Browsing by Subject "Hepatocyte"
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- PublicationOpen AccessCytotoxicity of Kuwait weathered lake crude oil on rat hepatocytes, a histological and ultrastructural study(Murcia : F. Hernández, 1998) Safer, A.M.; Meakins, R.; Akbar, L.; Abou-Salem, K.In the present study, the cytotoxic effects of Kuwaiti weathered crude oil and a potent carcinogen (DMBA) on rat liver cells were examined by light and electron microscopy at each of 4 sampling periods after the start of low dosing (0.5 and 0.2 mg/kg) of feed. Such effects were compared with those of olive oil and uncontaminated food-exposed controls. The results confirm a pronounced cell damage which statistically not significant (p<0.05). In crude oil, the organelle changes were variable and highly comparable to that of DMBA. The nuclei were mostly disintegrated while the cell showed demarcation of cytoplasmic vacuolization, lipid augmentation, and mitochondrial aberrations. The latter showed a remarkable association with the rough endoplasmic reticulum and lipid droplets, and appeared as decayed and diffused structures within the cell matrix. There was no comparable changes in the hepatocytes of animals fed with uncontaminated food except for the formation of lipid droplets in the olive oil-fed groups. Although the animals food was contaminated with Kuwaiti weathered oil formed in 1991 were exposed to extreme seasonal temperatures, yet the residues of such oil led to severe histopathological alterations in the liver cells which were similar to those of DMBA-treated cells. There is the need to pay attention to potential hazardous effects of the crude oil on environments.
- PublicationOpen AccessExpression and induction of anaphylatoxin C5a receptors in the rat liver(Murcia : F. Hernández, 2003) Schlaf, G.; Schmitz, M.; Rothermel, E.; Jungermann, K.; Schieferdecker, H.L.; Götze, O.The C5a-anaphylatoxin which is generated by limited proteolysis upon activation of the fifth component of complement may be induced by the classical, the alternative or the lectin pathway. C5a has been shown, under normal conditions, to induce the release of prostanoids from Kupffer cells (KC) and hepatic stellate cells (HSC) and thereby indirectly to increase glucose output from hepatocytes (HC). A direct action of C5a on HC would require the expression of the specific C5a receptor (C5aR). In studies using quantitative RT-PCR it was shown that non-stimulated HC lack C5aR, in contrast to KC, HSC and sinusoidal endothelial cells (SEC) all of which contained mRNA for the C5aR in decreasing amounts. FACS analyses, immunohisto- and immunocytochemistry as well as functional analyses confirmed the results of the RT-PCR assays. Under inflammatory situations the C5aR was found to be upregulated in various organs and tissues which included the liver. Interleukin-6 (IL-6) as a main inflammatory mediator in the liver induced a de novo expression of functional C5aR in HC in-vitro and invivo. In contrast, LPS failed to induce C5aR directly in cultured HC in-vitro but induced C5aR in HC in vivo and in co-cultures of HC and KC which release IL-6 upon stimulation with LPS. So far, the only known effector function of C5a on HSC was the induction of prostanoid release. In an approach to reveal new functions of C5aR in HSC, the cells responsible for liver fibrosis, it could be shown that C5a upregulated fibronectin-specific mRNA five-fold whereas entactin, collagen IV and the structure protein smooth muscle actin were not affected. In addition, C5a did not upregulate specific mRNA for the profibrotic cytokine TGF-ß1 in either isolated KC or HSC. Thus, C5a alone appears to have only a limited role in the induction of liver fibrosis.
- PublicationOpen AccessHepatocyte growth factorlscatter factor, a cytokine playing multiple and converse roles(Murcia : F. Hernández, 1997) Jiang, W.G.; Hiscox, S.Hepatocyte growth factor (HGF), otherwise known as scatter factor (SF), is a recently identified cytokine which exerts a wide spectrum of biological functions on a variety of cell types. Its receptor is encoded by the c-met proto-oncogene. HGFISF has been implicated in the regulation of mitogenesis, motogenesis, and morphogenesis. Over the past few years, the structure, function and signal transduction pathways of HGFISF and its receptor have become clearer. The cytokine is now know to play important roles in the regulation of both normal physiological processes as well as pathological ones. This review summarises recent progress involving HGFISF and its receptor and discusses their role in cell biology, organ regeneration, cancer and other processes. Hepatocyte growth factor (HGF) is a pleiotropic growth factor originally identified as a potent mitogen agent for rat hepatocytes. Subsequent studies have shown that it is mitogenic for a wide range of epithelial cells and not limited to hepatocytes. Its behaviour as a motogenic stimulator promoted its independent discovery and naming of scatter factor (SF). Analysis of cDNA and amino acid sequences have revealed that the two molecules are the same. A number of cytokine agents are known to stimulate cellular motility, however, it is the function of HGFISF as a potent motogenic, mitogenic and morpho-regulatory agent on the diverse variety of cell types that makes the discovery of HGFISF factor one of the most interesting stories in terms of identification of novel cytokines.
- PublicationOpen AccessHepatocyte nuclear phenotype, the cross-talk between anabolic androgenic steroids and exercise in transgenic mice(Murcia : F. Hernández, 2008) Fontana, Karina; Aldrovani, Marcela; Paoli, Flávia de; Oliveira, Helena C.F.; Campos Vidal, Benedicto de; da Cruz Hoflingl, M.A.The growing and indiscriminate use of high doses of anabolic androgenic steroid (AAS) among youth and athletes has raised serious concerns about its hepatotoxic effects. Herein, the influence of AAS in the nuclear phenotype of hepatocytes was investigated in sedentary and trained mice heterozygous for the human CETP (cholesteryl ester transfer protein) transgene and for LDL-receptor null allele (CETP+/-LDLr+/-) by image analysis. Five groups were assayed comprising treadmill exercised (Ex) and sedentary (Sed) mice, administered mesterolone (AAS) or gum arabic (GA) and a sedentary blank control: G1(SedAAS), G2(SedGA), G3(ExAAS), G4(ExGA), and G5(SedBL). To assess nuclear phenotypes, the state of chromatin supraorganization, DNA content and fragmentation (TUNEL assay), area and perimeter of hepatocytes were determined in Feulgen-stained liver imprints. In addition, the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) hepatic transaminases were measured. SedAAS-G1 showed the lowest chromatin condensation and highest Feulgen-DNA content, polyploid nuclei frequency, nuclear area and perimeter, suggesting gene activation. Contrarily, ExAAS-G3 showed a highest chromatin condensation, and a significant decrease of Feulgen-DNA content and decreased frequency of polyploid nuclei, which suggest gene silencing. Image analysis of the nuclear phenotype offered a coherent descriptive picture of the changing patterns of chromatin organization, which were shown to be congruent with the levels of Feulgen-DNA content, geometric nuclear parameters and hepatocyte activity. In this study, the image analysis permitted the monitoring of the nuclear response to mesterolone and physical exercise action in liver cells, the molecular mechanism of which is in prospect.
- PublicationOpen AccessHepatotoxicity induced by the anti-oxidant food additive, butylated hydroxytoluene BHT, in rats. An electron microscopical study(Murcia : F. Hernández, 1999) Safer, A.M.; Al-Nughamish, A.J.The anti-oxidant food additive, butylated hydroxytoluene (BHT), was fed to Sprague-Dawley rats at three concentrations: 0.2%, 0.4% and 0.8% for periods of 6, 12, 18 and 24 weeks, and the results were compared with corresponging groups treated with a potent carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA) groups, with olive oil, and with untreated control groups. BHT resulted in a significant increase in liver weight. The liver cells presented gradual vacuolization, cytoplasmic disintegration, "moth-eaten" appearance, ballooning degeneration, hepatocellular necrosis, aggregation of chromatin material around the periphery of the nuclear envelope, SER proliferation, RER clumping with broken cisternae, withered and autolyzed mitochondria, augmentation of lipid droplets and glycogen depletion. On the other hand, there was no sign of tumorigenicity. Whether or not BHT acts as a carcinogen in long-term administration may depend not only upon the organ system examined, but also on the strain of the animal used.
- PublicationOpen AccessHistological recovery of the hepatocytes is based on the redox system upregulation in the animal models of mutant superoxide dismutase (SOD)1-linked amyotrophic lateral sclerosis(Murcia : F. Hernández, 2006) Kato, Massuo J.; Kato, S.; Abe, Y.; Nishino, T.; Ohama, E.; Aoki, M.; Itoyama, Y.Histological rescue of superoxide dismutase1 (SOD1)-mutated hepatocytes from mutant SOD1 stress is investigated from the viewpoint of upregulation of the redox system [peroxiredoxin (Prx) and glutathione peroxidase (GPx)]. Histopathological and immunohistochemical studies using antibodies against PrxI/PrxII/GPxI were carried out on specimens from four different strains of animal models of mutant SOD1- linked familial amyotrophic lateral sclerosis (ALS). In the livers of the ALS animal models in the presymptomatic stage without motor neuron loss, both swollen and eosinophilic hepatocytes with vacuolation pathology were observed. After developing motor deficits, this swelling and vacuolation ceased to be apparent. In the terminal stage when severe motor neuron loss was observed, these hepatocytes recovered and appeared normal. In redox system-related immunohistochemical preparations, almost all of the normal hepatocytes expressed the redox system-related enzymes PrxI/PrxII/GPxI. In the presymptomatic stage, some hepatocytes did not express redox system-related enzymes. After clinical onset, over 75% of hepatocytes showed overexpression of PrxI/PrxII/GPxI, i. e., upregulation of the redox system. At the end stage, near normal PrxI/PrxII/GPxI expression was observed again in the hepatocytes. Redox system upregulation in SOD1- mutated hepatocytes rescues hepatocytes from the mutant SOD1 stress that leads to motor neuron death.
- PublicationOpen AccessImmunolocalization of metallothioneins in different tissues of turbot (Scophthalmus maximus) exposed to Cd(Murcia : F. Hernández, 2007) Alvarado, N.E.; Cancio, I.; Hylland, K.; Marigómez, I.; Soto, M.Metallothioneins (MT) were localized by immunochemistry in different organs and cell compartments of turbot exposed to sublethal concentrations (100 ppb) of Cd for 7 days. The polyclonal rabbit anti-cod MT antibody (NIVA, Norway) applied herein exhibited positive cross-reactivity with turbot MTs. Immunoreactive MTs were localized in the branchial epithelium, in the liver and in the kidney of turbot. In Cd exposed fishes MTs were demonstrated mainly in branchial chloride cells (CC) and to a lesser extend in the area where progenitor cells are located and in the cells of the respiratory epithelium (secondary lamellae). A higher staining intensity for MTs was observed in CC of the interlamelar space of the main branchial epithelium in comparison with control CC. MT-staining was also observed in the chondroblasts of the cartilage and in the erythrocytes within blood vessels both in control and Cd-exposed specimens. MT immunoreaction was high in the liver hepatocytes and weak in the epithelium of the proximal portion of the kidney in exposed turbot. The tegument, spleen and muscle were devoid of any immunolabelling in both treatments. Ultrastructural studies at the transmission electron microscope revealed that Cd-induced MTs were mainly located in the cytoplasm of gill CC, the lysosomes and the cytoplasm of hepatocytes and in the basal labyrinth of kidney proximal nephrocytes. The differential localization/induction of MTs in different cell types described hereby suggests that the quantification of the specific expression of MT may be used in biomonitoring programs as a biomarker of Cd exposure in aquatic environments.
- PublicationOpen AccessRegulation of hepatocyte glutathione content by hepatic sinusoidal cells activated with LPS: anatomical restrictions(Murcia : F. Hernández, 2009) Catalá, Myrian; Pagani, Raffaella; Portolés, M.TeresaThe liver is the main organ for the elimination of bacterial endotoxin involving Kupffer and parenchymal cells. This process is accompanied by the release of free radicals. Parenchymal cells possess especially high levels of glutathione, which make them a key point in the response to free radicals. Sinusoidal cells regulate hepatic function in a very important fashion through the release of cytokines and/or adhesion molecules. These facts suggest the importance of finding new in vitro experimental models representing an intermediate step towards in vivo models. The treatment with LPS of sinusoidal and parenchymal cell co-cultures on porous membranes provokes an intense reduction of parenchymal cell intracellular glutathione, which does not correspond to in vivo results. However, the addition of supernatants of LPS-treated sinusoidal cells to parenchymal cells renders increases in glutathione which agree better with in vivo results. We conclude that the regulation of liver hepatocyte glutathione content and NO release in the presence of LPS is strongly modulated by liver non parenchymal cells. The study of this phenomenon requires new in vitro models taking into account liver histophysiology and histopathology and anatomical restrictions in cell communication.
- PublicationOpen AccessShort term regulation of hepatocyte glutathione content by hepatic sinusoidal cells in co-culture(Murcia : F. Hernández, 2007) Catalá-Rodríguez, M.; Pagani, R.; Portolés, M.T.antioxidant mechanisms may constitute the primary mechanisms of a number of pathologies. The liver plays a central role in this balance: parenchymal hepatic cells contain and export especially high levels of the antioxidant glutathione and activated Kupffer cells release inflammation mediators and reactive oxygen species. There is growing evidence of a paracrine regulation of hepatic function by means of a fluent intercellular communication which must still be fully elucidated, especially in basal conditions. In vivo models provide often too complex results but, in vitro, tissue interactions are left aside; therefore it is important to find new experimental models to address cell communication studies. Here we propose the complementary use of three models to study liver glutathione system regulation in basal conditions: pure parenchymal cells primary cultures, addition of sinusoidal cell conditioned media to parenchymal cells and co-culture of sinusoidal cells using porous membranes. We have also developed a high specifity immunofluorescent method for the complete characterization of sinusoidal cell populations by flow cytometry and confocal microscopy. Our results show that Kupffer cells possess higher levels of reactive oxygen species than sinusoidal endothelial cells even in basal conditions. We also report that the glutathione content of hepatic parenchymal cells in basal conditions and suggest the existence of a paracrine circuit in the management of liver oxidative stress.
- PublicationOpen AccessThe hepatocytes of the brown trout (Salmo trutta f. fario):A quantitative study using design-based stereology(Murcia : F. Hernández, 2001) Rocha, E.; Monteiro, R.A.F.; Oliveira, M.H.; Silva, M.W.A stereological study was performed on brown trout hepatocytes aiming to disclose whether there are basic gender differences when minimal levels of sex hormones exist, and also to establish a platform for both interspecific comparisons and physiological correlations. We used the so-called "design-based stereology" (with no shape, size or orientation assumptions) and also some new related statistics. Twoyear- old brown trout were collected in April, and the livers were fixed by perfusion. From liver slicing to microscopical field selection, systematic sampling was used. Stereology was applied at light and electron microscopy. Target parameters were the relative and total hepatocyte number, the mean individual hepatocyte volume and surface, and also both relative and total volumes, and surfaces, either of organelles or of cell compartments. Observed variability was usually high, but the precision of estimates was proved to be globaily adequate facing the true biological variation amongst specimens. Females had more hepatocytes per liver ( 1 . 7 9 ~ 1 0v~s. 1 . 1 2~1 0~C) .o nsidering the individual hepatocytes, whereas no gender differences were detected in the cell volume, males had higher values of nuclear volume (199 vs. 151 pm3) and surface (170 vs. 131 pm2), endoplasmic reticulum volume I 1300 vs. 824 pm3), and microvilli volume (82 vs. 54 pm ) and surface (1445 vs. 975 pm2). However, when dealing with quantities per liver, gender differences were found only in the volumes of dense bodies (56 vs. 97 mm3) and of residual cytoplasm (169 vs. 341 mm3) - both volumes were higher in females. Functional implications of data are discussed, namely that females seem to have basic structural traits for coping with the later demands of breeding. Data also support that structural remodelling of hepatocytes occurs after breeding, urging to pursue seasonal studies (namely on lysosomes). We advanced the hypothesis that genders differ in microvilli surface just to maintain an optimal physiological surface-to-volume ratio. Interspecific similarities and differences were disclosed. For example, the number of hepatocytes/cm3 of parenchyma of brown trout was much lower than those reported in rainbow trout, but in both trouts femaies seem to have an higher cell number. In addition, when comparing the size of hepatocytes of brown trout with that from other fish and mammals it was suggested that major interspecific differences exist.
- PublicationOpen AccessTight junction proteins and signal transduction pathways in hepatocytes(Murcia : F. Hernández, 2009) Kojima, Takashi; Murata, Masaki; Yamamoto, Toshinobu; Lan, Mengdong; Imamura, Masafumi; Son, Seiichi; Takano, Ken-ichi; Yamaguchi, Hiroshi; Ito, Tatsuya; Tanaka, Satoshi; Chiba, Hideki; Hirata, Koichi; Sawada, NorimasaTight junctions of hepatocytes play crucial roles in the barrier to keep bile in bile canaliculi away from the blood circulation, which we call the bloodbilliary- barrier (Kojima et al., 2003). Tight junction proteins of hepatocytes are regulated by various cytokines and growth factors via distinct signal transduction pathways. They are also considered to participate in signal transduction pathways that regulate epithelial cell proliferation, gene expression, differentiation and morphogenesis. This review focuses on recent findings about the relationship between tight junction proteins and signal transduction pathways in hepatocytes.
- PublicationOpen AccessUltrastructural and hormonal metabolic studies of rat liver maintained in vitro by perfusion at 30° C and 37O C: a time course study by TEM, SEM and RIA(Murcia : F. Hernández, 1989) Hassan, Ibrahim M.; Al-Ali, Saad Y.; Hassan, MithalIsolated rat liver perfusion system has been extensively used for metabolic and functional studies. Results derived from the application of this system may reflect true biochemical changes but they may also be associated with some structural changes. This study was undertaken to correlate the cytological changes and functional integrity of isolated rat liver perfused in vitro at normal physiological temperature (37°C) and 30°C, using a non-recirculating system. The livers were perfused for 3 hours with modified Ham's F10 culture medium supplemented with thyroxine hormone (T4). The hepatocyte structural integrity was studied by light microscopy, transmission and scanning electron microscopy. The triiodothyronine (T3) and T4 hormones in the perfusion medium and the effluent fractions were assessed by radioimmunoassay. The livers perfused at 30°C remained morphologically intact at the ultrastructural level for 3 hours whilst at 37"C, hepatocytes in the centrilobular zone exhibited marked structural alterations. The percentage of T4 uptake was significantly higher (P < 0.01) in livers perfused at 30°C (50.8 * 7.7% vs 38 * 7.7%, 37"C), but the net T3 output (3.16 * 1.04 pg) and the conversion of T4 to T3 (4 * 0.62%) were significantly higher (P < 0.001) in livers perfused at 37°C in comparison to livers perfused at 30°C (1.61 * 0.84 pg and 1.68 2 0.76%, respectively). In conclusion, at 30°C the hepatic T4 uptake is not inhibited, but the rate of T4 to T3 conversion has decreased, additionally the livers remain morphologically well preserved throughout the experimental period. At 37"C, although T4 to T3 conversion is higher, structurally the livers could not be maintained intact for more than 2 hours. Therefore, isolated rat livers perfused in vitro at 30°C offer the best compromise for further morphological and metabolic studies.
- PublicationOpen AccessVitamin B12 Induces Hepatic Fatty Infiltration through Altered Fatty Acid Metabolism(Cell Physiol Biochem Press GmbH&Co. KG, Duesseldorf, ) Boachie, J.; Adaikalakoteswari, A.; Gazquez Garcia, A.; Zammit, V.; Larque Daza, E.; Saravanan, P.; FisiologíaBackground/Aims: Rise in global incidence of obesity impacts metabolic health. Evidence from human and animal models show association of vitamin B12 (B12) deficiency with elevated BMI and lipids. Human adipocytes demonstrated dysregulation of lipogenesis by low B12 viahypomethylation and altered microRNAs. It is known de novo hepatic lipogenesis plays a key role towards dyslipidaemia, however, whether low B12 affects hepatic metabolism of lipids is not explored. Methods: HepG2 was cultured in B12-deficient EMEM medium and seeded in different B12 media: 500nM(control), 1000pM(1nM), 100pM and 25pM(low) B12. Lipid droplets were examined by Oil Red O (ORO) staining using microscopy and then quantified by elution assay. Gene expression were assessed with real-time quantitative polymerase chain reaction (qRT-PCR) and intracellular triglycerides were quantified using commercial kit (Abcam, UK) and radiochemical assay. Fatty acid composition was measured by gas chromatography and mitochondrial function by seahorse XF24 flux assay. Results: HepG2 cells in low B12 had more lipid droplets that were intensely stained with ORO compared with control. The total intracellular triglyceride and incorporation of radio-labelled-fatty acid in triglyceride synthesis were increased. Expression of genes regulating fatty acid, triglyceride and cholesterol biosynthesis were upregulated. Absolute concentrations of total fatty acids, saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), trans-fatty acids and individual even-chain and oddchain fatty acids were significantly increased. Also, low B12 impaired fatty acid oxidation and mitochondrial functional integrity in HepG2 compared with control. Conclusion: Our data provide novel evidence that low B12 increases fatty acid synthesis and levels of individual fatty acids, and decreases fatty acid oxidation and mitochondrial respiration, thus resulting in dysregulation of lipid metabolism in HepG2. This highlights the potential significance of de novo lipogenesis and warrants possible epigenetic mechanisms of low B12.