthesis, urea cycle, phase I and II metabolic enzymes, and clotting factors. Biochemical activity of rocked spheroid hepatocytes was superior to monolayer culture of hepatocytes on tissue culture plastic and collagen. Conclusion: Spheroid formation by rocker technique was more rapid and more efficient than by rotational technique. Rocker-formed spheroids appear suitable for application in a bioartificial liver or as an in vitro liver tissue construct. (HEPATOLOGY 2009.) [source] Engineered measles virus as a novel oncolytic viral therapy system for hepatocellular carcinoma,HEPATOLOGY, Issue 6 2006Boris Blechacz The oncolytic measles virus Edmonston strain (MV-Edm), a nonpathogenic virus targeting cells expressing abundant CD46, selectively destroys neoplastic tissue. Clinical development of MV-Edm would benefit from noninvasive monitoring strategies to determine the speed and extent of the spread of the virus in treated patients and the location of virus-infected cells. We evaluated recombinant MV-Edm expressing carcinoembryonic antigen (CEA) or the human sodium iodide symporter (hNIS) for oncolytic potential in hepatocellular carcinoma (HCC) and efficiency in tracking viruses in vivo by noninvasive monitoring. CD46 expression in human HCC and primary hepatocytes was assessed by flow cytometry and immunohistochemistry. Infectivity, syncytium formation, and cytotoxicity of recombinant MV-Edm in HCC cell lines were evaluated by fluorescence microscopy, crystal violet staining, and the MTS assay. Transgene expression in HCC cell lines after infection with recombinant MV-Edm in vitro and in vivo was assessed by CEA concentration, 125I-uptake, and 123I-imaging studies. Toxicology studies were performed in IfnarKO×CD46 transgenic mice. The CD46 receptor was highly expressed in HCC compared to nonmalignant hepatic tissue. Recombinant MV-Edm efficiently infected HCC cell lines, resulting in extensive syncytium formation followed by cell death. Transduction of HCC cell lines and subcutaneous HCC xenografts with recombinant MV-Edm resulted in high-level expression of transgenes in vitro and in vivo. MV-Edm was nontoxic in susceptible mice. Intratumoral and intravenous therapy with recombinant MV-Edm resulted in inhibition of tumor growth and prolongation of survival with complete tumor regression in up to one third of animals. In conclusion, engineered MV-Edm may be a potent and novel cancer gene therapy system for HCC. MV-Edm expressing CEA or hNIS elicited oncolytic effects in human HCC cell lines in vitro and in vivo, enabling the spread of the virus to be monitored in a noninvasive manner. (HEPATOLOGY 2006;44:1465,1477.) [source] Interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes,HEPATOLOGY, Issue 4 2006Sean E. Doyle Interleukin-28A (IL-28A), IL-28B and IL-29 are a family of class II cytokines that stimulate antiviral responses through a heterodimeric receptor that is distinct from the type I interferon (IFN) receptor. To better understand how this newly described family of cytokines regulates the antiviral state, we compared various cellular responses elicited by IL-29 and IFN-,. Here we show that these cytokines stimulate similar patterns of signal transducer and activator of transcription 1 (STAT-1), -2, -3, and -5 phosphorylation and nearly identical patterns of gene expression when analyzed in two distinct cell types by microarray analysis. Interestingly, the IL-29 receptor is preferentially expressed on primary hepatocytes within normal liver and pegylated forms of IL-29 and IFN-, induced equivalent 2,5, oligoadenylate synthetase (OAS) and MX1 gene expression in this cell type. Pegylated IL-29 also produced a significant reduction in human hepatitis B and hepatitis C viral load in vitro and reduced the cytopathic effect caused by the fully replicating flavivirus, West Nile virus. In conclusion, IL-29 and IFN-, stimulate identical antiviral responses despite their utilization of different receptors. This fact, combined with significant receptor expression in hepatitis virus-infected livers, suggests that IL-29 may have therapeutic value against chronic viral hepatitis in human patients. (HEPATOLOGY 2006;44:896,906.) [source] S-adenosylhomocysteine sensitizes to TNF-, hepatotoxicity in mice and liver cells: A possible etiological factor in alcoholic liver diseaseHEPATOLOGY, Issue 4 2004Zhenyuan Song In alcoholic liver disease, tumor necrosis factor-, (TNF,) is a critical effector molecule, and abnormal methionine metabolism is a fundamental acquired metabolic abnormality. Although hepatocytes are resistant to TNF,-induced killing under normal circumstances, previous studies have shown that primary hepatocytes from rats chronically fed alcohol have increased TNF, cytotoxicity. Therefore, there must be mechanisms by which chronic alcohol exposure "sensitizes" to TNF, hepatotoxicity. S-adenosylhomocysteine (SAH) is product of methionine in transsulfuration pathway and a potent competitive inhibitor of most methyltransferases. In this study, we investigated the effects of increased SAH levels on TNF, hepatotoxicity. Our results demonstrated that chronic alcohol consumption in mice not only decreased hepatic S-adenosylmethionine levels but also increased hepatic SAH levels, which resulted in a significantly decreased S-adenosylmethionine-to-SAH ratio. This was associated with significant increases in hepatic TNF, levels, caspase-8 activity, and cell death. In vitro studies demonstrated that SAH-enhancing agents sensitized hepatocytes to TNF, killing, and the death was associated with increased caspase-8 activity, which was blocked by a caspase-8 inhibitor. In addition, increased intracellular SAH levels had no effect on nuclear factor ,B activity induced by TNF,. In conclusion, these results provide a new link between abnormal methionine metabolism and abnormal TNF, metabolism in alcoholic liver disease. Increased SAH is a potent and clinically relevant sensitizer to TNF, hepatotoxicity. These data further support improving the S-adenosylmethionine-to-SAH ratio and removal of intracellular SAH as potential therapeutic options in alcoholic liver disease. Supplementary material for this article can be found on the HEPATOLOGYwebsite (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2004;40:989,997.) [source] Bid-dependent generation of oxygen radicals promotes death receptor activation,induced apoptosis in murine hepatocytesHEPATOLOGY, Issue 2 2004Wen-Xing Ding Activation of tumor necrosis factor receptor 1 or Fas leads to the generation of reactive oxygen species, which are important to the cytotoxic effects of tumor necrosis factor , (TNF-,) or Fas ligand. However, how these radicals are generated following receptor ligation is not clear. Using primary hepatocytes, we found that TNF-, or anti,Fas antibody,induced burst of oxygen radicals was mainly derived from the mitochondria. We discovered that Bid,a pro-death Bcl-2 family protein activated by ligated death receptors,was the main intracellular molecule signaling the generation of the radicals by targeting to the mitochondria and that the majority of oxygen radical production was dependent on Bid. Reactive oxygen species contributed to cell death and caspase activation by promoting FLICE-inhibitory protein degradation and mitochondrial release of cytochrome c. For the latter part, the oxygen radicals did not affect Bak oligomerization but instead promoted mitochondrial cristae reorganization and membrane lipid peroxidation. Antioxidants could reverse these changes and therefore protect against TNF-, or anti,Fas-induced apoptosis. In conclusion, our studies established the signaling pathway from death receptor engagement to oxygen radical generation and determined the mechanism by which reactive oxygen species contributed to hepatocyte apoptosis following death receptor activation. (HEPATOLOGY 2004;40:403,413.) [source] Activation of the Raf-1/MEK/ERK cascade by bile acids occurs via the epidermal growth factor receptor in primary rat hepatocytesHEPATOLOGY, Issue 2 2002Yi-Ping Rao Bile acids have been reported to activate several different cell signaling cascades in rat hepatocytes. However, the mechanism(s) of activation of these pathways have not been determined. This study aims to determine which bile acids activate the Raf-1/MEK/ERK cascade and the mechanism of activation of this pathway. Taurodeoxycholic acid (TDCA) stimulated (+235%) the phosphorylation of p74 Raf-1 in a time (5 to 20 minutes) and concentration-dependent (10 to 100 ,mol/L) manner. Raf-1 and ERK activities were both significantly increased by most bile acids tested. Deoxycholic acid (DCA) was the best activator of ERK (3.6-fold). A dominant negative Ras (N17) construct expressed in primary hepatocytes prevented the activation of ERK by DCA. The epidermal growth factor receptor (EGFR)-specific inhibitor (AG1478) significantly inhibited (,81%) the activation of ERK by DCA. DCA rapidly (30 to 60 seconds) increased phosphorylation of the EGFR (,2-fold) and Shc (,4-fold). A dominant negative mutant of the EGFR (CD533) blocked the ability of DCA to activate ERK. In conclusion, these results show that DCA activates the Raf-1/MEK/ERK signaling cascade in primary hepatocytes primarily via an EGFR/Ras-dependent mechanism. [source] Different cellular localization, translocation, and insulin-induced phosphorylation of PKB, in HepG2 cells and hepatocytesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2002Noor Afshan Syed Abstract Protein kinase B (PKB), a serine/threonine protein kinase, prevents apoptosis and promotes cellular transformation. PKB activity is stimulated by insulin. In this report, we examined the relative amounts of expression, location, and translocation upon insulin stimulation of PKB, in normal primary hepatocytes and carcinoma cells, HepG2 cells. Non-phosphorylated PKB, was present in both types of unstimulated cells. The phosphorylated form of the enzyme was present in the nucleus of unstimulated HepG2 cells but not in normal hepatocytes. In the cytoplasm, PKB, was found in greater abundance in the hepatocytes as compared in HepG2 cells. Insulin induced the translocation of phosphorylated PKB, from the nucleus to the nuclear membrane in HepG2 cells. In contrast, insulin caused translocation and phosphorylation of PKB, from the cytosol to the plasma membrane in normal hepatocytes. In addition, there is a higher expression of PKB, in the HepG2 cells as compared to normal primary hepatocytes. These findings provide an important distinction between hepatocellular HepG2 cells and normal liver cells and suggest that the presence of constitutively active nuclear PKB in the transformed cells might be an important contributor in cell transformation and immortality of hepatoma cells. J. Cell. Biochem. 86: 118,127, 2002. © 2002 Wiley-Liss, Inc. [source] Temporary amelioration of bilirubin conjugation defect in Gunn rats by transplanting conditionally immortalized hepatocytesJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 6 2002BYUNG-HO KIM Abstract Background: Conditionally immortalized hepatocytes (CIH) have been used in hepatocyte transplantation as an alternative to primary hepatocytes to cope with the shortage of donor organs. However, CIH are known to undergo apoptosis at body temperature and survive in vivo for a short period. In the present study, we investigated whether CIH function or not and how long their function is maintained in vivo. Methods: Various CIH cell lines that were established with temperature-sensitive Simian virus 40 large T antigen were transplanted into the spleen of Gunn rats, which are defective in bilirubin uridine diphosphate glucuronoside transferase (BUGT). Then, we measured biological changes over 3 months. Results: Serum bilirubin of the syngeneic CIH recipients decreased by 30%, which was maintained for 8 weeks. Thereafter, it began to rise to basal levels. The recipients of allogeneic CIH showed a minor reduction of bilirubin, although this was not statistically significant. However, there was no significant change in the bilirubin level in recipients of BUGT-defective congeneic CIH throughout the study period. Bilirubin monoglucuronides in the bile were not detected in the recipients of BUGT-defective CIH. However, they appeared in recipients of non-defective CIH and made up approximately 41% of total bile pigments. Conclusions: Conditionally immortalized hepatocytes expressed hepatocyte function in vivo as well as in vitro, but the function lasted for a couple of months. According to our previous study, the limited functional duration may be related to the inevitable occurrence of apoptosis of these cells at body temperature. These data suggest that CIH can be used in hepatocyte transplantation only for temporary hepatic support. © 2002 Blackwell Publishing Asia Pty Ltd [source] Effect of interferons on P-glycoprotein-mediated rhodamine-123 efflux in cultured rat hepatocytesJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 10 2002Yukiko Akazawa Abstract The effect of interferon (IFN)-, and IFN-, on P-glycoprotein (P-gp)-mediated efflux of rhodamin-123(Rho-123), a typical substrate of P-gp, was studied in rat hepatocytes in primary culture. After treatment with IFN-,, IFN-,, or both for 3 days, steady-state levels of Rho-123, incorporated into the hepatocytes, were measured to evaluate the P-gp activity. Whereas IFN-, did not affect the intracellular level of Rho-123, IFN-, treatment caused a significant increase of the level, suggesting that IFN-, treatment suppresses the expression of P-gp or its activity. A combination of the two types of IFN exhibited a similar effect to that of IFN-, alone. The effect of IFN-, was still observed in the presence of H2O2, which enhances the expression and activity of P-gp. Immunoblot analysis using a monoclonal antibody C219 revealed, however, that P-gp expression was increased after treatment with IFN-,, but only slightly by IFN-, treatment. These results suggest that the enhanced Rho-123 uptake of rat primary hepatocytes induced by IFN-, does not result from reduced expression of P-gp but, rather, from impaired maturation or dysfunction of the efflux transporter. © 2002 Wiley-Liss Inc. and the American Pharmaceutical Association J Pharm Sci 91:2110,2115, 2002 [source] Mitochondrial S -Adenosyl- l -Methionine Transport is Insensitive to Alcohol-Mediated Changes in Membrane DynamicsALCOHOLISM, Issue 7 2009Anna Fernández Background:, Alcohol-induced liver injury is associated with decreased S -adenosyl- l -methionine (SAM)/S -adenosyl- l -homocysteine (SAH) ratio and mitochondrial glutathione (mGSH) depletion, which has been shown to sensitize hepatocytes to tumor necrosis factor (TNF). Aims:, As the effect of alcohol on mitochondrial SAM (mSAM) has been poorly characterized, our aim was to examine the status and transport of mSAM in relation to that of mGSH during alcohol intake. Methods:, Sprague,Dawley rats were pair fed Lieber,DeCarli diets containing alcohol for 1 to 4 weeks and liver fractionated into cytosol and mitochondria to examine the mSAM transport and its sensitivity to membrane dynamics. Results:, We found that cytosol SAM was depleted from the first week of alcohol feeding, with mSAM levels paralleling these changes. Cytosol SAH, however, increased during the first 3 weeks of alcohol intake, whereas its mitochondrial levels remained unchanged. mGSH depletion occurred by 3 to 4 weeks of alcohol intake due to cholesterol-mediated impaired transport from the cytosol. In contrast to this outcome, the transport of SAM into hepatic mitochondria was unaffected by alcohol intake and resistant to cholesterol-mediated perturbations in membrane dynamics; furthermore cytosolic SAH accumulation in primary hepatocytes by SAH hydrolase inhibition reproduced the mSAM depletion by alcohol due to the competition of SAH with SAM for mitochondrial transport. However, alcohol feeding did not potentiate the sensitivity to inhibition by SAH accumulation. Conclusions:, Alcohol-induced mSAM depletion precedes that of mGSH and occurs independently of alcohol-mediated perturbations in membrane dynamics, disproving an inherent defect in the mSAM transport by alcohol. These findings suggest that the early mSAM depletion may contribute to the alterations of mitochondrial membrane dynamics and the subsequent mGSH down-regulation induced by alcohol feeding. [source] Induction of Transferrin Receptor by Ethanol in Rat Primary Hepatocyte CultureALCOHOLISM, Issue 2004Masako Suzuki Background: It is not uncommon for alcoholics to have iron accumulation in the liver, a condition that may contribute to the development of alcoholic liver disease. Recently, we reported that the expression of transferrin receptor, which mediates cellular iron uptake, was increased in hepatocytes in patients with alcoholic liver disease. To elucidate the mechanism of the iron accumulation in hepatocytes in such disease, we examined whether ethanol exposure induced the transferrin receptor expression and increased the cellular iron uptake. Methods: Rat primary hepatocytes were isolated and cultured in the presence of 20 ,mol/liter of iron and 25 mmol/liter of ethanol. Results: Ethanol exposure to the hepatocytes demonstrated an ,2-fold increase in transferrin receptor expression for 24 hr, shown by Western blot analysis and 35S-methionine metabolic labeling, 19% increase in 59Fe-transferrin uptake by hepatocytes, and 20% increase in activity of iron regulatory protein examined by band shift assay. Conclusion: Ethanol exposure induced the transferrin receptor expression, partially through the activation of iron regulatory protein, and increased the transferrin-bound iron uptake in rat hepatocyte cultures. The induction of transferrin receptor by ethanol might be one of the mechanisms of iron accumulation in the hepatocytes in alcoholic liver disease. [source] Dual effects of hepatitis C virus Core protein on the transcription of cyclin-dependent kinase inhibitor p21 geneJOURNAL OF VIRAL HEPATITIS, Issue 4 2003H. J. Kwun Summary. Transcription of p21 was activated in hepatitis C virus (HCV) Core-expressing HepG2 cells where its upstream p53 was stabilized. However, this effect was not absolutely required for the activation of p21 by Core, as demonstrated in Hep3B cells. In addition, an opposite effect on the transcription of p21 was observed in NIH3T3 and primary hepatocytes, where p53 was not decreased by Core. To explain the p53-independent regulation of p21 by Core, we identified a Core-responsive element between positions ,74 and ,83 of the p21 promoter, exactly overlapped with a tumour growth factor , (TGF- ,)/butyrate responsive element. Furthermore, we demonstrated that Core could activate the p21 through the element by stimulating a butyrate pathway, whereas this was inhibited through a TGF- , pathway. The opposing effects of Core protein on the transcription of p21 might be important in understanding the progression of hepatic disease in HCV-positive patients. [source] Patterned Co-Culture of Primary Hepatocytes and Fibroblasts Using Polyelectrolyte Multilayer TemplatesMACROMOLECULAR BIOSCIENCE, Issue 3 2007Srivatsan Kidambi Abstract This paper describes the formation of patterned cell co-cultures using the layer-by-layer deposition of synthetic ionic polymers and without the aid of adhesive proteins/ligands such as collagen or fibronectin. In this study, we used synthetic polymers, namely poly(diallyldimethylammonium chloride) (PDAC) and sulfonated polystyrene (SPS) as the polycation and polyanion, respectively, to build the multilayer films. We formed SPS patterns on polyelectrolyte multilayer (PEM) surfaces either by microcontact printing PDAC onto SPS surfaces or vice-versa. To create patterned co-cultures on PEMs, we capitalize on the preferential attachment and spreading of primary hepatocytes on SPS as opposed to PDAC surfaces. In contrast, fibroblasts readily attached to both PDAC and SPS surfaces, and as a result, we were able to obtain patterned co-cultures of fibroblast and primary hepatocytes on synthetic PEM surfaces. We characterized the morphology and hepatic-specific functions of the patterned cell co-cultures with microscopy and biochemical assays. Our results suggest an alternative approach to fabricating controlled co-cultures with specified cell,cell and cell,surface interactions; this approach provides flexibility in designing cell-specific surfaces for tissue engineering applications. [source] Real time monitoring of drug metabolic enzyme response inside human hepatoma GS-3A4-HepG2 cells by means of electrochemical impedance measurementPOLYMERS FOR ADVANCED TECHNOLOGIES, Issue 5 2004Masaaki Kobayashi Abstract Cytochrome P-450s (CYPs) are important biopolymers for the maintenance of cellular function. If metabolic activity of the CYP in the cells can be estimated, so can the function of metabolism, which is closer to the organism. In this research, the method of measuring the drug metabolic activity inside the cell by making use of an electrochemical technique was examined. Human hepatoma GS-3A4-HepG2 cells of which the cytochrome P-4503A4 (CYP3A4) drug metabolic activity is found to be the same as that of primary hepatocytes were used in the experiment. The GS-3A4-HepG2 cells were cultured on an indium-tin oxide (ITO) electrode until they became confluent. Substrate testosterone and inhibitor ketoconazole of CYP3A4 were exposed to cells cultured on an ITO electrode, and the reaction was observed by noting the electrochemical impedance measurement. Impedance was decomposed into the resistance component and the reactance component, and each was examined in detail. As a result, according to testosterone concentration change, there was a remarkable time change in the reactance component. A similar impedance measurement was done by using human hepatoma HepG2 cells in which the drug metabolic activity had extremely decreased. Nevertheless, no time change in the reactance component that was noticed in GS-3A4-HepG2 cells was observed. Next, the amount of metabolite in the solution after impedance measurement was measured by means of liquid chromatography-tandem mass spectroscopy (LC-MS/MS). In the experiment with GS-3A4-HepG2 cells, a testosterone concentration-dependent correlation was observed between the reactance component change and the amount of metabolite. But, in the impedance measurement by ketoconazole, the change in reactance components was not observed in either the GS-3A4-HepG2 cells or the HepG2 cells. Ketoconazole and the heme iron in CYP3A4 effect the coordination bond, but ketoconazole was not metabolized by CYP3A4. It was confirmed that the time change in the reactance component which was caused by the testosterone was detected neither in the cells that take up the substrate, nor in the coordination bond between the CYP enzyme and the drug. Therefore, the time change in the remarkable reactance component observed by this electrochemical impedance measurement is dependent on drug metabolic activity. An electrochemical drug metabolic activity measuring method with the human hepatoma GS-3A4-HepG2 cells was able to be established. Copyright © 2004 John Wiley & Sons, Ltd. [source] Effects of glucose and insulin on HepG2-C3A cell metabolismBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Vidya V. Iyer Abstract HepG2, hepatocellular carcinoma cells, are used in drug toxicity studies and have also been explored for bioartificial livers. For these applications, the cells are under variable levels of nutrients and hormones, the effects of which on metabolism are poorly understood. In this study, HepG2-C3A cells were cultured under varying levels of glucose (high, low, and glucose-free) and insulin (without and with physiological levels of insulin) for 5 days. Cell growth was found to be comparable between high and low glucose media and lowest for glucose-free medium. Several features of central metabolism were affected profoundly by the medium glucose levels. Glucose consumption was greater for low glucose medium compared to high glucose medium, consistent with known glucose feedback regulation mechanisms. Urea productivity was highest in glucose-free medium. Further, it was seen that lactate acted as an alternative carbon source in the absence of glucose, whereas it acted as a sink for the high and low glucose media. Using a metabolic network flexibility analysis (MNFA) framework with stoichiometric and thermodynamic constraints, intracellular fluxes under varying levels of glucose and insulin were evaluated. The analysis indicates that urea production in HepG2-C3A cells arises via the arginase II pathway rather than from ammonia detoxification. Further, involvement of the putrescine metabolism with glutamine metabolism caused higher urea production in glucose-free medium consistent with higher glutamine uptake. MNFA indicated that in high and low glucose media, glycolysis, glutaminolysis, and oxidative phosphorylation were the main sources of energy (NADH, NADPH, and ATP). In the glucose-free medium, due to very low glycolytic flux, higher malate to pyruvate glutaminolytic flux and TCA cycle contributed more significantly to energy metabolism. The presence of insulin lowered glycerol uptake and corresponding fluxes involved in lipid metabolism for all glucose levels but otherwise exerted negligible effect on metabolism. HepG2-C3A cells thus show distinct differences from primary hepatocytes in terms of energy metabolism and urea production. This knowledge can be used to design media supplements and metabolically engineer cells to restore necessary hepatic functions to HepG2-C3A cells for a range of applications. Biotechnol. Bioeng. 2010;107: 347,356. © 2010 Wiley Periodicals, Inc. [source] Ammonia Removal Using Hepatoma Cells in Mammalian Cell CulturesBIOTECHNOLOGY PROGRESS, Issue 5 2000Yeon Sook Choi It was examined whether hepatocyte cell lines can be used for ammonia removal in mammalian cell cultures. It was found that there exists a critical ammonium concentration level for each hepatocyte cell to remove ammonia. Among the cells tested in this work, primary hepatocytes showed the strongest ammonia removal capability if ammonium concentration is higher than the critical level. However, primary hepatocytes lost the liver function gradually and finally died after 2,3 weeks. Because of this limitation, primary hepatocytes were not appropriate to be used for ammonia removal in long-term cultures. Hep G2 cells, which are immortal, also showed a strong ammonia removal activity. The ammonia removal activity of Hep G2 cells depended on the concentration of ammonium in the medium, as in the case of primary hepatocytes. However, urea could not be detected in the course of ammonia removal by Hep G2 cells. Instead of urea, Hep G2 cells secreted glutamine into the culture medium. The capacity for ammonia removal was higher in the absence than in the presence of glutamine. Thus we checked the activity of glutamine synthetase in the Hep G2 cells. The level of glutamine synthetase activity increased with the addition of ammonium chloride. This result accounts for the ammonium concentration dependency of Hep G2 cells in ammonia removal and glutamine synthesis. Furthermore Hep G2 cells could grow well in the absence of glutamine, which was necessarily required in mammalian cell cultures. These results prove that glutamine formation serves as the primary mechanism of detoxifying ammonia in hepatocyte cell lines as expected. In addition, it was demonstrated that ammonium level could be reduced 38% and that erythropoietin production increased 2-fold in the mixed culture of Hep G2 and recombinant CHO cells. [source] Inhibition of NF-,B activation by the histone deacetylase inhibitor 4-Me2N-BAVAH induces an early G1 cell cycle arrest in primary hepatocytesCELL PROLIFERATION, Issue 6 2007P. Papeleu The authors would like to draw the readers' attention to the fact that in the above article, an incorrect version of Table 1 was published. The correct version of Table 1 is printed below: [source] Inhibition of NF-,B activation by the histone deacetylase inhibitor 4-Me2N-BAVAH induces an early G1 cell cycle arrest in primary hepatocytesCELL PROLIFERATION, Issue 5 2007P. Papeleu 4-Me2N-BAVAH has been shown to induce histone hyperacetylation and to inhibit proliferation in Friend erythroleukaemia cells in vitro. However, the molecular mechanisms have remained unidentified. Materials and Methods:,In this study, we evaluated the effects of 4-Me2N-BAVAH on proliferation in non-malignant cells, namely epidermal growth factor-stimulated primary rat hepatocytes. Results and Conclusion:,We have found that 4-Me2N-BAVAH inhibits HDAC activity at non-cytotoxic concentrations and prevents cells from responding to the mitogenic stimuli of epidermal growth factor. This results in an early G1 cell cycle arrest that is independent of p21 activity, but instead can be attributed to inhibition of cyclin D1 transcription through a mechanism involving inhibition of nuclear factor-kappaB activation. In addition, 4-Me2N-BAVAH delays the onset of spontaneous apoptosis in primary rat hepatocyte cultures as evidenced by down-regulation of the pro-apoptotic proteins Bid and Bax, and inhibition of caspase-3 activation. [source] Cytidine deaminase APOBEC3B interacts with heterogeneous nuclear ribonucleoprotein K and suppresses hepatitis B virus expressionCELLULAR MICROBIOLOGY, Issue 1 2008Wei Zhang Summary The cytidine deaminase apolipoprotein B mRNA editing catalytic subunit-3 (APOBEC3) proteins have been identified as potent inhibitors of diverse retroviruses, retrotransposons and hepatitis B virus (HBV). The mechanism of APOBEC3 proteins in the control of HBV infection, however, is less clear. Here we report that APOBEC3B (A3B) displays dual inhibitory effects on both HBsAg and HBeAg expression as well as HBV core-associated DNA synthesis. Heterogeneous nuclear ribonucleoprotein K (hnRNP K), a positive regulator of HBV expression, has been identified as a major interaction partner of A3B protein. A3B protein inhibited the binding of hnRNP K to the enhancer II of HBV (Enh II), and S gene transcription of HBV. Moreover, A3B directly suppressed HBV S gene promoter activity. Individual variation in A3B expression was observed in both normal primary hepatocytes and liver tissues. Interestingly, A3B was able to inhibit CMV and SV40 promoter-mediated gene expression. In conclusion, A3B suppresses HBV replication in hepatocytes by inhibiting hnRNP K-mediated transcription and expression of HBV genes as well as HBV core DNA synthesis. In addition, A3B protein may be a broad antiviral host factor. Thus, regulated A3B expression may contribute to non-cytolytic HBV clearance in vivo. 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