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Primary Rat Hepatocytes (primary + rat_hepatocyte)
Selected AbstractsInteraction between halogenated aromatic compounds in the Ah receptor signal transduction pathwayENVIRONMENTAL TOXICOLOGY, Issue 5 2004Guosheng Chen Abstract Many toxic and biochemical responses to halogenated aromatic compounds (HACs) such as polychlorinated biphenyls (PCBs) and polychlorinated dibenzo- p -dioxins (PCDDs) are mediated through the aryl hydrocarbon receptor (AhR), which is an intracellular cytosolic target for HACs. Environmental exposure to HACs almost always involves complex mixtures of congeners, some of which can antagonize the action of potent HACs such as 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). In this work we studied TCDD and representative PCB congeners, alone and in mixture, for their effect on CYP1A gene transcription and protein levels in primary rat hepatocytes. Together with our previous work, our results suggest that formation of the Ah receptor-ligand-DRE (dioxin response element) complex is the principal point of divergence in the mechanism between an AhR agonist and an AhR antagonist. The coplanar PCBs 77 and 126 and the mono- ortho PCB 156 were full agonists toward CYP1A1 gene transcription and CYP1A protein levels, showing typical additive behavior with TCDD to the target molecule AhR. In contrast, the nonplanar PCB 153 antagonized the action of TCDD, even at concentrations that occupied a significant fraction of AhR molecules. Competitive inhibition explains the commonly reported decrease of ethoxyresorufin- O -deethylase (EROD) activity when PCBs are present in high concentrations and the antagonism of PCBs to the EROD activity of TCDD. The result is that Western blotting offers a much more reliable measure of CYP1A protein concentration than does the EROD assay, despite the greater convenience of the latter. © 2004 Wiley Periodicals, Inc. Environ Toxicol 19: 480,489, 2004. [source] Calcium influx mechanisms underlying calcium oscillations in rat hepatocytes,HEPATOLOGY, Issue 4 2008Bertina F. Jones The process of capacitative or store-operated Ca2+ entry has been extensively investigated, and recently two major molecular players in this process have been described. Stromal interacting molecule (STIM) 1 acts as a sensor for the level of Ca2+ stored in the endoplasmic reticulum, and Orai proteins constitute pore-forming subunits of the store-operated channels. Store-operated Ca2+ entry is readily demonstrated with protocols that provide extensive Ca2+ store depletion; however, the role of store-operated entry with modest and more physiological cell stimuli is less certain. Recent studies have addressed this question in cell lines; however, the role of store-operated entry during physiological activation of primary cells has not been extensively investigated, and there is little or no information on the roles of STIM and Orai proteins in primary cells. Also, the nature of the Ca2+ influx mechanism with hormone activation of hepatocytes is controversial. Hepatocytes respond to physiological levels of glycogenolytic hormones with well-characterized intracellular Ca2+ oscillations. In the current study, we have used both pharmacological tools and RNA interference (RNAi)-based techniques to investigate the role of store-operated channels in the maintenance of hormone-induced Ca2+ oscillations in rat hepatocytes. Pharmacological inhibitors of store-operated channels blocked thapsigargin-induced Ca2+ entry but only partially reduced the frequency of Ca2+ oscillations. Similarly, RNAi knockdown of STIM1 or Orai1 substantially reduced thapsigargin-induced calcium entry, and more modestly diminished the frequency of vasopressin-induced oscillations. Conclusion: Our findings establish that store-operated Ca2+ entry plays a role in the maintenance of agonist-induced oscillations in primary rat hepatocytes but indicate that other agonist-induced entry mechanisms must be involved to a significant extent. (HEPATOLOGY 2008.) [source] Nuclear translocation of UDCA by the glucocorticoid receptor is required to reduce TGF-,1,induced apoptosis in rat hepatocytes,HEPATOLOGY, Issue 4 2005Susana Solá Ursodeoxycholic acid (UDCA) inhibits classical mitochondrial pathways of apoptosis by either directly stabilizing mitochondrial membranes or modulating specific upstream targets. Furthermore, UDCA regulates apoptosis-related genes from transforming growth factor ,1 (TGF-,1),induced hepatocyte apoptosis by a nuclear steroid receptor (NSR),dependent mechanism. In this study, we further investigated the potential role of the glucocorticoid receptor (GR) in the antiapoptotic function of UDCA. Our results with short interference RNA (siRNA) technology confirmed that UDCA significantly reduces TGF-,1,induced apoptosis of primary rat hepatocytes through a GR-dependent effect. Immunoprecipitation assays and confocal microscopy showed that UDCA enhanced free GR levels with subsequent GR nuclear translocation. Interestingly, when a carboxy-terminus deleted form of GR was used, UDCA no longer increased free GR and/or GR translocation, nor did it protect against TGF-,1,induced apoptosis. In co-transfection experiments with GR response element reporter and overexpression constructs, UDCA did not enhance the transactivation of GR with TGF-,1. Finally, using a flourescently labeled UDCA molecule, the bile acid appeared diffuse in the cytosol but was aggregated in the nucleus of hepatocytes. Both siRNA assays and transfection experiments with either wild-type or mutant forms of GR showed that nuclear trafficking occurs through a GR-dependent mechanism. In conclusion, these results further clarify the antiapoptotic mechanism(s) of UDCA and suggest that GR is crucial for the nuclear translocation of this bile acid for reducing apoptosis. (HEPATOLOGY 2005;42:925,934.) [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] Endotoxin-stimulated macrophages decrease bile acid uptake in WIF-B cells, a rat hepatoma hybrid cell lineHEPATOLOGY, Issue 1 2000Ekkehard Sturm Endotoxemia leads to cytokine-mediated alterations of the hepatocellular sodium-taurocholate-cotransporting polypeptide (ntcp). We hypothesized that stimulated macrophages are essential transducers for down-regulating hepatocellular bile salt uptake in response to endotoxin (lipopolysaccharide [LPS]) exposure. Using an in vitro model, we exposed mouse macrophages (IC-21 cell line) to LPS for 24 hours. Concentrations of cytokines tumor necrosis factor-, (TNF-,), interleukin (IL)-1,, and IL-6 increased 10.6-fold, 12.5-fold, and 444-fold, respectively, in LPS-conditioned IC-21 medium (CM) versus unconditioned IC-21 medium (UM). WIF-B rat hepatoma hybrid cells were incubated with either CM or UM or treated directly with medium containing recombinant TNF-,, IL-1,, and IL-6. [3H]Taurocholate ([3H]TC) uptake decreased in WIF-B cells exposed to either TNF-, (54% of control), IL-1, (78%), IL-6 (55%) as single additives, or in triple combination (TCC) (43%). A virtually identical decrease was observed after exposing WIF-B cells to CM (52%, P < .001). LPS had no direct effect on [3H]TC uptake. CM treatment did not decrease L-alanine transport in WIF-B cells. Blocking antibodies against TNF-,, IL-1,, and IL-6 restored the diminished [3H]TC uptake in cells exposed to TCC and CM to 87% and 107% of controls, respectively. Northern blotting revealed that ntcp messenger RNA (mRNA) expression was significantly reduced in WIF-B cells after exposure to CM, and in primary rat hepatocytes exposed to CM or TNF-, (68%, 14%, and 29% of control, respectively). We conclude that macrophages and their ability to secrete the cytokines TNF-,, IL-1,, and IL-6 may be essential in mediating the endotoxin-induced cholestatic effect of decreased hepatocellular bile salt uptake. [source] Characterization of Cd-induced molecular events prior to cellular damage in primary rat hepatocytes in culture: Activation of the stress activated signal protein JNK and transcription factor AP-1JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 3 2004Chin-ju J. Hsiao Abstract The effect of Cadmium (Cd) on the expression of c-Jun N -terminal kinase (JNK), c-jun, and activator protein-1 (AP-1) has been investigated. We previously reported that Cd causes cell damage as indicated by increases in the cytotoxic parameters, lactate dehydrogenase and lipid peroxidation, and this damage was mediated by decreases in cellular concentration of glutathione. In the present study, we investigate the molecular events involved prior to the Cd-induced cellular toxicity and damage in primary rat hepatocytes. We propose that Cd, through the generation of reactive oxygen species (ROS) and prior to significant cellular damage, activates the stress activated signal protein JNK, regulates c-jun expression, and promotes the binding of a redox sensitive transcription factor AP-1. We show JNK activity and c-jun mRNA level significantly increased at 1 h and AP-1 DNA binding activity significantly enhanced at 3 h in the presence of 4 ,M cadmium chloride. Blocking the Cd induction of JNK activity, c-jun mRNA level, and AP-1 binding activity using the antioxidants N -acetyl cysteine (10 mM) or carnosol (0.5 ,g/mL) suggests a role for ROS. Blocking JNK activity and c-jun mRNA by SP600125 (20 ,M), a JNK inhibitor, supports the role of JNK in transmission of signals induced by Cd. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:133,142, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20018 [source] Differential regulation of P-glycoprotein genes in primary rat hepatocytes by collagen sandwich and drugsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2002Chow H. Lee Abstract P-glycoprotein (Pgp) is a small family of plasma membrane proteins, which are capable of transporting substrates across cell membranes. Class I and II Pgp are able to transport drugs and have been shown to mediate multidrug resistance (MDR). Class III Pgp is a long chain phospholipid transporter and does not mediate MDR. The regulation of all three Pgp genes is still poorly understood. For instance, it is not clear if the three Pgp genes are co-regulated or differentially regulated by external stimuli. This study examined the effect of drugs and collagen sandwich system on expression and transcription of all the three Pgp genes in primary rat hepatocytes. Consistent with previous findings, dramatic overexpression (25-fold) of Class II Pgp mRNA was seen, upon culturing of hepatocytes onto a single layered collagen gel. Hepatocytes sandwiched between two layers of collagen gel exhibited decreased (4.5-fold) Class II Pgp mRNA expression as compared to the single layer system. Treatment of hepatocytes cultured on the single layer collagen system with cytoskeletal disrupting (cytochalasin D, colchicine) but not cytoskeletal stabilizing (phalloidin, taxol) drugs, suppressed Class II Pgp expression. In all cases, no change in Class II Pgp transcription was observed as demonstrated by nuclear run-on studies. This suggests that collagen configuration and drugs affect Class II Pgp mRNA expression predominantly through post-transcriptional mechanisms. In contrast, parallel increases in mRNA expression and transcription of Class I Pgp gene were observed upon culturing of hepatocytes, in the collagen sandwich system, and treatment with some drugs (cytochalasin D, colchicine, and phalloidin). This suggests that Class I Pgp gene is regulated primarily via transcriptional mechanisms by these stimuli. On the other hand, Class III Pgp gene appears to be post-transcriptionally co-regulated with Class II Pgp gene by treatment with the drugs, while collagen configuration affected both transcription and post-transcription of Class III Pgp gene. Finally, dose-dependent studies using cycloheximide provided further evidence that the two MDR-associated genes are not co-regulated. This study has implications for future studies on the molecular mechanisms of Pgp gene regulation. J. Cell. Biochem. 86: 12,20, 2002. © 2002 Wiley-Liss, Inc. [source] Enhanced proliferation and differentiation of rat hepatocytes cultured with bone marrow stromal cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2001Toru Mizuguchi Liver transplantation is the only clinically effective method of treating acute liver failure. However, wider application of this therapeutic modality is restricted primarily by shortage of donor organs. In the search for alternative methods of liver replacement therapy, investigators have focused on transplantation of normal allogeneic hepatocytes and on the development of liver support systems utilizing isolated hepatocytes. Since all human livers suitable for cell harvest are being used for transplantation, hepatocyte therapy using human tissue would require growing of cells in vitro. Unfortunately, although hepatocytes have tremendous capacity to proliferate in vivo, their ability to grow in culture is severely limited. Stromal cells from bone marrow and other blood-forming organs have been found to support hematopoiesis. In this paper, we show that bone marrow-derived stromal cells (BMSCs) enhance proliferation and support differentiation of rat hepatocytes in culture. Further, we demonstrate that in hepatocyte/BMSC co-cultures, clonal expansion of small hepatocytes (SH) is increased. Using semipermeable membrane cultures, we established that direct cell,cell contact is necessary for stimulation of cell proliferation. We also show that BMSCs which are in direct contact with hepatocytes and SH colonies express Jagged1. This suggests a potential role for Notch signaling in the observed effects. Finally, we present evidence that the expression and activity of liver specific transcirption factors, CCAAT/enhancer binding proteins and liver specific key enzymes such as tryptophan 2,3-dioxygenase, are improved in hepatocyte/BMSC co-cultures. In conclusion, results of this study indicate that BMSCs could facilitate proliferation and differentiation of primary rat hepatocytes and their progenitors (SH) in vitro. © 2001 Wiley-Liss, Inc. [source] Azathioprine hepatotoxicity and the protective effect of liquorice and glycyrrhizic acidPHYTOTHERAPY RESEARCH, Issue 8 2006Yue-Ting Wu Abstract This study aimed to evaluate the responses of human hepatocytes to azathioprine hepatotoxicity in comparison with the well-studied azathioprine hepatotoxicity in rat hepatocytes and the effects of protective agents to suppress azathioprine hepatotoxicity. Azathioprine presented its hepatotoxicity at clinically relevant concentrations (lower than 10 µm) in primary rat hepatocytes after 48 h of treatment as shown by a severe decrease in cell viability as well as intracellular GSH depletion. However, primary human hepatocytes exhibited only significant intracellular GSH depletion after treatment with azathioprine at these clinically relevant concentrations, while a reduction in cell viability by 29% was only evidenced after 48 h of treatment with azathioprine at the high concentration of 50 µm. In addition, a monolayer culture of primary rat hepatocytes was used as an in vitro model to examine the protective effects of antihepatotoxic drugs including glutathione (GSH), N-acetylcysteine (NAC, a GSH precursor), liquorice and glycyrrhizic acid (GA), a major bioactive component of liquorice, against hepatotoxicity of 1 µm azathioprine. It was found that both liquorice and GA showed substantial protection according to assays of cell viability and intracellular GSH, while neither GSH nor NAC had such a protective function. Similarly, GA protected human hepatocytes from intracellular GSH depletion on exposure to 1 µm azathioprine. These results implied that GA or liquorice could be considered as potent protection agents against azathioprine hepatotoxicity. Copyright © 2006 John Wiley & Sons, Ltd. [source] Genotoxic potential of xenobiotic growth promoters and their metabolites,APMIS, Issue 2 2001Review article This paper reviews data reported in the literature as well as recent and unpublished studies from our laboratory on the metabolism and genotoxicity of the xenobiotic growth promoters 17,-trenbolone, melengestrol acetate and zeranol. In our metabolic study, the oxidative in vitro metabolites generated by hepatic microsomes from rats, bovine and humans were analyzed by HPLC and GC/MS. 17,-Trenbolone gave rise to at least 13 monohydroxylated products, whereas 12 mono- and dihydroxylated metabolites were obtained with melengestrol acetate and at least 5 with zeranol. The genotoxic potential of the parent compounds was studied using the following endpoints: induction of HPRT mutations in cultured V79 cells and of lacI mutations in E. coli; induction of micronuclei in V79 cells; and formation of DNA adducts in cultured primary rat hepatocytes. Negative results were obtained in most of these assay systems. Only the micronucleus induction was marginally positive with 17,-trenbolone and zeranol at near-cytotoxic concentrations. Commercial melengestrol acetate was found to contain an impurity causing apoptosis in V79 cells. The genotoxic potential of the numerous oxidative metabolites of the xenobiotic growth promoters remains to be studied. [source] Protective Effect of Ebselen on Aflatoxin B1 -Induced Cytotoxicity in Primary Rat HepatocytesBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 4 2000Cheng-Feng Yang Recent studies have shown that aflatoxin B1 enhances reactive oxygen species formation and causes oxidative damage, which may ultimately contribute to the cytotoxicity and carcinogenic effect of aflatoxin B1. Ebselen, 2-phenyl-1,2-benzoisoseleazol-3(H)-one, a synthetic seleno-organic compound has been shown to possess glutathione peroxidase-like activity and free radical scavenging ability. Thus present study was designed to investigate the protective effect of ebselen on aflatoxin B1 -induced cytotoxicity in primary rat hepatocytes. Aflatoxin B1 -induced cytotoxicity and lipid peroxidation were determined by lactate dehydrogenase leakage and malondialdehyde generation, respectively. Intracellular reactive oxygen species level was measured using the fluorescent probe 2,,7,-dichlorofluorescin diacetate, and the intracellular reduced glutathione concentration was determined with a fluorometric method. Ebselen was found to display a dose-dependent protective effect on lactate dehydrogenase leakage and malondialdehyde generation caused by aflatoxin B1 exposure. The results also demonstrate that ebselen efficiently inhibits the intracellular reactive oxygen species formation in aflatoxin B1 -treated hepatocytes in a dose and time-dependent manner. It was also noted that ebselen was able to increase the intracellular reduced glutathione concentration, both in the control and in aflatoxin B1 -treated hepatocytes. The protection of ebselen against aflatoxin B1 cytotoxicity, however, was not affected by lowering the concentration of intracellular reduced glutathione. The overall data indicate that ebselen possesses a potent protective effect against aflatoxin B1 -induced cytotoxicity, and the main mechanism involved in the protection may be its strong capability in inhibiting intracellular reactive oxygen species formation and preventing oxidative damage. [source] Application of Multivariate Analysis to Optimize Function of Cultured HepatocytesBIOTECHNOLOGY PROGRESS, Issue 2 2003Christina Chan Understanding the metabolic and regulatory pathways of hepatocytes is important for biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. To characterize intermediary metabolism in the hepatocytes, metabolic flux analysis (MFA) was applied to elucidate the changes in intracellular pathway fluxes of primary rat hepatocytes exposed to human plasma and to provide a comprehensive snapshot of the hepatic metabolic profile. In the current study, the combination of preconditioning and plasma supplementation produced distinct metabolic states. Combining the metabolic flux distribution obtained by MFA with methodologies such as Fisher discriminant analysis (FDA) and partial least squares or projection to latent structures (PLS) provided insights into the underlying structure and causal relationship within the data. With the aid of these analyses, patterns in the cellular response of the hepatocytes that contributed to the separation of the different hepatic states were identified. Of particular interest was the recognition of distal pathways that strongly correlated with a particular hepatic function. The hepatic functions investigated were intracellular triglyceride accumulation and urea production. This study illustrates a framework for optimizing hepatic function and a possibility of identifying potential targets for improving hepatic functions. [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] CDK2 regulation through PI3K and CDK4 is necessary for cell cycle progression of primary rat hepatocytesCELL PROLIFERATION, Issue 4 2007L. Wierød In response to mitogenic stimuli, CDK4 and CDK2 form complexes with cyclins D and E, respectively, and translocate to the nucleus in the late G1 phase. It is an on-going discussion whether mammalian cells need both CDK4 and CDK2 kinase activities for induction of S phase. Methods and results: In this study, we have explored the role of CDK4 activity during G1 progression of primary rat hepatocytes. We found that CDK4 activity was restricted by either inhibiting growth factor induced cyclin D1-induction with the PI3K inhibitor LY294002, or by transient transfection with a dominant negative CDK4 mutant. In both cases, we observed reduced CDK2 nuclear translocation and reduced CDK2-Thr160 phosphorylation. Furthermore, reduced pRb hyperphosphorylation and reduced cellular proliferation were observed. Ectopic expression of cyclin D1 alone was not sufficient to induce CDK4 nuclear translocation, CDK2 activity or cell proliferation. Conclusions: Thus, epidermal growth factor-induced CDK4 activity was necessary for CDK2 activation and for hepatocyte proliferation. These results also suggest that, in addition to regulating cyclin D1 expression, PI3K is involved in regulation of nuclear shuttling of cyclin-CDK complexes in G1 phase. [source] | |||||||||||||