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Sample records for isolated rat hepatocytes

  1. Insulin internalization in isolated rat hepatocytes

    SciTech Connect

    Galan, J.; Trankina, M.; Noel, R.; Ward, W. )

    1990-02-26

    This project was designed to determine whether neomycin, an aminoglycoside antibiotic, has a significant effect upon the pathways of ligand endocytosis in isolated rat hepatocytes. The pathways studied include receptor-mediated endocytosis and fluid-phase endocytosis. Neomycin causes a dose-dependent acceleration of {sup 125}I-insulin internalization. Since fluid-phase endocytosis can also be a significant factor in {sup 125}I-insulin internalization, lucifer yellow (LY), a marker for fluid-phase endocytosis, was incorporated into an assay similar to the {sup 125}I-insulin internalization procedure. In the presence of 5 mM neomycin, a significant increase in LY uptake was evident at 0.2 and 0.4 mg/ml of LY. At 0.8 mg/ml, a decrease in LY uptake was observed. The increased rate of {sup 125}I-insulin internalization in the presence of neomycin was intriguing. Since one action of neomycin is to inhibit phosphoinositidase C, it suggests that the phosphotidylinositol cycle may be involved in ligand internalization by hepatocytes. At low insulin concentrations, receptor-mediated uptake predominates. Fluid-phase uptake can become an important uptake route as insulin concentrations are increased. Since neomycin stimulates fluid-phase endocytosis, it must also be taken into account when measuring ligand internalization.

  2. Mechanisms of the statins cytotoxicity in freshly isolated rat hepatocytes.

    PubMed

    Abdoli, Narges; Heidari, Reza; Azarmi, Yadollah; Eghbal, Mohammad Ali

    2013-06-01

    Statins are potent drugs, used as lipid-lowering agents in cardiovascular diseases. Hepatotoxicity is one of the serious adverse effects of statins, and the exact mechanism of hepatotoxicity is not yet clear. In this study, the cytotoxic effects of the most commonly used statins, that is, atorvastatin, lovastatin, and simvastatin toward isolated rat hepatocytes, were evaluated. Markers, such as cell death, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and the amount of reduced and oxidized glutathione in the statin-treated hepatocytes, were investigated. It was found that the statins caused cytotoxicity toward rat hepatocytes dose dependently. An elevation in ROS formation, accompanied by a significant amount of lipid peroxidation and mitochondrial depolarization, was observed. Cellular glutathione reservoirs were decreased, and a significant amount of oxidized glutathione was formed. This study suggests that the adverse effect of statins toward hepatocytes is mediated through oxidative stress and the hepatocytes mitochondria play an important role in the statin-induced toxicity.

  3. Toxicity of ethacrynic acid in isolated rat hepatocytes.

    PubMed

    Yamamoto, K; Masubuchi, Y; Narimatsu, S; Kobayashi, S; Horie, T

    2002-04-01

    Ethacrynic acid, a loop diuretic drug, caused lipid peroxidation in isolated rat hepatocytes. The thiobarbituric acid reactive substances (TBARS) formation showed a good correlation with the leakage of glutamic-oxaloacetic acid transaminase (GOT) from the hepatocytes. The addition of antioxidants such as N, N'-diphenyl-p-phenylenediamine (DPPD) and promethazine to the isolated rat hepatocyte suspension containing ethacrynic acid prevented the lipid peroxidation and decreased the GOT leakage to some extent. SKF-525A inhibited the oxidative metabolism of ethacrynic acid and decreased the TBARS formation, suggesting that the lipid peroxidation was caused by the oxidative metabolism. The intracellular reduced glutathione markedly decreased in the hepatocyte suspension containing ethacrynic acid and the hepatocellular protein sulfhydryls were decreased, which was negatively correlated with the GOT leakage. Thus the ethacrynic acid-induced hepatotoxicity was found to be related to the lipid peroxidation and the decrease of cellular protein sulfhydryls.

  4. Enzymatic antioxidant defense in isolated rat hepatocytes exposed to cadmium.

    PubMed

    Skrzycki, M; Czeczot, H; Majewska, M; Podsiad, M; Karlik, W; Grono, D; Wiechetek, M

    2010-01-01

    The aim of the study was the evaluation of cadmium effects on the activity of antioxidant enzymes in rat hepatocytes. The studies were conducted with isolated rat hepatocytes incubated for 1 or 2 hours in a modified (deprived of carbonates with phosphates) Williams' E medium (MWE) in the presence of cadmium chloride (25, 50 and 200 microM). Hepatocytes incubated in the MWE medium without cadmium chloride were used as a control. The application of the modified Williams' E medium allowed for the appearance of cadmium compounds in a soluble form that is indispensable for suitable estimation of its toxic action. There were evaluated markers of the oxidative stress such as: concentration of thiobarbiturate reactive substances (TBARS)--proportional to the level of lipid peroxidation, concentration of reduced glutathione (GSH), and the activity of antioxidant enzymes, including superoxide dismutase (SOD1 and SOD2), catalase (CAT), total glutathione peroxidase (GSHPx), selenium--dependent glutathione peroxidase (SeGSHPx), glutathione transferase (GST) and glutathione reductase (GSHR). Alterations of antioxidant enzymes activity, the level of TBARS and GSH in isolated rat hepatocytes caused by cadmium in vitro, were shown to depend on the concentration and time of exposure of cells to this metal. The increased level of TBARS and GSH was observed as well as changes in the activity of antioxidant enzymes. The activity of SOD isoenzymes and CAT was increased, whereas GSHPx and GST were decreased. These results indicate that cadmium induces oxidative stress followed by alterations in the cellular antioxidant enzyme system in isolated rat hepatocytes.

  5. Morphology and function of isolated hepatocytes transplanted into rat spleen.

    PubMed

    Mito, M; Ebata, H; Kusano, M; Onishi, T; Saito, T; Sakamoto, S

    1979-12-01

    Hepatocytes isolated by the collagenase digestive method were transplanted into the spleens of syngeneic rats. Morphology and function of the hepatocytes in the spleen were investigated for 12 to 17 months after transplantation. The transplanted hepatocytes proliferated and reconfigured in the spleen without direct perfusion of portal venous blood and with the presence of an intact host liver. Fourteen to 17 months after transplantation, the hepatocytes which had formed a demarcated nodule occupied approximately 40% of the area of the splenic parenchyma without undifferentiation on microscopic examination. However, the weight of the hepatized spleen did not increase beyond the weight of a normal spleen and the weight of the host liver that had normal morphology also did not differ from a normal liver. Light and electron microscopic studies demonstrated differentiated cord structure and normal architecture for each heptocyte. Furthermore, the hepatized spleen synthesized albumin and glycogen as demonstrated by immunofluorescence and histochemical studies. Ammonia tolerance and indocyanine green clearance tests revealed functioning hepatocytes in the spleen proper. These results indicate that our experimental model lends itself well to investigations in cell growth mechanism and that hepatocellular transplantation has potential clinical application to compensate for impaired hepatic function.

  6. Molecular cytotoxic mechanisms of chlorpromazine in isolated rat hepatocytes.

    PubMed

    MacAllister, Stephanie L; Young, Cheryl; Guzdek, Anna; Zhidkov, Nickholas; O'Brien, Peter J

    2013-01-01

    Chlorpromazine (CPZ), a member of the largest class of first-generation antipsychotic agents, is known to cause hepatotoxicity in the form of cholestasis and hepatocellular necrosis in some patients. The mechanism of CPZ hepatotoxicity is unclear, but is thought to result from reactive metabolite formation. The goal of this research was to assess potential cytotoxic mechanisms of CPZ using the accelerated cytotoxicity mechanism screening (ACMS) technique with freshly isolated rat hepatocytes. This study identified CPZ cytotoxicity and inhibition of mitochondrial membrane potential (MMP) to be concentration-dependent. Furthermore, inhibition of cytochrome P450s (CYPs), including CYP2D1 and 1A2, delayed CPZ cytotoxicity, suggesting a role for CYP activation of CPZ to a toxic metabolite(s) in this model. Metabolism studies also demonstrated glucuronide and glutathione (GSH) requirement for CPZ detoxification in hepatocytes. Inactivating the 2-electron reduction pathway, NAD(P)H quinone oxidoreductase (NQO1), caused a significant increase in hepatocyte susceptibility to CPZ, indicating quinoneimine contribution to CPZ cytotoxicity. Nontoxic concentrations of peroxidase/H(2)O(2) (inflammatory model) increased cytotoxicity in CPZ-treated hepatocytes and caused additional mitochondrial toxicity. Inflammation further depleted GSH and increased oxidized glutathione (GSSG) levels. Results suggest activation of CPZ to reactive metabolites by 2 pathways in hepatocytes: (i) a CYP-catalyzed quinoneimine pathway, and (ii) a peroxidase-catalyzed oxidation of CPZ to CPZ radicals.

  7. Bioactivation of fluorotelomer alcohols in isolated rat hepatocytes.

    PubMed

    Martin, Jonathan W; Chan, Katie; Mabury, Scott A; O'Brien, Peter J

    2009-02-12

    Fluorotelomer alcohols (FTOHs; C(x)F(2x+1)C(2)H(4)OH) are intermediates in the production of specialty surfactants and stain-repellent polymers. The magnitude and pathways of human exposure to FTOHs are not understood, but FTOHs are present in ambient air and house dust, and FTOH-derivatives are used in food-contact applications. Previously, electrophilic FTOH biotransformation products were detected in rat hepatocytes, and liver lesions were found in FTOH exposed rodents. To begin elucidating the mechanism(s) of action, freshly isolated rat hepatocytes were incubated with FTOHs, or FTOH biotransformation products, and toxicity was followed in the presence or absence of carbonyl scavengers and metabolic enzyme modulators. The LC(50) depended on perfluorinated chain length, with the shortest (4:2 FTOH; x=4) and longest (8:2 FTOH; x=8) FTOHs tested being more toxic than the medium chain length FTOH (6:2 FTOH; x=6); a structure-toxicity relationship that is consistent with that for 2-alkenals. For hepatocytes treated with 8:2 FTOH, cytotoxicity corresponded to depletion of glutathione (GSH), increased protein carbonylation, and lipid peroxidation. Aminobenzotriazole, a P450 inhibitor, diminished cytotoxicity for all FTOHs tested, and decreased protein carbonylation and lipid peroxidation for 8:2 FTOH, indicating that a biotransformation product was responsible for FTOH cytotoxicity. Preincubation of hepatocytes with hydralazine or aminoguanidine decreased the cytotoxicity of 8:2 FTOH, suggesting that reactive aldehyde intermediates contributed to the cytotoxicity. A GSH-reactive alpha/beta-unsaturated acid metabolite was also more toxic than the corresponding FTOH, and may have contributed to the observed effects. Overall, these results suggested that FTOH toxicity was related to electrophilic aldehydes or acids through GSH depletion and protein carbonylation. Further research into the nature of protein modification is warranted for these current-use fluorochemicals.

  8. Effects of ethanol on antioxidant capacity in isolated rat hepatocytes

    PubMed Central

    Yang, Sien-Sing; Huang, Chi-Chang; Chen, Jiun-Rong; Chiu, Che-Lin; Shieh, Ming-Jer; Lin, Su-Jiun; Yang, Suh-Ching

    2005-01-01

    AIM: To investigate dose-response and time-course of the effects of ethanol on the cell viability and antioxidant capacity in isolated rat hepatocytes. METHODS: Hepatocytes were isolated from male adult Wistar rats and seeded into 100-mm dishes. Hepatocytes were treated with ethanol at concentrations between 0 (C), 10 (E10), 50 (E50), and 100 (E100) mmol/L (dose response) for 12, 24, and 36 h (time course). Then, lactate dehydrogenase (LDH) leakage, malondialdehyde (MDA) concentration, glutathione (GSH) level, and activities of glutathione peroxidase (GPX), glutathione reductase (GRD), superoxide dismutase (SOD), and catalase (CAT) were measured. RESULTS: Our data revealed that LDH leakage was significantly increased by about 30% in group E100 over those in groups C and E10 at 24 and 36 h, The MDA concentration in groups C, E10 and E50 were significantly lower than that in group E100 at 36 h. Furthermore, the concentration of MDA in group E100 at 36 h was significantly higher by 4.5- and 1.7-fold, respectively, than that at 12 and 24 h. On the other hand, the GSH level in group E100 at 24 and 36 h was significantly decreased, by 32% and 28%, respectively, compared to that at 12 h. The activities of GRD and CAT in group E100 at 36 h were significantly less than those in groups C and E10. However, The GPX and SOD activities showed no significant change in each group. CONCLUSION: These results suggest that long-time incubation with higher concentration of ethanol (100 mmol/L) decreased the cell viability by means of reducing GRD and CAT activities and increasing lipid peroxidation. PMID:16437627

  9. Cytotoxicity of ortho-phenylphenol in isolated rat hepatocytes.

    PubMed

    Nakagawa, Y; Moldéus, P; Moore, G A

    1992-01-22

    The effects of ortho-phenylphenol (OPP) and its metabolites, phenyl-hydroquinol (PHQ) and phenyl-benzoquinone (PBQ), on isolated rat hepatocytes were investigated. Addition of OPP (0.5-1.0 mM) to cells caused a dose-dependent cell death accompanied by the depletion of intracellular levels of ATP, glutathione (GSH) and protein thiols. GSH loss correlated with the formation of oxidized GSH. In addition, PHQ and especially PBQ (both at 0.5 mM) resulted in acute cell death with rapid depletion of ATP, GSH and protein thiols, and further low doses of PBQ (10-50 microM) elicited serious impairment of mitochondrial functions related to oxidative phosphorylation and Ca fluxes in isolated liver mitochondria. These results indicate that mitochondria are a target for these compounds and that OPP is itself toxic to hepatocytes even when metabolism is inhibited. The loss of cellular GSH and protein thiols accompanied by the impairment of mitochondrial function may be the main mechanisms of cytotoxicity induced by OPP and its metabolites.

  10. Isolated rat hepatocytes acquire iron from lactoferrin by endocytosis.

    PubMed

    McAbee, D D

    1995-10-15

    The iron-binding protein lactoferrin (Lf) present in blood is metabolized by the liver. Isolated rat hepatocytes vigorously endocytose bovine Lf via recycling Ca2(+)-dependent binding sites, but the uptake of iron from Lf by hepatocytes has not been examined. In this study, isolated rat hepatocytes were incubated with radiolabelled bovine Lf (125I-Lf, 59Fe-Lf or 125I-59Fe-Lf) at 37 degrees C, then washed at 4 degrees C in the presence of dextran sulphate with either Ca2+ or EGTA to distinguish between total bound and internal radioactivity respectively. Cells internalized 125I-Lf protein and Lf-bound 59Fe at maximal endocytic rates of 1700 and 480 mol.cell-1.s-1 respectively. When Lf was normalized for 59Fe content, these endocytic rates were equivalent and reflected an uptake potential of at least 3400 mol of iron.cell-1.s-1. Cells prebound with 125I-59Fe-Lf to Ca2+(-)dependent sites at 4 degrees C internalized more than 80% of both 125I-Lf protein and Lf-bound 59Fe approx. 6 min after warming to 37 degrees C at similar rates (125I-Lf: k(in) = 0.276 min-1, 59Fe: k(in) = 0.303 min-1). Within 4 h at 37 degrees C, cells had released 25% or less internalized Lf protein in the form of acid-soluble 125I-by-products but retained all the Lf-delivered 59Fe. Hyperosmotic disruption of clathrin-dependent endocytosis blocked the uptake of 125I-Lf and Lf-bound 59Fe. Incubation of cells with 125I-59Fe-Lf and a 100 molar excess of diferric transferrin reduced slightly the endocytosis of 125I-Lf protein and 59Fe accumulation. Treatment of cells with the ferric chelator desferrioxamine did not alter uptake of 125I-Lf protein or Lf-bound 59Fe, but the ferrous chelator bathophenanthroline disulphonate slightly elevated endocytosis of 125I-Lf protein and Lf-bound 59Fe. These findings indicate that Lf does not release its bound iron before endocytosis. It was concluded from this study that hepatocytes take up iron from Lf at high rates by a process that requires endocytosis of Lf

  11. Effects of Aronia melanocarpa Fruit Juice on Isolated Rat Hepatocytes

    PubMed Central

    Kondeva-Burdina, Magdalena; Valcheva-Kuzmanova, Stefka; Markova, Tsvetelina; Mitcheva, Mitka; Belcheva, Anna

    2015-01-01

    Background: Aronia melanocarpa (Michx.) Elliot fruits are very rich in polyphenols – procyanidins, flavonoids, and phenolic acids. Objective: On rat hepatocytes, isolated by two-stepped collagenase perfusion, we investigated the effect of A. melanocarpa fruit juice (AMFJ) in two models of liver toxicity caused by (i) metabolic bioactivation of carbon tetrachloride (CCl4), and (ii) tert-butyl hydroperoxide (t-BuOOH)-induced oxidative stress. Materials and Methods: Isolated rat hepatocytes are a suitable model for hepatotoxicity studies. We determined the main parameters of the functional and metabolic status of rat hepatocytes: Cell viability (measured by trypan blue exclusion) and the levels of lactate dehydrogenase (LDH), reduced glutathione (GSH), and malondialdehyde (MDA). These parameters were used to investigate the protective effects of AMFJ in the two toxicity models. The effects of AMFJ were compared with those of silymarin. The cells were treated either with AMFJ or silymarin at increasing concentrations of 5 μg/ml, 10 μg/ml, 30 μg/ml, 50 μg/ml, and 100 μg/ml which were used for measuring of IC50. Results: In both toxicity models – CCl4 and t-BuOOH, AMFJ showed statistically significant cytoprotective and antioxidant activities. AMFJ prevented the loss of cell viability and GSH depletion, decreased LDH leakage and MDA production. The effects of AMFJ at the concentrations of 5, 10, 30, and 50 μg/ml were similar to those of the same concentrations of silymarin, while the effect of the highest AMFJ concentration of 100 μg/ml was higher than that of the same silymarin concentration. The effects were concentration-dependent and more prominent in the t-BuOOH model, compared to those in the CCl4 model. Conclusion: The cytoprotective and antioxidant effects of AMFJ established in this study might be due to its polyphenolic ingredients, which could influence the cytochrome P450-mediated metabolism of the experimental hepatotoxic substances (CCl4 and t

  12. Stimulatory effect of the intestinal peptide PHI on glycogenolysis and gluconeogenesis in isolated rat hepatocytes.

    PubMed Central

    Felíu, J E; Marco, J

    1983-01-01

    The newly isolated peptide PHI provoked a dose-dependent stimulation of glycogenolysis and gluconeogenesis in isolated rat hepatocytes; at 1 microM-PHI, both processes were increased 1.6-fold as compared with basal values. These PHI-mediated effects were accompanied by the activation of glycogen phosphorylase and the inactivation of pyruvate kinase. PHI (1 microM) also caused a 2-fold increase in hepatocyte cyclic AMP. PMID:6312969

  13. Growth hormone acutely increases glucose output by hepatocytes isolated from hypophysectomized rats.

    PubMed

    Blake, W L; Clarke, S D

    1989-08-01

    A series of experiments using isolated rat hepatocytes was carried out to establish rat liver cells in suspension as a physiological model for examining GH responses, and to determine whether acute recombinant bovine GH (rbGH) treatment of rat liver cells increased glucose output and/or suppressed fatty acid synthesis from lactate. Rat liver cells were isolated by collagenase perfusion and incubated in short-term (less than 60 min) suspension. The amount of insulin, glucagon or vasopressin required to elicit a half-maximal response was within the physiological range of the circulating hormone. When hepatocytes from normal rats were acutely (less than 60 min) treated with 0, 0.1, 10, 100 or 1000 nmol rbGH/l, rates of hepatocyte glucose output and fatty acid synthesis were unaltered. In addition, acute rbGH treatment (1000 nmol/l) did not alter hepatocyte responsiveness to insulin or vasopressin. However, acute rbGH treatment of hepatocytes isolated from hypophysectomized rats significantly (P less than 0.05) increased the rate of glucose output twofold and moderately (P less than 0.10) enhanced fatty acid synthesis. The accelerated rate of glucose production was not accompanied by an increase in the amount of glycogen phosphorylase-a. The observations with liver cells from hypophysectomized rats are not consistent with a GH receptor-transducing mechanism which is like that for glucagon (adenylate cyclase-linked) or insulin (tyrosine kinase-linked).

  14. Effects of dehydroepiandrosterone (DHEA) on glucose metabolism in isolated hepatocytes from Zucker rats

    SciTech Connect

    Finan, A.; Cleary, M.P.

    1986-03-05

    DHEA has been shown to competitively inhibit the pentose phosphate shunt (PPS) enzyme glucose-6-phosphate dehydrogenase (G6PD) when added in vitro to supernatants or homogenates prepared from mammalian tissues. However, no consistent effect on G6PD activity has been determined in tissue removed from DHEA-treated rats. To explore the effects of DHEA on PPS, glucose utilization was measured in hepatocytes from lean and obese male Zucker rats (8 wks of age) following 1 wk of DHEA treatment (0.6% in diet). Incubation of isolated hepatocytes from treated lean Zucker rats with either (1-/sup 14/C) glucose or (6-/sup 14/C) glucose resulted in significant decreases in CO/sub 2/ production and total glucose utilization. DHEA-lean rats also had lowered fat pad weights. In obese rats, there was no effect of 1 wk of treatment on either glucose metabolism or fat pad weight. The calculated percent contribution of the PPS to glucose metabolism in hepatocytes was not changed for either DHEA-lean or obese rats when compared to control rats. In conclusion, 1 wk of DHEA treatment lowered overall glucose metabolism in hepatocytes of lean Zucker rats, but did not selectively affect the PPS. The lack of an effect of short-term treatment in obese rats may be due to differences in their metabolism or storage/release of DHEA in tissues in comparison to lean rats.

  15. Hepatocytes isolated from preneoplastic rat livers are resistant to ethacrynic acid cytotoxicity.

    PubMed

    Parody, Juan Pablo; Alvarez, María de Luján; Quiroga, Ariel; Ronco, María Teresa; Francés, Daniel; Carnovale, Cristina; Carrillo, María Cristina

    2007-08-01

    Glutathione S-transferases (GSTs) are involved in the detoxification of xenobiotics, such as several cytostatic drugs, through conjugation with glutathione (GSH). Pi class GST (GST P) liver expression is associated with preneoplastic and neoplastic development and contributes with the drug-resistance phenotype. Ethacrynic acid (EA) is an inhibitor of rat and human GSTs. In addition, causes lipid peroxidation in isolated rat hepatocytes. Therefore, we decided to evaluate the role of the GST/GSH system in isolated hepatocytes from preneoplastic rat livers (IP) in the presence of EA and determine the cytotoxicity of the drug. Our results showed a resistance to the toxic effects of EA since viability and cellular integrity values were significantly higher than control. Initial levels of thiobarbituric acid reactive substances (TBARS) in IP hepatocytes were significantly higher than control and the presence of EA did not change TBARS levels. A diminution in intracellular total GSH was observed by treating with EA isolated hepatocytes from both groups. However, the initial total GSH levels were higher in IP hepatocytes than in control. Immunoblotting analysis showed the presence of GST P in IP animals only. Although alpha and mu class isoenzymes levels were decreased in IP hepatocytes, total GST activity was 1.5-fold higher than in control. In addition, multidrug-resistance protein 2 (Mrp2) showed fivefold decreased levels in IP hepatocytes. In conclusion, increased total GSH, decreased Mrp2 levels and the presence of GST P could be critical factors involved in the resistance of IP hepatocytes to the toxicity of EA.

  16. Glucose production and storage in hepatocytes isolated from normal versus diabetic rats

    SciTech Connect

    Olivieri, M.C.; Dragland-Meserve, C.J.; Parker Botelho, L.H.

    1987-05-01

    The rates of glucose production and storage were compared in hepatocytes isolated from normal versus insulin-resistant diabetic rats. A single low-dose (40 mg/kg) IV injection of streptozotocin to 250 g rats resulted in a Type II diabetic animal model which was hyperglycemic with normal insulin levels. Addition of 8 mM /sup 14/C-lactate and 2 mM pyruvate to hepatocytes resulted in a linear increase in total glucose production (/sup 14/C-glucose and unlabeled glucose) and incorporation into glycogen measured over 120 min. The rate of gluconeogenesis was estimated from the production of /sup 14/C-glucose and the rate of glycogenolysis was estimated from the production of unlabeled glucose in cells incubated in the presence or absence of /sup 14/C-labelled substrate. There was not significant difference in total glucose production in hepatocytes isolated from normal versus diabetic rats, however, the contribution from gluconeogenesis versus glycogenolysis was significantly different. Following a 1 h incubation of cells from normal rats, 42% of the total glucose production was due to gluconeogenesis and 58% was due to glycogenolysis. In cells from diabetic rats, 83% of total glucose production was from gluconeogenesis and 17% from glycogenolysis. Also, incubation with /sup 14/C-lactate/pyruvate resulted in a 3.3-fold increase in /sup 14/C-glucose incorporation into glycogen in hepatocytes isolated from normal rats compared to diabetic rats. These data suggest that alterations occur in the rate-limiting enzymes responsible for glucose production and storage in hepatocytes isolated from a rat model of insulin-resistant Type II diabetes.

  17. Interaction of propionate and carnitine metabolism in isolated rat hepatocytes

    SciTech Connect

    Brass, E.P.; Beyerinck, R.A.

    1987-05-01

    Propionate (P) and its metabolic products P-CoA and methylmalonyl-CoA can disrupt normal hepatic metabolism. Carnitine (Cn) has been shown to partially restore cellular function in the presence of P. This effect of Cn may result from removal of propionyl groups as propionylcarnitine (P-Cn). The present study examined the kinetics of P-Cn formation in rat hepatocytes, and the consequence of P-Cn formation on P and Cn metabolism. /sup 14/C-P was converted to CO/sub 2/, glucose and P-Cn in the hepatocyte system. Increasing concentrations of Cn up to 10.0 mM increased P-Cn formation from P without affecting CO/sub 2/ or glucose formation. Thus, 10.0 mM Cn increased total P metabolism by 40%. Metabolism of P was associated with a decrease in Cn concentration and an increase in short chain acylcarnitines (SCCn). In the absence of added Cn, 60 min incubation with P decreased Cn from 6.8 to 2.5 ..mu..M with a corresponding increase in SCCn. This effect of P to deplete free Cn was not seen to the same degree with butyrate in place of P. Similar increases in the formation of SCCn in the presence of P at the expense of free Cn were seen when the incubation Cn concentration was increased to 50 ..mu..M or 150 ..mu..M. HPLC methodologies to study specific acylcarnitines demonstrated the accumulation of large amounts of P-Cn in the incubations containing P, accounting for the depletion of free Cn.

  18. Protective effect of metallothionein on cadmium toxicity in isolated rat hepatocytes.

    PubMed Central

    Din, W S; Frazier, J M

    1985-01-01

    An isolated rat hepatocyte preparation was used to study the cellular toxicity of cadmium and the protective effects of metallothionein on cadmium-induced toxicity. Exposure of primary suspension cultures of isolated rat hepatocytes to Cd2+ (0-35.7 microM) for 15 min resulted in a dose-dependent reduction in the synthesis of cellular proteins during a subsequent 6 h incubation. Such inhibition could not be correlated with cellular lethality or gross membrane damage. Pre-induction of metallothionein in hepatocytes by zinc treatment in vivo of donor rats protected hepatocytes in vitro from cadmium-induced inhibition of protein synthesis. The protective effects in zinc-pre-induced hepatocytes are not due to alterations in the level of total cellular cadmium, but could be accounted for by the redistribution of intracellular cadmium in the presence of high levels of zinc-metallothionein. The data suggest that metallothionein exerts its protective effect by a kinetic detoxification mechanism, i.e. a decrease in reactive intracellular cadmium. PMID:4052053

  19. Cytotoxic effects of postharvest fungicides, ortho-phenylphenol, thiabendazole and imazalil, on isolated rat hepatocytes.

    PubMed

    Nakagawa, Y; Moore, G A

    1995-01-01

    The cytotoxic effects of ortho-phenylphenol (OPP), imazalil (IMZ) and thiabendazole (TBZ) on isolated rat hepatocytes were investigated. Addition of IMZ and OPP to hepatocyte suspensions at a concentration of 0.75 mM resulted in acute cell death, accompanied by depletion of intracellular levels of glutathione and protein thiols. Both compounds rapidly depleted cellular ATP which consistently preceded the cell death. In addition, the cell death caused by IMZ was accompanied by the accumulation of intracellular malondialdehyde, indicating initiation of lipid peroxidation. During a 3-hr incubation period, TBZ did not affect these parameters. In mitochondria isolated from rat liver, IMZ and OPP impaired respiration related to oxidative phosphorylation. Based on these results, the order of toxic potency is IMZ > OPP > TBZ.

  20. Effects of carbon tetrachloride on isolated rat hepatocytes. Inhibition of protein and lipoprotein secretion.

    PubMed Central

    Gravela, E; Albano, E; Dianzani, M U; Poli, G; Slater, T F

    1979-01-01

    The effects of carbon tetrachloride on protein and lipoprotein secretion, and on lipid peroxidation, have been investigated in isolated rat hepatocytes. It was found that although the free-radical scavenger promethazine completely suppressed the increased peroxidation produced by carbon tetrachloride, it had no effect on the inhibitory action of carbon tetrachloride on lipoprotein secretion. In consequence, the latter effect of carbon tetrachloride does not appear to be mediated through a peroxidative stage. PMID:444227

  1. Relationship between metabolism and cytotoxicity of ortho-phenylphenol in isolated rat hepatocytes.

    PubMed

    Nakagawa, Y; Tayama, S; Moore, G A; Moldéus, P

    1992-04-01

    The relationship between the metabolism and the cytotoxicity of ortho-phenylphenol (OPP) was investigated using isolated rat hepatocytes. Addition of OPP (0.5-1.0 mM) to the hepatocytes caused a dose-dependent toxicity; 1.0 mM OPP caused acute cell death. Pretreatment of hepatocytes with SKF-525A (50 microM, a non-toxic level) enhanced the cytotoxicity of OPP (0.5-1.0 mM). This was accompanied by inhibition of OPP metabolism. Conversely, OPP at low concentrations (0.5 or 0.75 mM) was converted sequentially to phenyl-hydroquinol (PHQ) and then to glutathione (GSH) conjugate in the cells. The concentrations of both metabolites, especially PHQ-GSH conjugate, were very low in hepatocytes exposed to 1.0 mM OPP alone as well as with SKF-525A. The cytotoxicity induced by 0.5 mM OPP was enhanced by the addition of diethylmaleate (1.25 mM) which continuously depletes cellular GSH. In contrast, additions to hepatocytes of 5 mM of dithiothreitol, cysteine, N-acetyl-L-cysteine or ascorbic acid significantly inhibited the cytotoxicity induced by 0.5 mM PHQ; GSH, protein thiols and ATP losses were also prevented. Further, these compounds depressed the rate of PHQ loss in hepatocyte suspensions. These results indicate that the acute cytotoxicity caused by the high dose (1.0 mM) of OPP is associated with direct action by the parent compound; at low doses (0.5-0.75 mM) of OPP, the prolonged depletion of GSH in hepatocytes enhances the cytotoxicity induced by PHQ.

  2. [Permeability of isolated rat hepatocyte plasma membranes for molecules of dimethyl sulfoxide].

    PubMed

    Kuleshova, L G; Gordienko, E A; Kovalenko, I F

    2014-01-01

    We have studied permeability of isolated rat hepatocyte membranes for molecules of dimethyl sulfoxide (DMSO) at different hypertonicity of a cryoprotective medium. The permeability coefficient of hepatocyte membranes κ1 for DMSO molecules was shown to be the differential function of osmotic pressure between a cell and an extracellular medium. Ten-fold augmentation of DMSO concentration in the cryoprotective medium causes the decrease of permeability coefficients κ1 probably associated with the increased viscosity in membrane-adjacent liquid layers as well as partial limitations appeared as a result of change in cell membrane shape after hepatocyte dehydration. We have found out that in aqueous solutions of NaCl (2246 mOsm/l) and DMSO (2250 mOsm/l) the filtration coefficient L(p) in the presence of a penetrating cryoprotectant (L(pDMSO) = (4.45 ± 0.04) x 10(-14) m3/Ns) is 3 orders lower compared to the case with electrolyte (L(pNaCl) = (2.25 ± 0.25) x 10(-11) m3/Ns). This phenomenon is stipulated by the cross impact of flows of a cryoprotectant and water at the stage of cell dehydration. Pronounced lipophilicity of DMSO, geometric parameters of its molecule as well as the presence of large aqueous pores in rat hepatocyte membranes allow of suggesting the availability of two ways of penetrating this cryoprotectant into the cells by non-specific diffusion through membrane lipid areas and hydrophilic channels.

  3. Activation of phospholipase A/sub 2/ by carbon tetrachloride in isolated rat hepatocytes

    SciTech Connect

    Glende, E.A. Jr.; Pushpendran, C.K.

    1986-03-05

    Freshly isolated rat hepatocytes were incubated with /sup 3/H-arachidonic acid or /sup 14/C-ethanolamine for 1 hour in order to label cellular lipids. Thin-layer chromatographic analysis indicated that of the /sup 3/H-arachidonate incorporated into lipid nearly 50% was found in phosphatidylcholine and 15% in phosphatidylethanolamine. /sup 14/C-Ethanolamine was incorporated mainly into phosphatidylethanolamine. Hepatocytes labeled as such were exposed to carbon tetrachloride (CCl/sub 4/) for periods up to 4 hours. Phospholipase A/sub 2/ of these preparations was determined by measuring either the release of /sup 4/H-arachidonic acid from cellular phospholipids prelabeled with /sup 3/H-arachidonic acid or measuring the formation of /sup 14/C-lysophosphatidylethanolamine from cellular lipids prelabeled with /sup 14/C-ethanolamine. Through the use of hexane-partition extraction and thin-layer chromatographic analysis of hepatocyte lipid extracts it was found that CCl, stimulated phospholipase A/sub 2/ activity in a dose- an time-dependent manner. Carbon tetrachloride at concentrations of 0.23 to 1.3 mM produced a 1.4- to 5.3-fold increase in phospholipase activity which was initiated within 30 to 60 minutes of incubation at 37/sup 0/. A role for phospholipase activation as a secondary mechanism of CCl/sub 4/-induced hepatocyte injury is proposed.

  4. Protective effects of Sesamum indicum extract against oxidative stress induced by vanadium on isolated rat hepatocytes.

    PubMed

    Hosseini, Mir-Jamal; Shahraki, Jafar; Tafreshian, Saman; Salimi, Ahmad; Kamalinejad, Mohammad; Pourahmad, Jalal

    2016-08-01

    Vanadium toxicity is a challenging problem to human and animal health with no entirely understanding cytotoxic mechanisms. Previous studies in vanadium toxicity showed involvement of oxidative stress in isolated liver hepatocytes and mitochondria via increasing of ROS formation, release of cytochrome c and ATP depletion after incubation with different concentrations (25-200 µM). Therefore, we aimed to investigate the protective effects of Sesamum indicum seed extract (100-300 μg/mL) against oxidative stress induced by vanadium on isolated rat hepatocytes. Our results showed that quite similar to Alpha-tocopherol (100 µM), different concentrations of extract (100-300 μg/mL) protected the isolated hepatocyte against all oxidative stress/cytotoxicity markers induced by vanadium in including cell lysis, ROS generation, mitochondrial membrane potential decrease and lysosomal membrane damage. Besides, vanadium induced mitochondrial/lysosomal toxic interaction and vanadium reductive activation mediated by glutathione in vanadium toxicity was significantly (P < 0.05) ameliorated by Sesamum indicum extracts. These findings suggested a hepato-protective role for extracts against liver injury resulted from vanadium toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 979-985, 2016.

  5. Cytoprotective effects of taurine against toxicity induced by isoniazid and hydrazine in isolated rat hepatocytes.

    PubMed

    Heidari, Reza; Babaei, Hossein; Eghbal, Mohammad Ali

    2013-06-01

    Isoniazid is one of the most commonly used drugs to treat tuberculosis. Its administration is associated with a high incidence of hepatotoxicity. The aim of this study was to establish the protective effects of taurine against cytotoxicity induced by isoniazid and its suspected toxic metabolite hydrazine in isolated rat hepatocytes by measuring reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial depolarisation, reduced glutathione (GSH), and oxidised glutathione (GSSG). Isoniazid caused no significant ROS formation in normal hepatocytes, but in glutathione-depleted cells it was considerable. Hydrazine caused ROS formation and lipid peroxidation in both intact and glutathione-depleted cells. Both isoniazid and hydrazine caused mitochondrial membrane depolarisation. Hydrazine lowered cellular GSH reserve and increased GSSG. Taurine (200 μmol L(-1)) and N-acetylcysteine (200 μmol L(-1)) effectively countered the toxic effects of isoniazid and/or hydrazine by decreasing ROS formation, lipid peroxidation, and mitochondrial damage. Taurine prevented depletion of GSH and lowered GSSG levels in hydrazine-treated cells. This study suggests that the protective effects of taurine against isoniazid and its intermediary metabolite hydrazine cytotoxicity in rat hepatocytes could be attributed to antioxidative action.

  6. Venlafaxine-Induced Cytotoxicity Towards Isolated Rat Hepatocytes Involves Oxidative Stress and Mitochondrial/Lysosomal Dysfunction

    PubMed Central

    Ahmadian, Elham; Babaei, Hossein; Mohajjel Nayebi, Alireza; Eftekhari, Aziz; Eghbal, Mohammad Ali

    2016-01-01

    Purpose: Depression is a public disorder worldwide. Despite the widespread use of venlafaxine in the treatment of depression, it has been associated with the incidence of toxicities. Hence, the goal of the current investigation was to evaluate the mechanisms of venlafaxine–induced cell death in the model of the freshly isolated rat hepatocytes. Methods: Collagenase-perfused rat hepatocytes were treated with venlafaxine and other agents. Cell damage, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential decline, lysosomal damage, glutathione (GSH) level were analyzed. Moreover, rat liver mitochondria were isolated through differential centrifugation to assess respiratory chain functionality. Results: Our results demonstrated that venlafaxine could induce ROS formation followed by lipid peroxidation, cellular GSH content depletion, elevated GSSG level, loss of lysosmal membrane integrity, MMP collapse and finally cell death in a concentration-dependent manner. N-acetyl cysteine, taurine and quercetine significantly decreased the aforementioned venlafaxine-induced cellular events. Also, radical scavenger (butylatedhydroxytoluene and α-tocopherol), CYP2E1 inhibitor (4-methylpyrazole), lysosomotropic agents (methylamine and chloroquine), ATP generators (L-gluthamine and fructose) and mitochondrial pore sealing agents (trifluoperazine and L-carnitine) considerably reduced cytotoxicity, ROS generation and lysosomal leakage following venlafaxine treatment. Mitochondrion dysfunction was concomitant with the blockade of the electron transfer complexes II and IV of the mitochondrial respiratory system. Conclusion: Therefore, our data indicate that venlafaxine induces oxidative stress towards hepatocytes and our findings provide evidence to propose that mitochondria and lysosomes are of the primary targets in venlafaxine-mediated cell damage. PMID:28101459

  7. Protective effect of NAC against malathion-induced oxidative stress in freshly isolated rat hepatocytes

    PubMed Central

    Mostafalou, Sara; Abdollahi, Mohammad; Eghbal, Mohammad Ali; Saeedi Kouzehkonani, Nazli

    2012-01-01

    Purpose: Induction of oxidative stress by Organophosphate compounds (OPs) has been previously reported. In the present work, the mechanism of protective effects of N-acetylcysteine as a glutathion (GSH) prodrug against malathion–induced cell toxicity was investigated. In this work, freshly isolated rat hepatocytes were used to determine the effect of NAC on malathion-induced cytotoxicity, formation of reactive oxygen species (ROS) and mitochondrial dysfunction. Methods: Rat hepatocytes were isolated using collagenase perfusion and then cell viability, mitchondrial membrane potential (MMP) and ROS formation were determined using trypan blue exclusion, Rhodamine 123 fluorescence and fluorogenic probe, 2', 7' -dichlorofluorescin diacetate (DCFH-DA), respectively. Results: Despite the protective effect of NAC on malathion-induced cell toxicity and MMP dysfunction, its efficacy against ROS formation was not adequate to completely protect the cells. Conclusion: Cytotoxic effects of malathion regardless of its cholinergic feature, is started with gradual free radical production but, the main factor that causes cell death, is mitochondrial dysfunction, so that reduction of ROS formation alone is not sufficient for cell survival, and the maintenance of mitochondrial integrity through different mechanisms is the most ameliorative factor specially at high levels of cell damage, as NAC seemed to protect cells with various fashions apart from ROS scavenging in concentrations higher than malathion’s LC50. PMID:24312774

  8. Effect of. cap alpha. -ketobutyrate on the metabolism of pyruvate and palmitate in isolated rat hepatocytes

    SciTech Connect

    Brass, E.P.

    1986-05-01

    Alpha-ketobutyrate (..cap alpha..KB), an intermediate in the catabolism of threonine and methionine, is decarboxylated to propionyl-CoA. The authors have reported that propionate (PROP) inhibits oxidative metabolism in rate hepatocytes. Based on these observations, the present study examined the effects of ..cap alpha..KB on pyruvate and palmitate metabolism in hepatocytes isolated from fed rats. Similar to PROP, ..cap alpha..KB (10mM) inhibited palmitate oxidation and this inhibition was diminished when 10mM carnitine (CN) was added (35 +/- 6% inhibition without CN, 22 +/- 8% with CN). ..cap alpha..KB inhibited the conversion of 3-/sup 14/C-pyruvate to glucose and CO/sub 2/. Inhibition of pyruvate metabolism by ..cap alpha..KB was concentration-dependent. At equal concentrations, ..cap alpha..KB inhibited pyruvate metabolism to a greater extent than PROP. Addition of CN partially reversed the effects of PROP on pyruvate metabolism, but not those of ..cap alpha..KB despite the generation of propionylcarnitine when ..cap alpha..KB and CN were included in the incubation. These results demonstrate that accumulation of ..cap alpha..KB can impair normal hepatocyte metabolism. While some of the effects of ..cap alpha..KB can be explained on the basis of propionyl-CoA formation, ..cap alpha..KB has effects on pyruvate metabolism not explainable by this mechanism.

  9. Effect of curcumin analog on gamma-radiation-induced cellular changes in primary culture of isolated rat hepatocytes in vitro.

    PubMed

    Srinivasan, M; Sudheer, A Ram; Rajasekaran, K N; Menon, Venugopal P

    2008-10-22

    The present study was aimed to evaluate the radioprotective effect of curcumin analog, on gamma-radiation-induced toxicity in primary cultures of isolated rat hepatocytes. Hepatocytes were isolated from the liver of rats by collagenase perfusion. The DNA damage was analysed by single cell gel electrophoresis (comet assay). An increase in the severity of DNA damage was observed with the increase in gamma-radiation dose at 1-4 Gy in cultured rat hepatocytes. The levels of lipid peroxidative indices like thiobarbituric acid reactive substances (TBARSs) were increased significantly, whereas the levels of reduced glutathione (GSH) and antioxidant enzymes were significantly decreased in gamma-irradiated groups. The maximum damage to hepatocytes was observed at 4Gy gamma-irradiation. Pretreatment with different concentrations of curcumin analog (1.38, 6.91 and 13.82 microM) shows a significant decrease in the levels of TBARS and DNA damage. Pretreatment with curcumin analog prevents the loss of enzymic and non-enzymic antioxidants like GSH upon gamma-irradiation. The maximum protection of hepatocytes was observed at 6.91 microM of curcumin analog pretreatment. Thus, our result shows that pretreatment with curcumin analog protects the hepatocytes against gamma-radiation-induced cellular damage.

  10. Specificity of an Na+ -dependent taurocholate transport site in isolated rat hepatocytes.

    PubMed

    Hardison, W G; Bellentani, S; Heasley, V; Shellhamer, D

    1984-05-01

    The structural specificity of the taurocholate uptake mechanism was studied by determining the ability of 100 microM structural analogues of taurocholate to inhibit the uptake of 5 microM [14C]-taurocholate into freshly isolated rat hepatocytes. Taurocholate was a more potent inhibitor than cholate. The fewer the number of hydroxyl groups, the more potent the inhibitors. Neither hydroxyl group orientation (alpha or beta) nor esterification with formic acid strongly influenced inhibitory potency. Replacement of hydroxyl groups with keto groups, however, diminished inhibitory potency. These facts suggested that distortion of the sterol backbone may be an important factor in diminishing inhibitory potency. Charge on the nonsteroid moiety affected potency. Both neutral and negatively charged compounds inhibited more strongly than positively charged compounds. As important as charge, however, was length of the nonsteroidal moiety. Inhibitory potency increased progressively as length beyond C-17 increased up to 11 A. Inhibitory potency was reduced again, however, with an extremely long end group of 18 A. The inhibitors do not act as cytotoxins since cellular oxygen consumption was not diminished by any of them. Furthermore, inhibition was specific for the taurocholate uptake mechanism. The three most potent inhibitory compounds inhibited L-alanine uptake little or not at all. The kinetics of inhibition, determined for three selected compounds, were consistent with competitive inhibition. The structural specificity of the rat hepatocyte sodium-dependent taurocholate uptake site is similar to that of the guinea pig ileum uptake site.

  11. Inhibition of glycolysis by 5-amino-4-imidazolecarboxamide riboside in isolated rat hepatocytes.

    PubMed Central

    Vincent, M F; Bontemps, F; Van den Berghe, G

    1992-01-01

    5-Amino-4-imidazolecarboxamide riboside (AICAriboside; Z-riboside), the nucleotide corresponding to AICAribotide (AICAR or ZMP), an intermediate of the 'de novo' pathway of purine nucleotide biosynthesis, has been shown to inhibit gluconeogenesis in isolated rat hepatocytes [Vincent, Marangos, Gruber & Van den Berghe (1991) Diabetes 40, 1259-1266]. We now report that glycosis is also inhibited and even more sensitive to AICAriboside in these cells. In hepatocyte suspensions from fasted rats, production of lactate from 15 mM-glucose was half-maximally inhibited by 25-50 microM-AICAriboside. AICAriboside influenced two regulatory steps of glycolysis: (1) it decreased the release of 3H2O from [2-3H]glucose and the concentrations of both glucose 6-phosphate and fructose 6-phosphate, indicating that it diminished the phosphorylation of glucose by glucokinase; (2) it decreased the concentration of fructose 2,6-bisphosphate (Fru-2,6-P2), the main physiological stimulator of liver 6-phosphofructo-1-kinase. Further studies showed that AICAriboside induced an inactivation of 6-phosphofructo-2-kinase, the enzyme that produces Fru-2,6-P2, without affecting the concentration of cyclic AMP. Similarly to the inhibiton of gluconeogenesis by AICAriboside, the inhibition of glycolysis became apparent after an approx. 10 min latency and persisted when the cells were washed after addition of AICAriboside, strongly suggesting that the effects were also exerted by the Z-nucleotides, which accumulate after addition of AICAriboside to hepatocytes. An increased uptake of lactate was evident when 50-200 microM-AICAriboside was added 15 min after addition of glucose. This can be explained by the higher sensitivity of glycolysis, as compared with gluconeogenesis, to inhibition by AICAriboside, and reveals the simultaneous operation of both processes. PMID:1531010

  12. In vitro study of lovastatin interactions with amiodarone and with carbon tetrachloride in isolated rat hepatocytes

    PubMed Central

    Krasteva, AZ; Mitcheva, MK; Kondeva-Burdina, MS; Descatoire, VA

    2007-01-01

    AIM: To investigate the interactions at a metabolic level between lovastatin, amiodarone and carbon tetrachloride in isolated rat hepatocytes. METHODS: For cell isolation two-step collagenase liver perfusion was performed. Lovastatin was administered alone in increasing concentrations (1 μmol/L, 3 μmol/L, 5 μmol/L and 10 μmol/L) and in combination with CCl4 (86 μmol/L). The cells were also pretreated with 14 μmol/L amiodarone and then the other two compounds were added. RESULTS: Lovastatin promoted concentration-dependent significant toxicity estimated by decrease in cell viability and GSH level by 45% and 84%, respectively. LDH-activity increased by 114% and TBARS content by 90%. CCl4 induced the expected severe damage on the examined parameters. CCl4 induced toxicity was attenuated after lovastatin pretreatment, which was expressed in less increased values of LDH activity and TBARS levels, as well as in less decreased cell viability and GSH concentrations. However, the pretreatment of hepatocytes with amiodarone abolished the protective effect of lovastatin. CONCLUSION: We suggest that the observed cytopro-tective effect was due to interactions between lovastatin, CCl4 and amiodarone at a metabolic level. PMID:17465501

  13. Mechanism of p-hydroxybenzoate ester-induced mitochondrial dysfunction and cytotoxicity in isolated rat hepatocytes.

    PubMed

    Nakagawa, Y; Moldéus, P

    1998-06-01

    The relationship between the metabolism and the cytotoxic effects of the alkyl esters of p-hydroxybenzoic acid (parabens) has been studied in freshly isolated rat hepatocytes. Incubation of hepatocytes with propyl-paraben (0.5 to 2.0 mM) elicited a concentration- and time-dependent cell death that was enhanced when enzymatic hydrolysis of propyl-paraben to p-hydroxybenzoic acid was inhibited by a carboxylesterase inhibitor, diazinon. The cytotoxicity was accompanied by losses of cellular ATP, total adenine nucleotide pools, and reduced glutathione, independently of lipid peroxidation and protein thiol oxidation. In the comparative toxic effects based on cell viability, ATP level, and rhodamine 123 retention, butyl- and isobutyl-parabens were more toxic than propyl- and isopropyl-parabens, and ethyl- and methyl-parabens and p-hydroxybenzoic acid were less toxic than propyl-paraben. The addition of propyl-paraben to isolated hepatic mitochondria reduced state 3 respiration with NAD+-linked substrates (pyruvate plus malate) and/or with an FAD-linked substrate (succinate plus rotenone), whereas state 3 respiration with ascorbate plus tetramethyl-p-phenylenediamine (cytochrome oxidase-linked respiration) was not affected significantly by propyl-paraben. Further, the addition of these parabens caused a concentration-dependent increase in the rate of state 4 oxygen consumption, indicating an uncoupling effect. The rate of state 3 oxygen consumption was inhibited by propyl-paraben, butyl-paraben, and their chain isomers. These results indicate that a) propyl-paraben-induced cytotoxicity is mediated by the parent compound rather than by its metabolite p-hydroxybenzoic acid; b) the toxicity is associated with ATP depletion via impairment of mitochondrial function related to membrane potential and/or oxidative phosphorylation; and c) the toxic potency of parabens to hepatocytes or mitochondria depends on the relative elongation of alkyl side-chains esterified to the carboxyl

  14. Some biochemical observations on gluconeogenesis from propionate in hepatocytes isolated from normal and biotin-deficients rats.

    PubMed

    Djabal, A; Mangeot, M; Cherruau, B; Lemonnier, A

    1985-01-01

    Propionate and pyruvate added to isolated normal and biotin-deficient adult rat hepatocytes increase the production of glucose. This production decreases about 30% on biotin deficiency. Malonate inhibits gluconeogenesis from propionate showing the metabolic transformation of propionyl-CoA via the Krebs cycle. Neither glucagon nor dibutyryl-cyclic AMP significantly stimulate gluconeogenesis.

  15. Efficient amelioration of carbon tetrachloride induced toxicity in isolated rat hepatocytes by Syzygium cumini Skeels extract.

    PubMed

    Veigas, Jyothi M; Shrivasthava, Richa; Neelwarne, Bhagyalakshmi

    2008-09-01

    Syzygium cumini, Indian black plum or Java plum, is a rich source for anthocyanins (230 mg/100g DW) showing high antioxidant activity in vitro. In the following study it is further demonstrated that S. cumini peel extract rich in anthocyanins (SCA) offers considerable protection against carbon tetrachloride (CCl(4))-induced damage in rat hepatocytes. SCA itself being non-toxic to primary rat hepatocytes at concentrations ranging from 50 to 500 ppm, was found to suppress CCl(4)-induced LDH leakage by 54% at 50ppm, thereby improving the cell viability by 39%. The SCA significantly reversed the CCl(4) induced changes in cellular glutathione (GSH) level, lipid peroxidation and activity of the antioxidant enzyme glutathione peroxidase. Exposure of hepatocytes to SCA after CCl(4) treatment was found to elevate GSH and GPx activities by 2-folds, whereas the activities of catalase and superoxide dismutase were not significantly affected. The fruit pulp extract (SPE) was less effective in offering protection to rat hepatocytes, particularly in terms of total GSH content and a consequent increase in lipid peroxidation although the higher GPx activity suggests the probable involvement of GSH as a substrate for GPx. These observations suggest that the fruit peel extract of S. cumini, is largely responsible for the reversal of CCl(4)-induced oxidative damage in rat hepatocytes. Both peel and pulp extract appear to offer protection to rat hepatocytes through GPx along with other biological pathways independent of catalase and superoxide dismutase.

  16. Metabolism of symmetrical N-nitroso-di-n-alkylamines in isolated hepatocytes from control and EtOH treated rats

    SciTech Connect

    Gorsky, L.D.; Hollenberg, P.F.

    1986-05-01

    N-nitrosodialkylamines are known to be carcinogenic in laboratory animals giving rise to hepatocellular carcinomas and a variety of other neoplasms in different organs of the body. The liver is the main target organ for tumor production in the rat; however, the biological effects of the C/sub 1/ (N-nitrosodimethylamine) through C/sub 4/ (N-nitrosodi-n-butylamine) series of symmetrical N-nitrosodialkylamines are dependent on the route of administration, the absolute dose, and the dosing regimen. For those reasons the authors have chosen to study the metabolism of N-nitrosodialkylamines in isolated rat hepatocytes. These studies were designed to compare the metabolism of the N-nitrosodialkylamines in hepatocytes isolated from control and EtOH-treated rates. For instance, after incubation of /sup 14/C-diethylnitrosamine (15 ..mu..M) with hepatocytes from control animals for 3 hours, 9% of the dose was metabolized to CO/sub 2/, 2% was found associated with the acid precipitable fraction and no radioactivity was associated with the DNA. In contrast to these results, however, in similar studies using hepatocytes isolated from EtOH-treated rats, 25% of the dose was metabolized to CO/sub 2/, 2.9% was incorporated into the acid precipitable fraction and 0.8% was associated with the DNA. These data demonstrate that EtOH treatment of rats results in increased hepatocellular metabolism of diethyl nitrosamine including the generation of DNA alkylating intermediates.

  17. Cytoprotective Effects of Melatonin Against Amitriptyline-Induced Toxicity in Isolated Rat Hepatocytes

    PubMed Central

    Taziki, Shohreh; Sattari, Mohammad Reza; Dastmalchi, Siavoush; Eghbal, Mohammad Ali

    2015-01-01

    Purpose: Amitriptyline, one of the commonly used tricyclic antidepressants, caused rare but severe hepatotoxicity in patients who received it continuously. Previous findings showed that the intermediate metabolites of amitriptyline produced by CYP450 are involved in hepatic injury. Melatonin is an antiaging and antioxidant hormone synthesized from pineal gland. The aim of present study was to evaluate the protective role of melatonin in an in vitro model of isolated rat hepatocytes. Methods: Markers such as cell viability, reactive oxygen species formation, lipid peroxidation, mitochondrial membrane potential, and hepatocytes glutathione content were evaluated every 60 minutes for 180 minutes. Results: Present results indicated that administration of 1mM of melatonin effectively reduced the cell death, ROS formation and lipid peroxidation, mitochondrial membrane potential collapse, and reduced cellular glutathione content caused by amitriptyline. Conclusion: Our results indicated that melatonin is an effective antioxidant in preventing amitriptyline-induced hepatotoxicity. We recommend further in vivo animal and clinical trial studies on the hepatoprotective effects of melatonin in patients receiving amitriptyline. PMID:26504754

  18. The metabolism of aflatoxin B1 by hepatocytes isolated from rats following the in vivo administration of some xenobiotics

    SciTech Connect

    Metcalfe, S.A.; Neal, G.E.

    1983-01-01

    Isolated rat hepatocytes, an intact cellular system capable of performing phase I and phase II metabolism, have been used to investigate metabolism of aflatoxin B1. These cells were found to metabolise (/sup 14/C)aflatoxin B1 to aflatoxins M1 and Q1, and to radiolabelled polar material, presumably conjugates, as analysed by h.p.l.c., t.l.c. and radioactive determination. In vivo administration of the mixed function oxidase inducers, phenobarbitone and 3-methylcholanthrene, resulted in enhanced hepatocyte phase I (microsomal) metabolism of aflatoxin B1. In contrast to metabolism of AFB1 by in vitro subcellular systems increased production of polar material (conjugated metabolites) derived from (/sup 14/C)aflatoxin B1 was also detected in hepatocytes isolated from these pretreated animals. Formation of aflatoxin Q1 by isolated hepatocytes appeared to be mediated by cytochrome P450-linked enzymes whereas cytochrome P448-linked enzymes were apparently involved in aflatoxin M1 production. Chronic feeding of aflatoxin B1 to rats enhanced hepatocyte production of conjugated material only and did not elevate cellular cytochrome P450 levels, thus suggesting that aflatoxin B1 is not an inducer of its own primary metabolism.

  19. The biosynthesis of ascorbate protects isolated rat hepatocytes from cumene hydroperoxide-mediated oxidative stress.

    PubMed

    Chan, Tom S; Shangari, Nandita; Wilson, John X; Chan, Helen; Butterworth, Roger F; O'Brien, Peter J

    2005-04-01

    Most animals synthesize ascorbate. It is an essential enzymatic cofactor for the synthesis of a variety of biological molecules and also a powerful antioxidant. There is, however, little direct evidence supporting an antioxidant role for endogenously produced ascorbate. Recently, we demonstrated that incubation of rat hepatocytes with 1-bromoheptane or phorone simultaneously depleted glutathione (GSH) and triggered rapid ascorbate synthesis. The present study investigates the hypothesis that endogenous ascorbate synthesis can confer protection against oxidative stress. Rat and guinea pig hepatocytes were depleted of GSH with 1-bromoheptane and subsequently treated with the oxidative stressor cumene hydroperoxide (CHP) in the presence or absence of the ascorbate synthesis inhibitor sorbinil. In rat hepatocytes, ascorbate content increased linearly (from 15.1 to 35.8 nmol/10(6) cells) over a 105-min incubation. Prior depletion of GSH increased CHP-induced cellular reactive oxygen species (ROS) production, lipid peroxidation, and cell death in rat and guinea pig hepatocytes. Inhibiting ascorbate synthesis, however, further elevated ROS production (2-fold), lipid peroxidation (1.5-fold), and cell death (2-fold) in rat hepatocytes only. This is the first time that endogenous ascorbate synthesis has been shown to decrease cellular susceptibility to oxidative stress. Protection by endogenously produced ascorbate may therefore need to be addressed when extrapolating data to humans from experiments using rodents capable of synthesizing ascorbate.

  20. Uncoupling effect of polyunsaturated fatty acid deficiency in isolated rat hepatocytes:effect on glycerol metabolism.

    PubMed Central

    Piquet, M A; Fontaine, E; Sibille, B; Filippi, C; Keriel, C; Leverve, X M

    1996-01-01

    The effects of a 4-week deficiency in polyunsaturated fatty acids (PUFA) in isolated rat hepatocytes have been investigated for oxidative phosphorylation and fatty acid, dihydroxyacetone (DHA) or glycerol metabolism. Oxygen uptake was significantly increased (by 20%) with or without fatty acid addition (octanoate or oleate) in the PUFA-deficient group compared with controls. The effect persisted after oligomycin addition but not after that of potassium cyanide, leading to the conclusion that, in these intact cells, the mitochondria were uncoupled. The PUFA-deficient group exhibited a significant decrease in the cytosolic ATP/ADP ratio, whereas the mitochondrial ratio was not affected. PUFA deficiency led to a 16% decrease in DHA metabolism owing to a 34% decrease in glycerol kinase activity; the significant decrease in the ATP/ADP ratio was accompanied by an increase in the fractional glycolytic flux. In contrast, glycerol metabolism was significantly enhanced in the PUFA-deficient group. The role of the glycerol 3-phosphate dehydrogenase step in this stimulation was evidenced in hepatocytes perifused with glycerol and octanoate in the presence of increased concentrations of 2,4-dinitrophenol (Dnp): uncoupling with Dnp led to an enhancement of glycerol metabolism, as found in PUFA deficiency, although it was more pronounced than in controls. The matrix/cytosol gradients for redox potential and ATP/ADP ratio were lower in cells from PUFA-deficient rats, suggesting a decreased mitochondrial membrane potential in accordance with the uncoupling effect. Moreover, a doubling of the mitochondrial glycerol 3-phosphate dehydrogenase activity in the PUFA-deficient group compared with controls led us to conclude that the activation of glycerol metabolism is the consequence of two mitochondrial effects: uncoupling and an increase in glycerol 3-phosphate dehydrogenase activity. PMID:8760348

  1. Contribution of several amino acids and lactate to gluconeogenesis in hepatocytes isolated from rats fed various diets

    SciTech Connect

    Kaloyianni, M.; Freedland, R.A. )

    1990-01-01

    The contribution under various nutritional regimens of several amino acids and lactate to gluconeogenesis was estimated by measuring the glucose formation from 14C-labeled substrates. Isolated rat hepatocytes were incubated for 60 min in a Krebs-Ringer bicarbonate buffer pH 7.4 containing lactate, pyruvate and all the amino acids at concentrations similar to their physiological levels found in rat plasma, with one precursor labeled in each flask. In all conditions, lactate was the major glucose precursor, providing over 60% of the glucose formed. Glutamine and alanine were the major amino acid precursors of glucose, contributing 9.8% and 10.6% of the glucose formed, respectively, in hepatocytes isolated from starved rats. Serine, glycine and threonine also contributed to gluconeogenesis in the starved liver cells at 2.6, 2.1 and 3.8%, respectively, of the glucose formed. The rate of glucose formation from the isolated hepatocytes of the starved rats and those fed either high protein or high fat was higher than that from rats fed a nonpurified diet.

  2. Effect of melatonin administration on parameters related to oxidative damage in hepatocytes isolated from old Wistar rats.

    PubMed

    Castillo, Carmen; Salazar, Veronica; Ariznavarreta, Carmen; Vara, Elena; Tresguerres, Jesus A F

    2005-05-01

    Aging induces changes in several organs and tissues, such as the liver, and this process might be due to oxidative damage caused by free radicals and inflammatory mediators. Melatonin is a secretory product with well-known antioxidant properties. The aim of this study was to investigate the effect of melatonin administration on age-induced alterations in hepatocytes. Twenty-two-month old male Wistar rats were treated with oral melatonin for 10 wk. At the end of the treatment, hepatocytes were isolated and cultured, and different parameters were measured in both cells and medium. Aging induced a significant increase in lipid peroxidation, nitric oxide, carbon monoxide and cyclic guanosyl-monophosphate, as well as a reduction in adenosine triphosphate content and phosphatidylcholine synthesis when compared to young animals. Melatonin administration significantly ameliorated all these age-related changes in males. Melatonin administration seems to exert beneficial effects against age-induced changes in hepatocytes.

  3. Involvement of oxidative stress and mitochondrial/lysosomal cross-talk in olanzapine cytotoxicity in freshly isolated rat hepatocytes.

    PubMed

    Eftekhari, Aziz; Azarmi, Yadollah; Parvizpur, Alireza; Eghbal, Mohammad Ali

    2016-01-01

    1. Olanzapine (OLZ) is a widely used atypical antipsychotic agent for the treatment of schizophrenia and other disorders. Serious hepatotoxicity and elevated liver enzymes have been reported in patients receiving OLZ. However, the cellular and molecular mechanisms of the OLZ hepatotoxicity are unknown. 2. In this study, the cytotoxic effect of OLZ on freshly isolated rat hepatocytes was assessed. Our results showed that the cytotoxicity of OLZ in hepatocytes is mediated by overproduction of reactive oxygen species (ROS), mitochondrial potential collapse, lysosomal membrane leakiness, GSH depletion and lipid peroxidation preceding cell lysis. All the aforementioned OLZ-induced cellular events were significantly (p < 0.05) prevented by ROS scavengers, antioxidants, endocytosis inhibitors and adenosine triphosphate generators. Also, the present results demonstrated that CYP450 is involved in OLZ-induced oxidative stress and cytotoxicity mechanism. 3. It is concluded that OLZ hepatotoxicity is associated with both mitochondrial/lysosomal involvement following the initiation of oxidative stress in hepatocytes.

  4. Stimulation by glucose of gluconeogenesis in hepatocytes isolated from starved rats.

    PubMed Central

    Rigoulet, M; Leverve, X M; Plomp, P J; Meijer, A J

    1987-01-01

    Control properties of the gluconeogenic pathway in hepatocytes isolated from starved rats were studied in the presence of glucose. The following observations were made. (1) Glucose stimulated the rate of glucose production from 20 mM-glycerol, from a mixture of 20 mM-lactate and 2 mM-pyruvate, or from pyruvate alone; no stimulation was observed with 20 mM-alanine or 20 mM-dihydroxyacetone. Maximal stimulation was obtained between 2 and 5 mM-glucose, depending on the conditions. At concentrations above 6 mM, gluconeogenesis declined again, so that at 10 mM-glucose the glucose production rate became equal to that in its absence. (2) With glycerol, stimulation of gluconeogenesis by glucose was accompanied by oxidation of cytosolic NADH and reduction of mitochondrial NAD+ and was insensitive to the transaminase inhibitor amino-oxyacetate; this indicated that glucose accelerated the rate of transport of cytosolic reducing equivalents to the mitochondria via the glycerol 1-phosphate shuttle. (3) With lactate plus pyruvate (10:1) as substrates, stimulation of gluconeogenesis by glucose was almost additive to that obtained with glucagon. From an analysis of the effect of glucose on the curves relating gluconeogenic flux and the steady-state intracellular concentrations of gluconeogenic intermediates under various conditions, in the absence and presence of glucagon, it was concluded that addition of glucose stimulated both phosphoenolpyruvate carboxykinase and pyruvate carboxylase activity. PMID:3663184

  5. Fatty acid and cholesterol synthesis from specifically labeled leucine by isolated rat hepatocytes.

    PubMed

    Mathias, M M; Sullivan, A C; Hamilton, J G

    1981-10-01

    Hepatocytes isolated from female rats meal-fed a high-glucose diet were incubated in Krebs-Henseleit bicarbonate medium containing 16.5 mM glucose, 3H2O, and 14C-labeled amino acids (-)-Hydroxycitrate depressed the incorporation of 3H2O and [14C] alanine into fatty acids and cholesterol. Incorporation of [U-14C]leucine into lipids was not affected but incorporation of 3H2O into lipids was decreased significantly by (-)-hydroxycitrate. (-)-Hydroxycitrate depressed the incorporation of radioactivity from [2-14C]leucine into fatty acids and cholesterol by 61 and 38%, respectively, and stimulated the incorporation of radioactivity from [4,5-3H]leucine 35 and 28%. As [2-14C]leucine labels the acetyl-CoA pool and [4,5-3H]leucine labels the acetoacetate pool, it was concluded that mitochondrial 3-hydroxy-3-methylglutaryl-CoA is not incorporated intact into cholesterol, and that acetoacetate can be activated effectively in the liver cytosol for support of cholesterol and fatty acid synthesis.

  6. Regulation of amino acid transport in isolated rat hepatocytes during development

    SciTech Connect

    Leoni, S.; Spagnuolo, S.; Dini, L.; Devirgiliis, L.C.

    1987-01-01

    The effect of amino acid depletion or supplementation and the effect of glucagon and insulin on the amino acid transport mediated by system A were investigated by determining the uptake of either 2-amino (1-/sup 14/C)isobutyric acid (AIB) or N-methyl 2-amino (1-/sup 14/C)isobutyric acid (MeAIB) in rat hepatocytes, freshly isolated at different stages of pre- and postnatal development. The data obtained show that the Na/sup +/ -dependent uptake was higher at the earliest developmental stages, and steadily decreased until the adult level. The hormones increased AIB and MeAIB uptake enhancing the V/sub max/, while the K/sub m/ was unchanged. This effect was evident in cells from adult and 18-20-day-old fetuses, while no response was present before the 18th day of fetal life and in the prenatal period. Actinomycin D or cycloheximide abolished this hormone-dependent increase. A decrease in AIB and MeAIB transport after incubation in an amino acid-rich medium was demonstrated at all ages tested, but was particularly evident in the prenatal life. The increase in the activity of the system following amino acid starvation was shown to be mostly dependent from de novo protein synthesis in the fetal life; on the contrary in the adult the increase appeared to be more linked to the release from transinhibition of the transport.

  7. Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes

    SciTech Connect

    Frost, S.J.; Raja, R.H.; Weigel, P.H. )

    1990-11-13

    125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 degrees C indicates a Kd = 1.8 x 10(-7) M and 1.3 x 10(6) molecules of HA (Mr approximately 30,000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to approximately 6.2 x 10(5). Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength above causes an approximately 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4{degrees}C increased greater than 10-fold at pH 5.0 as compared to pH 7.

  8. Effects of tert-butyl hydroperoxide on Ca(2+) ATPase activity in isolated rat hepatocytes and its reversal by antioxidants.

    PubMed

    Singh, Sangram; Agarwal, Richa; Jamal, Farrukh; Mehrotra, Sudhir; Singh, Rakesh

    2012-09-01

    Calcium ions play an importantrole in various physiological processes such as nerve impulse transmission, muscle contraction, hormone action, blood clotting. They ions act as an intracellular second messenger, relaying information within cells to regulate their activity. To understand the mechanism of hepatotoxicity of t-BHP, studies were carried out using freshly isolated rat hepatocytes. The effect of t-BHP on Ca(2+) accumulation and Ca(2+) uptake by rat hepatocytes was monitored using 45Ca(2+). It caused decrease in 15% accumulation of 45Ca(2+) in comparison to the control group. t-BHP also significantly decreased the Ca(2+) ATPase activity in isolated hepatocytes .This decrease in Ca(2+) ATPase activity by t-BHP was reversed 40% by naturally occurring antioxidant glutathione (GSH) and 20% by the synthetic antioxidant butylated hydroxy toluene (BHT). These results indicate that the hepatotoxic action of t-BHP involves oxidative stress as evident by the protection accorded by various antioxidants employed in the study as well as impairment of intracellular calcium homeostasis which can lead to liver cell injury.

  9. Insulin resistance in uremia. Characterization of lipid metabolism in freshly isolated and primary cultures of hepatocytes from chronic uremic rats.

    PubMed Central

    Caro, J F; Lanza-Jacoby, S

    1983-01-01

    We have studied the mechanism(s) of hyperlipidemia and liver insulin sensitivity in a rat model of severe chronic uremia (U). Basal lipid synthesis was decreased in freshly isolated hepatocytes from U when compared with sham-operated ad lib.-fed controls (alfC). Basal lipid synthesis in pair-fed controls (pfC) was in between U and alfC. Similarly, the activity of liver acetyl CoA carboxylase, fatty acid synthetase, citrate cleavage enzyme, malate dehydrogenase, and glucose-6-phosphate dehydrogenase was diminished in U. Muscle and adipose tissue lipoprotein lipase was also decreased. Insulin stimulated lipid synthesis in freshly isolated hepatocytes from alfC. Hepatocytes from U and pfC were resistant to this effect of insulin. To ascertain if the insulin resistance in U was due to starvation (chow intake 50% of alfC) or to uremia itself, the U and pfC were intragastrically fed an isocaloric diet via a Holter pump the last week of the experimental period. Hepatocytes from orally fed U and pfC were also cultured for 24 h in serum-free medium. While freshly isolated and cultured U hepatocytes remained insulin resistant, those from pfC normalized, in vivo and in vitro, when they were provided with enough nutrients. Conclusions: (a) Hyperlipidemia in uremia is not due to increased synthesis, but to defect(s) in clearance. (b) Insulin does not stimulate lipid synthesis in uremia. This finding, along with our recent demonstration that insulin binding and internalization are not decreased in the uremic liver, suggests that a post-binding defect(s) in the liver plays an important role in the mechanism(s) of insulin resistance in uremia. (c) Cultured hepatocytes from uremic rats remain insulin resistant. This quality renders these cells useful in studying the postinsulin binding events responsible for the insulin-resistant state in the absence of complicating hormonal and substrate changes that occur in vivo. PMID:6350367

  10. Cytoprotective Effects of Organosulfur Compounds against Methimazole Induced Toxicity in Isolated Rat Hepatocytes

    PubMed Central

    Heidari, Reza; Babaei, Hossein; Eghbal, Mohammad Ali

    2013-01-01

    Purpose: Methimazole is a drug widely used in hyperthyroidism. However, life threatening hepatotoxicity has been associated with its clinical use. No protective agent has been found to be effective against methimazole induced hepatotoxicity yet. Hence, the capacity of organosulfur compounds to protect rat hepatocytes against cytotoxic effects of methimazole and its proposed toxic metabolite, N-methylthiourea was evaluated. Methods: Hepatocytes were prepared by the method of collagenase enzyme perfusion via portal vein. Cells were treated with different concentrations of methimazole, N methylthiourea, and organosulfur chemicals. Cell death, protein carbonylation, reactive oxygen species formation, lipid peroxidation, and mitochondrial depolarization were assessed as toxicity markers and the role of organosulfurs administration on them was investigated. Results: Methimazole caused a decrease in cellular glutathione content, mitochondrial membrane potential (ΔΨm) collapse, and protein carbonylation. In addition, an increase in reactive oxygen species (ROS) formation and lipid peroxidation was observed. Treating hepatocytes with N methylthiourea caused a reduction in hepatocytes glutathione reservoirs and an elevation in carbonylated proteins, but no significant ROS formation, lipid peroxidation, or mitochondrial depolarization was observed. N-acetyl cysteine, allylmercaptan, and diallyldisulfide attenuated cell death and prevented ROS formation and lipid peroxidation caused by methimazole. Furthermore, organosulfur compounds diminished methimazole induced mitochondrial damage and reduced the carbonylated proteins. In addition, these chemicals showed protective effects against cell death and protein carbonylation induced by methimazole metabolite. Conclusion: Organosulfur chemicals extend their protective effects against methimazole-induced toxicity by attenuating oxidative stress caused by this drug and preventing the adverse effects of methimazole and/or its

  11. Isolation and characterization of multivesicular bodies from rat hepatocytes: an organelle distinct from secretory vesicles of the Golgi apparatus

    PubMed Central

    1985-01-01

    Hepatocytes of estradiol-treated rats, which express many low density lipoprotein receptors, rapidly accumulate intravenously injected low density lipoprotein in multivesicular bodies (MVBs). We have isolated MVBs and Golgi apparatus fractions from livers of estradiol-treated rats. MVB fractions were composed mainly of large vesicles, approximately 0.55 micron diam, filled with remnantlike very low density lipoproteins, known to be taken up into hepatocytes by receptor- mediated endocytosis. MVBs also contained numerous small vesicles, 0.05- 0.07 micron in diameter, and had two types of appendages: one fingerlike and electron dense and the other saclike and electron lucent. MVBs contained little galactosyltransferase or arylsulfatase activity, and content lipoproteins were largely intact. Very low density lipoproteins from Golgi fractions, which are derived to a large extent from secretory vesicles, were larger than those of MVB fractions and contained newly synthesized triglycerides. Membranes of MVBs contained much more cholesterol and less protein than did Golgi membranes. We conclude that two distinct lipoprotein-filled organelles are located in the bile canalicular pole of hepatocytes. MVBs, a major prelysosomal organelle of low density in the endocytic pathway, contain remnants of triglyceride-rich lipoproteins, whereas secretory vesicles of the Golgi apparatus contain nascent very low density lipoproteins. PMID:3988801

  12. Evaluating the Role of Drug Metabolism and Reactive Intermediates in Trazodone-Induced Cytotoxicity toward Freshly-Isolated Rat Hepatocytes.

    PubMed

    Najibi, A; Heidari, R; Zarifi, J; Jamshidzadeh, A; Firoozabadi, N; Niknahad, H

    2016-11-01

    Background: Trazodone is an antidepressant agent widely administered for the treatment of depressive disorders. On the other hand, several cases of hepatic injury have been reported after Trazodone administration. Although the precise mechanism(s) of trazodone-induced liver injury is not known, some investigations proposed the role of reactive intermediates in this complication. This study was designed to investigate the role of reactive metabolites in hepatocytes injury induced by trazodone. Methods: Isolated rat hepatocytes were prepared by the method of collagenase enzyme perfusion via the portal vein. Cells were treated with trazodone, its cytotoxic metabolite, and different enzyme inhibitors and cytoprotective agents. Results: It was found that trazodone was toxic towards hepatocytes and caused 50% cell death after 2 h of incubation at a dose of 450 µM. The trazodone postulated reactive metabolite; m-chlorophenyl piperazine (m-CPP) was less toxic and caused 50% cell death at a dose of 750 µM at a similar time period. Cellular glutathione (GSH) depletion and lipid peroxidation were detected when hepatocytes were treated with trazodone and/or m-CPP. Depleting hepatocytes GSH beforehand, increased cytotoxicity of both trazodone and m-CPP. Troleandomycin as the CYP3A4 inhibitor prevented cytotoxicity of trazodone but slightly affected m-CPP-induced cell injury. Inhibition of CYP2D6 by quinidine and cimetidine increased the cytotoxicity of both trazodone and m-CPP. Antioxidants and ATP suppliers slightly prevented cytotoxicity of trazodone and m-CPP. Conclusion: As inhibitors of CYP3A4 and 2D6 affected trazodone cytotoxicity, it is suggested that trazodone -induced cytotoxicity, at least in part, is mediated by its reactive metabolites.

  13. Cytotoxic effects of 3,4-methylenedioxy-N-alkylamphetamines, MDMA and its analogues, on isolated rat hepatocytes.

    PubMed

    Nakagawa, Yoshio; Suzuki, Toshinari; Tayama, Sumiko; Ishii, Hidemi; Ogata, Akio

    2009-01-01

    The amphetamine-derived designer drugs have been illegally used worldwide as recreational drugs, some of which are known to be hepatotoxic in humans. To compare their cytotoxic effects, 3,4-methylenedioxy-N-methamphetamine (MDMA) and its related analogues, N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB), 3,4-(methylenedioxyphenyl)-2-butanamine (BDB) and 2-methylamino-1-(3,4-methylenedioxyphenyl)-propane-1-one (methylone) were studied in freshly isolated rat hepatocytes. MBDB caused not only concentration (0-4.0 mM)- and time (0-2 h)-dependent cell death accompanied by the formation of cell blebs, and the loss of cellular ATP and adenine nucleotide pools, and reduced glutathione levels, but also the accumulation of oxidized glutathione. Of the other analogues examined, the cytotoxicity of MBDB and BDB was greater than that of MDMA and methylone, suggesting that hepatotoxicity is generally induced by these drugs. In addition, DNA damage and the induction of reactive oxygen species were greater after the incubation of hepatocytes with MBDB (2 and 4 mM) than after that with MDMA. In isolated liver mitochondria, MBDB/BDB resulted in a greater increase in the rate of state 4 oxygen consumption than did MDMA/methylone, indicating an uncoupling effect and a decrease in the rate of state 3 oxygen consumption in a concentration dependent manner. Furthermore, MBDB resulted in mitochondrial swelling dependent on the mitochondrial permeability transition (MPT); the effect of MDMA was less than that of MBDB. Taken collectively, these results suggest that (1) the onset of cytotoxicity caused by designer drugs such as MBDB and MDMA is linked to mitochondrial failure dependent upon the induction of the MPT accompanied by mitochondrial depolarization and depletion of ATP through uncoupling of oxidative phosphorylation in rat hepatocytes, and (2) MBDB and MDMA elicit DNA damage, suggesting that nuclei as well as mitochondria are target sites of these compounds.

  14. Metabolic Rate Constants for Hydroquinone in F344 Rat and Human Liver Isolated Hepatocytes: Application to a PBPK model.

    SciTech Connect

    Poet, Torka S.; Wu, Hong; English, J C.; Corley, Rick A.

    2004-11-15

    Hydroquinone (HQ) is an important industrial chemical that also occurs naturally in foods and in the leaves and bark of a number of plant species. Exposure of laboratory animals to HQ may result in a species-, sex-, and strain-specific nephrotoxicity. The sensitivity of male F344 vs. female F344 and Sprague-Dawley rats or B6C3F1 mice appears to be related to differences in the rates of formation and further metabolism of key nephrotoxic metabolites. Metabolic rate constants for the conversion of HQ through several metabolic steps to the mono-glutathione conjugate and subsequent detoxification via mercapturic acid were measured in suspension cultures of hepatocytes isolated from male F344 rats and humans. An in vitro mathematic kinetic model was used to analyze each metabolic step by simultaneously fitting the disappearance of each substrate and the appearance of subsequent metabolites. An iterative, nested approach was used whereby downstream metabolites were considered first and the model was constrained by the requirement that rate constants determined during analysis of individual metabolic steps must also satisfy the complete, integrated metabolism scheme, including competitive pathways. The results from this study indicated that the overall capacity for metabolism of HQ and its mono-glutathione conjugate is greater in hepatocytes from humans than those isolated from rats, suggesting a greater capacity for detoxification of the glutathione conjugates. Metabolic rate constants were applied to an existing physiologically based pharmacokinetic model and the model was used to predict total glutathione metabolites produced in the liver. The results showed that body burdens of these metabolites will be much higher in rats than humans.

  15. Cytoprotective effects of silafibrate, a newly-synthesised siliconated derivative of clofibrate, against acetaminophen-induced toxicity in isolated rat hepatocytes.

    PubMed

    Nafisi, Sara; Heidari, Reza; Ghaffarzadeh, Mohammad; Ziaee, Mojtaba; Hamzeiy, Hossein; Garjani, Alireza; Eghbal, Mohammad Ali

    2014-06-01

    Acetaminophen (N-acetyl para amino phenol, APAP) is a widely used antipyretic and analgesic drug responsible for various drug-induced liver injuries. This study evaluated APAP-induced toxicity in isolated rat hepatocytes alongside the protective effects of silafibrate and N-acetyl cysteine (NAC). Hepatocytes were isolated from male Sprague-Dawley rats by collagenase enzyme perfusion via the portal vein. This technique is based on liver perfusion with collagenase after removing calcium ions (Ca2+) with a chelator. Cells were treated with different concentrations of APAP, silafibrate, and NAC. Cell death, reactive oxygen species (ROS) formation, lipid peroxidation, and mitochondrial depolarisation were measured as toxicity markers. ROS formation and lipid peroxidation occurred after APAP administration to rat hepatocytes. APAP caused mitochondrial depolarisation in isolated cells. Administration of silafibrate (200 μmol L-1) and/or NAC (200 μmol L-1) reduced the ROS formation, lipid peroxidation, and mitochondrial depolarisation caused by APAP. Cytotoxicity induced by APAP in rat hepatocytes was mediated by oxidative stress. In addition, APAP seemed to target cellular mitochondria during hepatocyte damage. The protective properties of silafibrate and/or NAC against APAP‑induced hepatic injury may have involved the induction of antioxidant enzymes, protection against oxidative stress and inflammatory responses, and alteration in cellular glutathione content.

  16. Proliferative and oxidative response of hepatocytes (Hep) and hepatic stellate cells (HSC) isolated from rats exposed to ketogenic diet.

    PubMed

    Wójcik, M; Wessely-Szponder, J; Kosior-Korzecka, U

    2014-01-01

    Ketogenic diet (KD) is considered in the context of its anti-epileptic effects, but its influence on liver dysfunction has not been elucidated yet. The study was aimed to investigate the activity of hepatocytes (Hep) and hepatic stellate cells (HSC) isolated from rats fed with KD, in respect of NO and superoxide generation by these cells as well as their proliferative activity in vitro. We also sought to characterize the plasma FFA profiles in control and ketogenic rats. Hep and HSC were isolated by the collagenase perfusion method and separated by the Percoll gradient centrifugation. After the 4th, 8th and 12th day of incubation, the media were collected for further analysis. NO generation increased within the time of incubation both in Hep and HSC isolated from KD-rats. In HSC group NO production raised significantly from 2.65 ± 0.07 μM/10(6) cells on 4th day of incubation to 5.49 ± 1.2 μM/10(6) cells on 12th day of incubation. In respect to O2⁻· generation experimental Hep and HSC provide considerably higher quantities of this free radical until 12th day of incubation (2.5 ± 0.07 and 3.2 ± 0.3 nM/10(6) cells, respectively). Although KD exerts anti-proliferative effect on hepatocytes, in respect to HSC it intensifies their proliferative activity. Furthermore, as we estimated on the basis of NO and O2⁻. generation both Hep and HSC exposed to KD are the source of free radicals.

  17. Epinephrine effects on mitochondrial Krebs cycle are not mediated by typical adrenergic receptors in isolated rat hepatocytes

    SciTech Connect

    Mohan, C.; Memon, R.A.; Bessman, S.P. )

    1990-02-26

    Oxidation of 2,3-{sup 14}C succinate (suc) carbons in the intra-mitochondrial Krebs cycle was used as a probe to investigate the effects of epinephrine (epi) on isolated rat hepatocytes. Hepatocytes were incubated at 30{degrees}C in Krebs-Henseleit bicarbonate buffer, pH 7.4, with 0.5 mM concentration of each of the 20 natural amino acids, 0.5 mm concentration of each of the 20 natural amino acids, 2,3-{sup 14}C suc and epi (10 uM), phenylephrine (pheni) (10uM) or isoproterenol (10 uM). Epi and phepi caused a significant increase in {sup 14}CO{sub 2} formation from 2,3-{sup 14}C suc, however, phentolamine, an {infinity}-antagonist, failed to inhibit this increased oxidation of suc carbons. Isoproterenol had no effect on hepatocyte metabolism and propranolol, a {beta}-antagonist, failed to cause any reduction in basal or epi stimulated oxidation of 2,3-{sup 14}C carbons. Unlike insulin, neither epi nor phepi had any significant effect on the anabolic utilization of suc carbons for protein or lipid synthesis. Anabolic channeling of Krebs cycle intermediates into amino acids was reduced by epi treatment of hepatocytes. Although epi treatment can enhance the oxidation of substrate through the Krebs cycle reactions, only insulin is capable of channeling these substrates into anabolic reactions. Data presented also suggest that epi effects on mitochondrial Krebs cycle oxidation are mediated through an atypical {infinity}-adrenergic receptor which is unresponsive to inhibition by non-selective {infinity}-antagonists.

  18. Activation of factor X by rat hepatocytes

    SciTech Connect

    Willingham, A.K.; Matschiner, J.T.

    1986-05-01

    Synthesis and secretion of blood coagulation factor X was studied in hepatocytes prepared by perfusion of rat livers with collagenase. Hepatocytes were incubated in the presence of vitamin K and /sup 3/H-leucine for up to 4h at 37/sup 0/C. Factor X was isolated from the incubation medium by immunochemical techniques and analyzed by SDS-PAGE. The recovered /sup 3/H-labeled proteins migrated, after reduction of disulfides, as two polypeptide chains with apparent molecular weights (M/sub r/) of approximately 42,000 and 22,000 representing the heavy and light chains of factor X respectively. The apparent M/sub r/ of the heavy chain was about 10,000 daltons lighter than seen with the heavy chain of factor X isolated from rat plasma and was more characteristic of the heavy chain of factor Xa. When the levels of factor X secreted by hepatocytes were determined by clotting assays, activity was present as factor Xa. Also, when purified plasma factor X was added to incubations of hepatocytes (>95% parenchymal cells) the added factor X was rapidly converted to factor Xa. Plasma membranes prepared from isolated hepatocytes or from liver homogenates contained an enzyme that converted factor X to factor Xa in a calcium dependent reaction. The physiological significance of a factor X activating enzyme on hepatocyte plasma membranes is not clear.

  19. Fatty acid and amino acid modulation of glucose cycling in isolated rat hepatocytes.

    PubMed Central

    Gustafson, L A; Neeft, M; Reijngoud, D J; Kuipers, F; Sauerwein, H P; Romijn, J A; Herling, A W; Burger, H J; Meijer, A J

    2001-01-01

    We studied the influence of glucose/glucose 6-phosphate cycling on glycogen deposition from glucose in fasted-rat hepatocytes using S4048 and CP320626, specific inhibitors of glucose-6-phosphate translocase and glycogen phosphorylase respectively. The effect of amino acids and oleate was also examined. The following observations were made: (1) with glucose alone, net glycogen production was low. Inhibition of glucose-6-phosphate translocase increased intracellular glucose 6-phosphate (3-fold), glycogen accumulation (5-fold) without change in active (dephosphorylated) glycogen synthase (GSa) activity, and lactate production (4-fold). With both glucose 6-phosphate translocase and glycogen phosphorylase inhibited, glycogen deposition increased 8-fold and approached reported in vivo rates of glycogen deposition during the fasted-->fed transition. Addition of a physiological mixture of amino acids in the presence of glucose increased glycogen accumulation (4-fold) through activation of GS and inhibition of glucose-6-phosphatase flux. Addition of oleate with glucose present decreased glycolytic flux and increased the flux through glucose 6-phosphatase with no change in glycogen deposition. With glucose 6-phosphate translocase inhibited by S4048, oleate increased intracellular glucose 6-phosphate (3-fold) and net glycogen production (1.5-fold), without a major change in GSa activity. It is concluded that glucose cycling in hepatocytes prevents the net accumulation of glycogen from glucose. Amino acids activate GS and inhibit flux through glucose-6-phosphatase, while oleate inhibits glycolysis and stimulates glucose-6-phosphatase flux. Variation in glucose 6-phosphate does not always result in activity changes of GSa. Activation of glucose 6-phosphatase flux by fatty acids may contribute to the increased hepatic glucose production as seen in Type 2 diabetes. PMID:11535127

  20. Mechanisms of trazodone-induced cytotoxicity and the protective effects of melatonin and/or taurine toward freshly isolated rat hepatocytes.

    PubMed

    Taziki, Shohreh; Sattari, Mohammad Reza; Eghbal, Mohammad Ali

    2013-10-01

    It has been reported that the bioactive intermediate metabolites of trazodone might cause hepatotoxicity. This study was designed to investigate the exact mechanism of hepatocellular injury induced by trazodone as well as the protective effects of taurine and/or melatonin against this toxicity. Freshly isolated rat hepatocytes were used. Trazodone was cytotoxic and caused cell death with LC50 of 300 µm within 2 h. Trazodone caused an increase in reactive oxygen species (ROS) formation, malondialdehyde accumulation, depletion of intracellular reduced glutathione (GSH), rise of oxidized glutathione disulfide (GSSG), and a decrease in mitochondrial membrane potential, which confirms the role of oxidative stress in trazodone-induced cytotoxicity. Preincubation of hepatocytes with taurine prevented ROS formation, lipid peroxidation, depletion of intracellular reduced GSH, and increase of oxidized GSSG. Taurine could also protect mitochondria against trazodone-induced toxicity. Administration of melatonin reduced the toxic effects of trazodone in isolated rat hepatocytes.

  1. Evidence for multiple pathways of sup 125 I-insulin internalization in isolated rat hepatocytes

    SciTech Connect

    Moss, A.L.

    1988-01-01

    Insulin internalization has been characterized frequently as occurring by the coated pit pathway of receptor-mediated endocytosis. The present study in rat hepatocytes demonstrates that insulin internalization is, in part, receptor-mediated, but also occurs by nonreceptor-mediated or fluid-phase endocytosis. Endocytosis was probed with four perturbations: depletion of metabolic energy with anoxia, inhibition of endocytosis with phenylarsine oxide, disruption of coated pits with hyperosmolar sucrose, and inhibition of receptor recycling or ligand-receptor dissociation with monensin. Internalization of {sup 125}I-epidermal growth factor and {sup 125}I-asialofetuin was compared to {sup 125}I-insulin internalization. Pretreatment of cells with anoxia or hyperosmolarity inhibited {sup 125}I-insulin internalization by 40%; pretreatment with phenylarsine oxide resulted in inhibition by 54%. Monensin has no effect on uptake or degradation of a high insulin concentration, but inhibited degradation of a low insulin concentration resulting in intracellular accumulation of insulin. In contract, all four perturbations inhibited {sup 125}I-asialofetuin internalization by greater than 90%. Phenylarsine oxide almost completely abolished {sup 125}I-epidermal growth factor uptake; the other perturbations caused partial inhibition. Competition studies demonstrated that insulin internalization was receptor-mediated over a wide concentration range.

  2. Zonal differences in ethanol-induced impairments in receptor-mediated endocytosis of asialoglycoproteins in isolated rat hepatocytes

    SciTech Connect

    Casey, C.A.; Kragskow, S.L.; Sorrell, M.F.; Tuma, D.J. )

    1991-02-01

    We have shown previously that ethanol-induced defects in receptor-mediated endocytosis of asialoorosomucoid occurred as early as 1 wk after ethanol feeding. This study was undertaken as an initial attempt to establish a possible role of defective receptor-mediated endocytosis in liver injury by investigating whether differences exist in the effects of ethanol on receptor-mediated endocytosis in hepatocytes isolated from different regions of the liver. Perivenule cells, present in the distal half of the liver, are thought to be more susceptible to ethanol-induced liver injury than are the periportal cells located in the proximal half of the liver acini. For these studies, we fed male Sprague-Dawley rats for 7 days with liquid diets containing either ethanol (36% of calories) or isocaloric carbohydrate. Perivenule and periportal hepatocytes were then isolated using a digitonin-collagenase perfusion method. In control animals, cells isolated from the perivenule region bound significantly more ligand than did cells from the periportal region. Amounts of ligand internalized and degraded were also greater in perivenule than in periportal cells in these animals. After ethanol feeding, cells isolated from both the perivenule and periportal regions bound significantly less ligand than their respective controls. This impairment in surface and total binding was more pronounced in perivenule than in periportal cells. Internalization and degradation of the ligand were also more adversely affected in the centrilobular region as shown by decreases of greater than 60% in perivenule cells and by only 20% to 30% in periportal cells of ethanol-fed animals compared with controls.

  3. Mitigation of statins-induced cytotoxicity and mitochondrial dysfunction by L-carnitine in freshly-isolated rat hepatocytes

    PubMed Central

    Abdoli, N.; Azarmi, Y.; Eghbal, M.A.

    2015-01-01

    Statins are widely used as anti hyperlipidemic agents. Hepatotoxicity is one of their adverse effects appearing in some patients. No protective agents have yet been developed to treat statins-induced hepatotoxicity. Different investigations have suggested L-carnitine as a hepatoprotective agent against drugs-induced toxicity. This study was designed to evaluate the effect of L-carnitine on the cytotoxic effects of statins on the freshly-isolated rat hepatocytes. Hepatocytes were isolated from male Sprague-Dawley rats by collagenase enzyme perfusion via portal vein. Cells were treated with the different concentrations of statins (simvastatin, lovastatin and atorvastatin), alone or in combination with L-carnitine. Cell death, reactive oxygen species (ROS) formation, lipid peroxidation, and mitochondrial depolarization were assessed as toxicity markers. Furthermore, the effects of statins on cellular reduced and oxidized glutathione reservoirs were evaluated. In accordance with previous studies, an elevation in ROS formation, cellular oxidized glutathione and lipid peroxidation were observed after statins administration. Moreover, a decrease in cellular reduced glutathione level and cellular mitochondrial membrane potential collapse occurred. L-carnitine co-administration decreased the intensity of aforementioned toxicity markers produced by statins treatment. This study suggests the protective role of L-carnitine against statins-induced cellular damage probably through its anti oxidative and reactive radical scavenging properties as well as its effects on sub cellular components such as mitochondria. The mechanism of L-carnitine protection may be related to its capacity to facilitate fatty acid entry into mitochondria; possibly adenosine tri-phosphate or the reducing equivalents are increased, and the toxic effects of statins toward mitochondria are encountered. PMID:26487891

  4. Comparison of the effects of metals on cellular injury and lipid peroxidation in isolated rat hepatocytes

    SciTech Connect

    Stacey, N.H.; Klaassen, C.D.

    1981-01-01

    Various mechanisms, including increases in lipid peroxidation, have been proposed to account for metal-induced cellular injury. By comparing several metals in the same cell population, it is possible to determine whether a correlation exists between ability to produce cell injury and ability to alter parameters pertaining to a particular mechanism. Of particular interest in this study was the relation between metal-induced cytotoxicity and increases in lipid peroxidation. The effects of Cr, Mn, Zn, Ni, Pb, Se, V, Fe, Cd, Hg, and Cu, at final concentrations of 1 to 1000 ..mu..M, on the viability of isolated hepatocytes were therefore examined by assessing the loss of intracellular K/sup +/ and aspartate aminotransferase (AST). Simultaneously, the ability of the metals to induce lipid peroxidation, as measured by an increase in thiobarbituric acid (TBA) reactants, was assessed. Hg and Cu required the lowest concentration to produce cellular injury, while Cd produced less dramatic changes in cell viability and Fe at 1000 ..mu..M produced only a small decrease in intracellular K/sup +/. The largest absolute increases in lipid peroxidation were found in the presence of V, followed by Fe and Hg, with Cd and Se causing the smallest increase in TBA reactants. These observations suggest that the lipid peroxidation associated with Cd and Hg is not necessarily responsible for the loss of cell viability induced by these two metals.

  5. Modeling population kinetics of free fatty acids in isolated rat hepatocytes using Markov Chain Monte Carlo.

    PubMed

    Pavan, Alessandra; Thomaseth, Karl; Valerio, Anna

    2003-01-01

    The aim of this study is the characterization, by means of mathematical models, of the activity of isolated hepatic rat cells as regards the conversion of free fatty acids (FFA) to ketone bodies (KB). A new physiologically based compartmental model of FFA metabolism is used within a context of population pharmacokinetics. This analysis is based on a hierarchical model, that differs from standard model formulations, to account for the fact that some data sets belong to the same animal but have been collected under different experimental conditions. The statistical inference problem has been addressed within a Bayesian context and solved by using Markov Chain Monte Carlo (MCMC) simulation. The results obtained in this study indicate that, although hormones epinephrine and insulin are important metabolic regulatory factors in vivo, the conversion of FFA to KB by isolated hepatic rat cells is not significantly affected by epinephrine and only little influenced by insulin. So we conclude that in vivo, the interaction of these two hormones with other compounds not considered in this study plays a fundamental role in ketogenesis. From this study it appears that mathematical models of metabolic processes can be successfully employed in population kinetic studies using MCMC methods.

  6. Cryopreservation of primarily isolated porcine hepatocytes with UW solution.

    PubMed

    Kunieda, Takemi; Maruyama, Masanobu; Okitsu, Teru; Shibata, Norikuni; Takesue, Michihiko; Totsugawa, Toshinori; Kosaka, Yoshikazu; Arata, Takashi; Kobayashi, Kazuya; Ikeda, Hideaki; Oshita, Mizuko; Nakaji, Shuhei; Ohmoto, Kenji; Yamamoto, Shinichiro; Kurabayashi, Yuzuru; Kodama, Makoto; Tanaka, Noriaki; Kobayashi, Naoya

    2003-01-01

    Development of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, requires a large amount of functional hepatocytes as needed. To achieve this development, establishing an excellent cryopreservation method of hepatocytes is an extremely important issue. Therefore, we performed a comparative review of cryoprotective effects of various cryopreservation solutions using primarily isolated porcine hepatocytes. Porcine hepatocytes were isolated with a four-step dispase and collagenase perfusion method. The obtained hepatocytes with the initial viabilities of 76%, 84%, and 96% were assigned to the following four groups for cryopreservation at -80 degrees C: Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) + 12% dimethyl sulfoxide (DMSO) (group A), University of Wisconsin (UW) solution + 12% DMSO (group B), Cell Banker 1 (group C), and Cell Banker 2 (group D). The hepatocytes in each group were thawed at 3 days, 10 days, and 5 months of cryopreservation and subjected to comparative analyses, including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer. These parameters were the most favorable in the hepatocytes cryopreserved with UW solution. Approximately 5% of thawed cryopreserved porcine hepatocytes expressed LacZ activity after lentiviral transduction. Intrasplenic transplantation of UW solution-cryopreserved hepatocytes improved the survival of rats treated with D-galactosamine. UW solution maintained the functions of cryopreserved porcine hepatocytes.

  7. Inhibition by 2,5-anhydromannitol of glycolysis in isolated rat hepatocytes and in Ehrlich ascites cells.

    PubMed Central

    Riquelme, P T; Kneer, N M; Wernette-Hammond, M E; Lardy, H A

    1985-01-01

    2,5-Anhydromannitol decreases lactate formation and 3H2O formation from [5-3H]glucose in isolated rat hepatocytes metabolizing high concentrations of glucose. The inhibition of glycolysis is accompanied by a slight decrease in the cellular content of fructose-6-P and a more substantial decrease in the cellular content of fructose-1,6-P2, with no change in the content of glucose-6-P. The 3H2O release data and changes in hexosephosphate distribution indicate possible inhibitions at phosphofructokinase-1 and phosphoglucose isomerase. 2,5-Anhydromannitol also inhibits glycolysis in Ehrlich ascites cells, but the tumor cells, unlike hepatocytes, must be treated with 2,5-anhydromannitol prior to exposure to glucose to obtain the inhibition. The decrease in 3H2O formation from [5-3H]glucose and the metabolite pattern that results from the addition of low concentrations (less than or equal to 0.25 mM) of 2,5-anhydromannitol indicate an inhibition at phosphofructokinase-1 that cannot be attributed to a decrease in the cellular content of fructose-2,6-P2. Higher concentrations (greater than or equal to 0.5 mM) of 2,5-anhydromannitol cause a substantial decrease in the cellular content of ATP that is accompanied by decreases in the content of glucose-6-P and fructose-6-P and transient increases in fructose-1,6-P2. In Ehrlich ascites cells, 2,5-anhydromannitol is metabolized to 2,5-anhydromannitol mono- and bisphosphate. The inhibition of glycolysis caused by 2,5-anhydromanitol decreases with time, because the phosphorylated metabolites formed during the preliminary incubation in the absence of glucose are rapidly dephosphorylated during the incubation in the presence of glucose. PMID:3155858

  8. [Differences in dynamics of insulin and insulin-like growth I (IGF-I) receptors internalization in isolated rat hepatocytes].

    PubMed

    Kolychev, A P; Ternovskaya, E E; Arsenieva, A V; Shapkina, E V

    2013-01-01

    Insulin and IGF-I are two related peptides performing in the mammalian body functionally different roles of the metabolic and growth hormones, respectively. Internalization of the insulin-receptor complex (IRC) is the most important chain of mechanism of the action of hormone. To elucidate differences in the main stages of internalization of the two related hormones, the internalization dynamics of 125I-insulin and 125I-IGF-I was traced in isolated rat hepatocytes at 37 and 12 degrees C. There were established marked differences in the process of internalization of labeled hormones, which is stimulated by insulin and IGF-I. At 37 degrees C the insulin-stimulated internalization, unlike the process initiated by IGF-I, did not reach the maximal level for 1 h of incubation. However, essential differences in the internalization course of these two related peptide were obvious at the temperature of 12 degrees C. The internalization level of insulin receptors at 12 degrees C decreased by one third in spite of a significant increase of the insulin receptor binding on the hepatocytes plasma membrane. At 12 degrees C a slight decrease of the proportion of intracellular 125I-IGF-I correlated with a decrease in the 125I-IGF-I binding to receptors on the cell membrane. Internalization of IGF-I receptors was not affected by low temperature, as neither its level, nor the rate changed at 12 degrees C. The paradoxical decrease of the insulin-stimulated internalization at low temperature seems to represent a peculiar "inhibition mechanism" of immersion of IRC into the cell, which leads to accumulation of the complexes on the cell surface and possibly to a readjustment of the insulin biological activity. The resistance of internalization of the IGF-I receptor to cold seems to be related to the more ancient origin of this mechanism in the poikilothermal vertebrates.

  9. Separation and characterization of the aldehydic products of lipid peroxidation stimulated by carbon tetrachloride or ADP-iron in isolated rat hepatocytes and rat liver microsomal suspensions.

    PubMed Central

    Poli, G; Dianzani, M U; Cheeseman, K H; Slater, T F; Lang, J; Esterbauer, H

    1985-01-01

    Carbonyl products were separated and identified in suspensions of rat liver microsomal fractions and in isolated hepatocytes, after stimulation of lipid peroxidation by incubation with the pro-oxidants CCl4 and ADP-iron. The carbonyl products were allowed to react with 2,4-dinitrophenylhydrazine, and the derivatives were extracted and separated by t.l.c. into three zones of non-polar materials, and one fraction of polar derivatives that remained at the origin. Separation of the individual non-polar hydrazones in each zone by h.p.l.c. demonstrated that zone I prepared from microsomal fraction or hepatocytes incubated with CCl4 or ADP-iron contained mainly 4-hydroxyhex-2-enal, 4-hydroxynon-2-enal and 4-hydroxynona-2,5-dienal. Zone III consisted mainly of the alkanals propanal, pentanal and hexanal, the 2-alkenals propenal, pent-2-enal, hex-2-enal, hept-2-enal, oct-2-enal and non-2-enal, the ketones butanone, pentan-2-one and pentan-3-one, and deca-2,4-dienal. Incubation of a microsomal fraction with ADP-iron was much more effective in producing malonaldehyde and other carbonyl products than an incubation with CCl4. Despite such quantitative differences, there were no obvious qualitative differences in the h.p.l.c. spectra obtained from zones I and III. However, the stoichiometric evaluation of fatty acid loss and the production of malonaldehyde and other carbonyls suggests that the pathways of lipid peroxidation triggered by CCl4 and ADP-iron are different. The accumulation of carbonyl products of lipid peroxidation in isolated hepatocytes is strongly affected by their metabolism; in particular, 4-hydroxyalkenals were found to be metabolized very rapidly. Nonetheless, both CCl4 and ADP-iron produced stimulation in the production of malonaldehyde and non-polar carbonyl production. After incubation of rat hepatocytes with CCl4 or ADP-iron it was found that approx. 50% of the total amount of non-polar carbonyls produced during incubation escaped into the external medium

  10. Protective effects of ferulic acid and related polyphenols against glyoxal- or methylglyoxal-induced cytotoxicity and oxidative stress in isolated rat hepatocytes.

    PubMed

    Maruf, Abdullah Al; Lip, HoYin; Wong, Horace; O'Brien, Peter J

    2015-06-05

    Glyoxal (GO) and methylglyoxal (MGO) cause protein and nucleic acid carbonylation and oxidative stress by forming reactive oxygen and carbonyl species which have been associated with toxic effects that may contribute to cardiovascular disease, complications associated with diabetes mellitus, Alzheimer's and Parkinson's disease. GO and MGO can be formed through oxidation of commonly used reducing sugars e.g., fructose under chronic hyperglycemic conditions. GO and MGO form advanced glycation end products which lead to an increased potential for developing inflammatory diseases. In the current study, we have investigated the protective effects of ferulic acid and related polyphenols e.g., caffeic acid, p-coumaric acid, methyl ferulate, ethyl ferulate, and ferulaldehyde on GO- or MGO-induced cytotoxicity and oxidative stress (ROS formation, protein carbonylation and mitochondrial membrane potential maintenance) in freshly isolated rat hepatocytes. To investigate and compare the protective effects of ferulic acid and related polyphenols against GO- or MGO-induced toxicity, five hepatocyte models were used: (a) control hepatocytes, (b) GSH-depleted hepatocytes, (c) catalase-inhibited hepatocytes, (d) aldehyde dehydrogenase (ALDH2)-inhibited hepatocytes, and (e) hepatocyte inflammation system (a non-toxic H2O2-generating system). All of the polyphenols tested significantly decreased GO- or MGO-induced cytotoxicity, ROS formation and improved mitochondrial membrane potential in these models. The rank order of their effectiveness was caffeic acid∼ferulaldehyde>ferulic acid>ethyl ferulate>methyl ferulate>p-coumaric acid. Ferulic acid was found to decrease protein carbonylation in GSH-depleted hepatocytes. This study suggests that ferulic acid and related polyphenols can be used therapeutically to inhibit or decrease GO- or MGO-induced hepatotoxicity.

  11. Effect of crilvastatin, a new cholesterol lowering agent, on unesterified LDL-cholesterol metabolism into bile salts by rat isolated hepatocytes.

    PubMed Central

    Clerc, T; Sbarra, V; Diaconescu, N; Lafont, H; Jadot, G; Laruelle, C; Chanussot, F

    1995-01-01

    1. The aim of these experiments was to determine the effect of crilvastatin, a new cholesterol lowering agent, on the metabolism of unesterified low density lipoprotein (LDL)-cholesterol by rat freshly isolated hepatocytes. This preclinical model was developed as an alternative to in vivo experiments, to mimic the metabolic effects of a molecule on its target cells and to define optimal conditions for future experimentation on human hepatocytes. 2. Cells were obtained from normolipidaemic or hypercholesterolaemic rats, hypercholesterolaemia was nutritionally induced. Incubations were performed in a medium containing 600 microM taurocholate and 50 microM or 300 microM crilvastatin. 3. This molecule was shown in vitro to be carried by physiological transporters, i.e., albumin-bile salt micellar associations and LDL. Crilvastatin induced a significance increase in the synthesis and secretion by hepatocytes of bile salts resulting from the metabolism of unesterified LDL-cholesterol in both normolipidaemic and hypercholesterolaemic rats. Stimulation involved non-conjugated as well as tauro- and glyco-conjugated bile salts. These findings corroborate preliminary studies showing in vivo that crilvastatin enhances the secretion of bile acids by stimulating the uptake and incorporation of LDL-cholesterol by the liver. PMID:7735689

  12. Transport in isolated rat hepatocytes of the phospho-oligosaccharide that mimics insulin action. Effects of adrenalectomy and glucocorticoid treatment.

    PubMed Central

    Alvarez, J F; Sánchez-Arias, J A; Guadaño, A; Estévez, F; Varela, I; Felíu, J E; Mato, J M

    1991-01-01

    The addition to intact cells of an inositol phospho-oligosaccharide (POS), which is the polar head-group of an insulin-sensitive glycosylphosphatidylinositol, mimics and may mediate some of the biological effects of this hormone. Here we report the existence of a POS transport system in hepatocytes. This POS transport system is specific and time- and dose-dependent. Insulin-resistance caused by dexamethasone administration to rats was accompanied by a decrease in the hepatocyte POS transport system. In contrast, bilateral adrenalectomy provoked a significant increase in the transport of POS. Both the temporal uptake of POS and the regulation of this process by conditions known to modify the sensitivity to insulin suggest that this novel transport system might be involved in the insulin signalling mechanism. PMID:2006906

  13. Mitochondrial and peroxisomal fatty acid oxidation in liver homogenates and isolated hepatocytes from control and clofibrate-treated rats.

    PubMed

    Mannaerts, G P; Debeer, L J; Thomas, J; De Schepper, P J

    1979-06-10

    Mitochondrial and peroxisomal fatty acid oxidation were compared in whole liver homogenates. Oxidation of 0.2 mM palmitoyl-CoA or oleate by mitochondria increased rapidly with increasing molar substrate:albumin ratios and became saturated at ratios below 3, while peroxisomal oxidation increased more slowly and continued to rise to reach maximal activity in the absence of albumin. Under the latter condition mitochondrial oxidation was severely depressed. In homogenates from normal liver peroxisomal oxidation was lower than mitochondrial oxidation at all ratios tested except when albumin was absent. In contrast with mitochondrial oxidation, peroxisomal oxidation did not produce ketones, was cyanide-insensitive, was not dependent on carnitine, and was not inhibited by (+)-octanoylcarnitine, malonyl-CoA and 4-pentenoate. Mitochondrial oxidation was inhibited by CoASH concentrations that were optimal for peroxisomal oxidation. In the presence of albumin, peroxisomal oxidation was stimulated by Triton X-100 but unaffected by freeze-thawing; both treatments suppressed mitochondrial oxidation. Clofibrate treatment increased mitochondrial and peroxisomal oxidation 2- and 6- to 8-fold, respectively. Peroxisomal oxidation remained unchanged in starvation and diabetes. Fatty acid oxidation was severely depressed by cyanide and (+)-octanoylcarnitine in hepatocytes from normal rats. Hepatocytes from clofibrate-treated rats, which displayed a 3- to 4-fold increase in fatty acid oxidation, were less inhibited by (+)-octanoylcarnitine. Hydrogen peroxide production was severalfold higher in hepatocytes from treated animals oxidizing fatty acids than in control hepatocytes. Assuming that all H2O2 produced during fatty acid oxidation was due to peroxisomal oxidation, it was calculated that the contribution of the peroxisomes to fatty acid oxidation was less than 10% both in cells from control and clofibrate-treated animals.

  14. Metabolism of 2,4-dinitro[14C]toluene by freshly isolated Fischer-344 rat primary hepatocytes.

    PubMed

    Bond, J A; Rickert, D E

    1981-01-01

    Fischer-344 rat hepatocytes display a capacity for both oxidation and reduction of 2,4-dinitrotoluene (2,4-DNT), a potent hepatocarcinogen. The major metabolite detected by high-pressure liquid chromatography was 2,4-dinitrobenzyl alcohol (2,4-DNBalc), which accounted for 75-80% of the total metabolites formed. The apparent KM and Vmax for 2,4-DNBalc formation was 58.0 microM and 25.5 nmoles/10(6) cells/30 min, respectively. Formation of 2,4-DNBalc was enhanced by treatment of rats with Aroclor 1254 (6-fold), phenobarbital (3.5-fold), and 3-methylcholanthrene (3.5-fold). In vitro additions of either SKF 525-A or 7,8-benzoflavone inhibited the formation of 2,4-DNBalc. Hepatocytes incubated under decreased oxygen concentrations (15%, 10%, 5% O2 in N2) displayed higher levels of reductive metabolism at 2,4-DNT (up to 5-fold) than when incubated in air. Concomitant decreases in oxidative metabolism of 2,4-DNT were observed in these experiments. Hepatocyte metabolism of 2-amino-4-nitrotoluene and 4-amino-2-nitrotoluene, two major products of rat cecal metabolism of 2,4-DNT, to 2-(N-acetyl)amino-4-nitrotoluene and 4-(N-acetyl)amino-2-nitrololuene, respectively, was observed. The results of this investigation suggest that hepatic reductive metabolism of 2,4-DNT probably plays a minor role in the overall metabolic scheme of 2,4-DNT.

  15. The role of prolactin and progesterone in the regulation of lipogenesis in maternal and foetal rat liver in vivo and in isolated hepatocytes during the last day of gestation.

    PubMed Central

    Lorenzo, M; Roncero, C; Benito, M

    1986-01-01

    The administration of progesterone on day 21 of gestation increases the rates of lipogenesis in the liver in vivo and in hepatocytes isolated from rats on day 22 of pregnancy. Bromocriptine administration increases the rates of hepatic lipogenesis in vivo, but has no effect on lipid synthesis in hepatocytes under the same treatment conditions. Concurrently, the administration of progesterone or bromocriptine on day 21 to the mother increases the rates of lipogenesis in the foetal liver in vivo on day 22. The rates of lipid synthesis in foetal isolated hepatocytes are increased by progesterone administration, but remain unchanged by bromocriptine. PMID:3800972

  16. The involvement of cytochrome P450 peroxidase in the metabolic bioactivation of cumene hydroperoxide by isolated rat hepatocytes.

    PubMed

    Anari, M R; Khan, S; O'Brien, P J

    1996-09-01

    Organic hydroperoxides are believed to be primarily detoxified in cells by the GSH peroxidase/GSSG reductase system and activated to cytotoxic radical species by non-heme iron. However, organic hydroperoxides seem to be bioactivated by cytochrome P450 (P450) in isolated hepatocytes as various P450 (particularly P450 2E1) inhibitors inhibited cumene hydroperoxide (CumOOH) metabolism and attenuated subsequent cytotoxic effects including antimycin A-resistant respiration, lipid peroxidation, iron mobilization, ATP depletion, and cell membrane disruption. CumOOH metabolism was also faster in P450 1A-induced hepatocytes and was inhibited by the P450 1A inhibitor alpha-naphthoflavone. The ferric chelator deferoxamine also prevented cytotoxicity even after CumOOH had been metabolized but had no effect on CumOOH metabolism. This emphasizes the toxicological significance of the iron released following hydroperoxide metabolic activation by cytochrome P450. The radical trap, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), had no effect on CumOOH metabolism but prevented CumOOH-induced antimycin A-resistant respiration, lipid peroxidation, iron mobilization, and loss of membrane integrity. These results suggest that CumOOH is metabolically activated by some P450 enzymes (e.g., P450 2E1) in hepatocytes to form reactive radical metabolites or oxidants that cause lipid peroxidation and cytotoxicity.

  17. Intrasplenic transplantation of allogeneic hepatocytes prolongs survival in anhepatic rats.

    PubMed

    Arkadopoulos, N; Lilja, H; Suh, K S; Demetriou, A A; Rozga, J

    1998-11-01

    To examine whether hepatocytes transplanted in the spleen can function as an ectopic liver, we performed hepatocyte transplantation in rats that were rendered anhepatic. Total hepatectomy was performed by using a novel single-stage technique. Following hepatectomy, Group 1 rats (n = 16) were monitored until death to determine survival time without prior intervention. Group 2 anhepatic rats (n = 20) were sacrificed at various times to measure blood hepatocyte growth factor (HGF) and transforming growth factor beta1 (TGF-beta1) levels. Group 3 (n = 16) rats received intrasplenic injection of isolated hepatocytes (2.5 x 10(7) cells/rat) followed by total hepatectomy after 3 days. Group 4 (n = 12) sham-transplanted rats received intrasplenic saline infusion, and after 3 days they were rendered anhepatic. Group 2, 3, and 4 rats were maintained on daily Cyclosporine A (10 mg/kg; intramuscularly). Group 1 anhepatic rats survived for 22.4 +/- 5.2 hours (standard deviation). The anhepatic state was associated with a progressive and statistically significant rise in blood HGF and TGF-beta1 levels. Rats that received hepatocyte transplantation before total hepatectomy had a significantly longer survival time than sham-transplanted anhepatic controls (34.1 +/- 8.5 vs. 15.5 +/- 4.8 hrs, P < .01). Additionally, at 12 hours post-hepatectomy, transplanted rats had significantly lower blood ammonia, prothrombin time, international normalized ratio, and TGF-beta1 levels when compared with sham-transplanted controls. In conclusion, intrasplenic transplantation of allogeneic hepatocytes prolonged survival, improved blood chemistry, and lowered blood TGF-beta1 levels in rats rendered anhepatic.

  18. Amodiaquine-induced toxicity in isolated rat hepatocytes and the cytoprotective effects of taurine and/or N-acetyl cysteine

    PubMed Central

    Heidari, R.; Babaei, H.; Eghbal, M.A.

    2014-01-01

    Amodiaquine is an antimalarial drug used in the prophylaxis and treatment of this disease. However, hepatotoxicity as a life-threatening adverse effect is associated with its clinical use. We evaluated amodiaquine-induced toxicity in isolated rat hepatocytes as an in vitro model for studying drug-induced hepatotoxicity. This study attempts to investigate the protective effects of taurine and N-acetyl cysteine against the cytotoxicity induced by amodiaquine. Hepatocytes were prepared by the method of collagenase enzyme perfusion via portal vein. This technique is based on liver perfusion with collagenase after removal of calcium ion (Ca2+) with a chelator (ethylene glycol tetraacetic acid (EGTA) 0.5 mM). Cells were treated with different concentrations of amodiaquine, taurine and N-acetyl cysteine. Cell death, protein carbonylation, reactive oxygen species formation, lipid peroxidation, and mitochondrial depolarization were assessed as toxicity markers. Amodiaquine cytotoxic mechanism involved protein carbonylation as well as reactive oxygen species formation and lipid peroxidation. In addition, mitochondria seem to be a target for amodiaquine to induce cellular damage. Administration of taurine (200 μM) and/or N-acetyl cysteine (200 μM) reduced oxidative stress, lipid peroxidation and protein carbonylation caused by amodiaquine. Furthermore, amodiaquine-induced mitochondrial injury was significantly mitigated by taurine and/or N-acetyl cysteine. In glutathione-depleted cells, only N-acetyl cysteine protected hepatocytes against amodiaquine, and taurine showed no protective properties in this situation. Taurine and N-acetyl cysteine protect hepatocytes against amodiaquine probably via their antioxidant properties and counteracting oxidative stress. PMID:25657778

  19. Hormone controlled phosphorylation and degradation of CYP2B1 and CYP2E1 in isolated rat hepatocytes.

    PubMed

    Johansson, I; Eliasson, E; Ingelman-Sundberg, M

    1991-01-15

    Addition of adrenalin to primary rat hepatocytes caused a 3- and 2-fold increase in [32P]-incorporation into CYP2E1 and CYP2B1, respectively. Adrenalin also increased the rate of CYP2E1 degradation at similar concentrations as needed for phosphorylation of the protein (r = 0.93), but did not influence the degradation rate of CYP2B1. Ethanol (75 mM) completely protected from adrenalin dependent phosphorylation and degradation of CYP2E1, but did not influence CYP2B1 on these parameters. Examination of para-nitrophenol hydroxylase revealed that ethanol stabilized the catalytically active form of CYP2E1. Insulin treatment caused a stabilization of CYP2E1, but did not affect CYP2B1 degradation. It is concluded that degradation of CYP2E1 is the subject of hormonal control, whereas CYP2B1 decomposition is accomplished in a different and a less regulated manner.

  20. Prediction of the pharmacokinetics of atorvastatin, cerivastatin, and indomethacin using kinetic models applied to isolated rat hepatocytes.

    PubMed

    Paine, Stuart W; Parker, Alison J; Gardiner, Philip; Webborn, Peter J H; Riley, Robert J

    2008-07-01

    The disposition of atorvastatin, cerivastatin, and indomethacin, established substrates of rat hepatic basolateral uptake transporters, has been evaluated in suspended rat hepatocytes. Cell and media concentration-time data were simultaneously fitted to a model incorporating active uptake, permeation, binding, and metabolism. Use of the model to estimate the ratio of intracellular to extracellular steady-state free drug concentrations demonstrated the strong influence of active uptake on the kinetics of atorvastatin (18:1) and cerivastatin (8:1), in comparison with indomethacin (3.5:1). Indomethacin, however, was shown to have a higher uptake clearance (599 +/- 101 microl/min/10(6) cells) than atorvastatin (375 +/- 45 microl/min/10(6) cells) and cerivastatin (413 +/- 47 microl/min/10(6) cells). The high passive permeability of indomethacin (237 +/- 63 microl/min/10(6) cells) clearly negated the effect of the active transport on the overall disposition. An analogous physiological model was constructed that allowed prediction of the in vivo pharmacokinetics, including the free intracellular concentration in liver. Hepatic clearance was well predicted by the model, in contrast to predictions based on standard methods. Volume of distribution was well predicted for indomethacin and predicted reasonably for atorvastatin and cerivastatin and higher than might be expected for an acid compound. Furthermore, the terminal half-life predictions for all three compounds were within 2-fold of the observed values. The ability to estimate the free-intracellular hepatic concentration of uptake substrates has major benefits in terms of predicting pharmacokinetics, potential CYP-mediated drug-drug interactions, and efficacy of hepatically targeted therapeutics.

  1. The calcium ionophore A23187 is a potent stimulator of the vitamin D3-25 hydroxylase in hepatocytes isolated from normocalcaemic vitamin D-depleted rats.

    PubMed Central

    Benbrahim, N; Dubé, C; Vallieres, S; Gascon-Barré, M

    1988-01-01

    The role played by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and/or by calcium on the C-25 hydroxylation of vitamin D3 (D3) was studied in hepatocytes isolated from D-depleted rats which were divided into four treatment groups: Group 1 served as controls, Group 2 received calcium gluconate, Groups 3 and 4 were infused with 1,25(OH)2D3 at 7 and 65 pmol/24 h x 7 days respectively. The treatments normalized serum calcium in all but the controls which remained hypocalcaemic, while serum 1,25(OH)2D3 remained low in Groups 1 and 2 but increased to physiologic and supraphysiologic levels in Groups 3 and 4. The data show that basal D3-25 hydroxylase activities were not significantly affected by any of the treatments. Addition of CaCl2, EGTA, or Quin-2 in vitro revealed that relative to basal values, EGTA strongly inhibited the enzyme activity in all groups (P less than 0.0001), except in G 1; Quin-2 and CaCl2 had no significant effect on the activity of the enzyme in any of the groups. Addition of 1,25(OH)2D3 or A23187 in vitro in the presence of CaCl2 revealed that 1,25(OH)2D3 did not significantly affect enzyme activity, while A23187 was found to stimulate its activity in vitamin D-depleted animals, but most specifically in Group 2 (P less than 0.001); low serum calcium (Group 1) dampened (P less than 0.01), and 1,25(OH)2D3 treatment in vivo totally blunted (P less than 0.001) the response to A23187. The data suggest that 1,25(OH)2D3 supplementation in vivo has per se little or no effect on the basal D3-25 hydroxylase activity. The data show, however, that the magnitude of the response to various challenges in vitro is greatly influenced by the conditioning in vivo of the animals. They also show that A23187 can be a potent stimulator of the enzyme activity, which allowed us to demonstrate a significant reserve for the C-25 hydroxylation of D3 which is well expressed in hepatocytes obtained from D-depleted calcium-supplemented rats. PMID:2848514

  2. Lycopene as a natural protector against gamma-radiation induced DNA damage, lipid peroxidation and antioxidant status in primary culture of isolated rat hepatocytes in vitro.

    PubMed

    Srinivasan, M; Sudheer, A Ram; Pillai, K Raveendran; Kumar, P Raghu; Sudhakaran, P R; Menon, V P

    2007-04-01

    The present study was designed to evaluate the radioprotective effect of lycopene, a naturally occurring dietary carotenoid, on gamma-radiation induced toxicity in cultured rat hepatocytes. The cellular changes were estimated using lipid peroxidative indices like thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), ceruloplasmin, vitamins A, E, C and uric acid. The DNA damage was analysed by single cell gel electrophoresis (comet assay). The increase in the severity of DNA damage was observed with the increase in gamma-radiation dose (1, 2 and 4 Gy) in cultured rat hepatocytes. TBARS were increased significantly whereas the levels of GSH, vitamins C, E and A, ceruloplasmin, uric acid and antioxidant enzymes were significantly decreased in gamma-irradiated groups. The maximum damage to hepatocytes was observed at 4 Gy irradiation. Pretreatment with lycopene (1.86, 9.31 and 18.62 microM) showed a significant decrease in the levels of TBARS and DNA damage. The antioxidant enzymes increased significantly along with the levels of GSH, vitamins A, E, C, uric acid and ceruloplasmin. The maximum protection of hepatocytes was observed at 9.31 muM of lycopene pretreatment. Thus, our results show that pretreatment with lycopene offers protection against gamma-radiation induced cellular damage and can be developed as an effective radioprotector during radiotherapy.

  3. Distribution of labeled products from (1-/sup 14/C), (U-/sup 14/C) and (16-/sup 14/C)-palmitate in isolated rat hepatocytes and liver mitochondria

    SciTech Connect

    Chatzidakis, C.; Otto, D.A.

    1986-05-01

    Fatty acids (FA) labeled in different carbon positions are used to study the distribution of labeled oxidation products. With rat hepatocytes (Hep) the authors observed significant differences in the distribution of label into products from (1-/sup 14/C) and (U-/sup 14/C)-palmitate (P). The total recovery of label in products (/sup 14/CO/sub 2/ + acid soluble fraction (ASF)) was identical between the two labeled FA. However, /sup 14/CO/sub 2/ production from (U-/sup 14/C)-P was only 40% of that from (1-/sup 14/C)-P. A recent report showed that approximately = 95% of succinate (Suc) utilized by Hep does not complete one full turn through the citric acid cycle. The authors observed that /sup 14/CO/sub 2/ evolution from (2,3-/sup 14/C)-Suc was approximately = 9% of that from (1,4-/sup 14/C)-Suc, indicating that the differences in label distribution between (1-/sup 14/C) and (U-/sup 14/C)-P are partially due to less /sup 14/CO/sub 2/ production from label in the even carbon positions of the FA with consequently more label remaining in the ASF. The /sup 14/CO/sub 2/ production from (16-/sup 14/C)-P was only 4% of that from (1-/sup 14/C)-P a value less than expected from the Suc experiments. Ketone bodies (KB) comprised 78% of total labeled products from (16-/sup 14/C)-P as compared to 28% from (1-/sup 14/C)-P and 41% from (U-/sup 14/C)-P, giving support to the previously reported preferential use of the omega-C/sub 2/ unit for KB synthesis without entry into the acetyl-CoA pool. Studies with isolated rat liver mitochondria gave results similar to those with Hep, indicating minimal involvement of perioxisomal ..beta..-oxidation.

  4. Effects of methylmercury on primary cultured rat hepatocytes: Cell injury and inhibition of growth factor stimulated DNA synthesis

    SciTech Connect

    Tanno, Keiichi; Fukazawa, Toshiyuki; Tajima, Shizuko; Fujiki, Motoo )

    1992-08-01

    Many more studies deal with the toxicity of methylmercury on nervous tissue than on its toxicity to the liver. Methylmercury accumulates in the liver in higher concentrations than brain and the liver has the primary function of detoxifying methylmercury. According to recent studies, hepatocyte mitochondrial membranes are destroyed by methylmercury and DNA synthesis is inhibited by methylmercury during hepatocyte regeneration. Methylmercury alters the membrane ion permeability of isolate skate hepatocytes, and inhibits the metal-sensitive alcohol dehydrogenase and glutathione reductase of primary cultured rat hepatocytes. However, little is known about the effect of methylmercury on hepatocyte proliferation in primary cultured rat hepatocytes. We therefore used the primary cultured rat hepatocytes to investigate the effects of methylmercury on cell injury and growth factor stimulate DNA synthesis. The primary effect of methylmercury is to inhibit hepatocyte proliferation rather than to cause direct cell injury. 16 refs., 4 figs.

  5. Characteristics of the accumulation of methotrexate polyglutamate derivatives in Ehrlich ascites tumor cells and isolated rat hepatocytes

    SciTech Connect

    Fry, D.W.; Gewirtz, D.A.; Yalowich, J.C.; Goldman, I.D.

    1983-01-01

    The intracellular synthesis and retention of polygammaglutamyl derivatives of methotrexate and their interactions with H/sub 2/ folate reductase was evaluated. Methotrexate polyglutamates were detected within 15 minutes in hepatocytes exposed to 1 microM methotrexate, and continued to accumulate for at least 60 minutes producing a large transmembrane gradient. These derivatives appeared to be preferentially retained within the cell. In studies with the Ehrlich ascites tumor accumulation of methotrexate polyglutamates was increased over 5-fold by the addition of 5 mM L-glutamine or L-glutamate and exhibited a positive correlation with the extracellular concentration of methotrexate. When Ehrlich ascites tumor cells were exposed to 10 microM methotrexate and 5 mM L-glutamine intracellular polyglutamates were detected within 10 minutes and their levels increased linearly over 4 hours. As these derivatives accumulated, there was a decline in intracellular methotrexate due at least in part to a replacement of methotrexate on H/sub 2/ folate reductase by polyglutamates and subsequent efflux of the previously bound methotrexate from the cell. When polyglutamate derivatives were in excess of the H/sub 2/ folate reductase binding capacity and extracellular methotrexate removed, methotrexate rapidly exited the cell whereas the majority of its metabolites were retained and eventually saturated the major portion of the enzyme. These studies indicate that (1) intracellular methotrexate is rapidly converted to polygammaglutamyl derivatives, (2) these metabolites effectively compete with methotrexate for binding sites on H/sub 2/ folate reductase, (3) these derivatives are retained within the cell more effectively than methotrexate, and (4) vincristine and probenecid may be potentially useful for selectively increasing methotrexate polyglutamates in tumor cells.

  6. Quantitative analysis of flux along the gluconeogenic, glycolytic and pentose phosphate pathways under reducing conditions in hepatocytes isolated from fed rats.

    PubMed Central

    Crawford, J M; Blum, J J

    1983-01-01

    Hepatocytes were isolated from the livers of fed rats and incubated with a mixture of glucose (10 mM), ribose (1 mM), mannose (4 mM), glycerol (3 mM), acetate (1.25 mM), and ethanol (5 mM) with one substrate labelled with 14C in any given incubation. Incorporation of label into CO2, glucose, glycogen, lipid glycerol and fatty acids, acetate and C-1 of glucose was measured at 20 and 40 min after the start of the incubation. The data (about 48 measurements for each interval) were used in conjunction with a single-compartment model of the reactions of the gluconeogenic, glycolytic and pentose phosphate pathways and a simplified model of the relevant mitochondrial reactions. An improved method of computer analysis of the equations describing the flow of label through each carbon atom of each metabolite under steady-state conditions was used to compute values for the 34 independent flux parameters in this model. A good fit to the data was obtained, thereby permitting good estimates of most of the fluxes in the pathways under consideration. The data show that: net flux above the level of the triose phosphates is gluconeogenic; label in the hexose phosphates is fully equilibrated by the second 20 min interval; the triose phosphate isomerase step does not equilibrate label between the triose phosphates; substrate cycles are operating at the glucose-glucose 6-phosphate, fructose 6-phosphate-fructose 1,6-bisphosphate and phosphoenolpyruvate-pyruvate-oxaloacetate cycles; and, although net flux through the enzymes catalysing the non-oxidative steps of the pentose phosphate pathway is small, bidirectional fluxes are large. PMID:6411069

  7. Disappearance of GFP-Positive Hepatocytes Transplanted into the Liver of Syngeneic Wild-Type Rats Pretreated with Retrorsine

    PubMed Central

    Maeda, Hiromichi; Shigoka, Masatoshi; Wang, Yongchun; Fu, Yingxin; Wesson, Russell N.; Lin, Qing; Montgomery, Robert A.; Enzan, Hideaki; Sun, Zhaoli

    2014-01-01

    Background and Aim Green fluorescent protein (GFP) is a widely used molecular tag to trace transplanted cells in rodent liver injury models. The differing results from various previously reported studies using GFP could be attributed to the immunogenicity of GFP. Methods Hepatocytes were obtained from GFP-expressing transgenic (Tg) Lewis rats and were transplanted into the livers of wild-type Lewis rats after they had undergone a partial hepatectomy. The proliferation of endogenous hepatocytes in recipient rats was inhibited by pretreatment with retrorsine to enhance the proliferation of the transplanted hepatocytes. Transplantation of wild-type hepatocytes into GFP-Tg rat liver was also performed for comparison. Results All biopsy specimens taken seven days after transplantation showed engraftment of transplanted hepatocytes, with the numbers of transplanted hepatocytes increasing until day 14. GFP-positive hepatocytes in wild-type rat livers were decreased by day 28 and could not be detected on day 42, whereas the number of wild-type hepatocytes steadily increased in GFP-Tg rat liver. Histological examination showed degenerative change of GFP-positive hepatocytes and the accumulation of infiltrating cells on day 28. PCR analysis for the GFP transgene suggested that transplanted hepatocytes were eliminated rather than being retained along with the loss of GFP expression. Both modification of the immunological response using tacrolimus and bone marrow transplantation prolonged the survival of GFP-positive hepatocytes. In contrast, host immunization with GFP-positive hepatocytes led to complete loss of GFP-positive hepatocytes by day 14. Conclusion GFP-positive hepatocytes isolated from GFP-Tg Lewis rats did not survive long term in the livers of retrorsine-pretreated wild-type Lewis rats. The mechanism underlying this phenomenon most likely involves an immunological reaction against GFP. The influence of GFP immunogenicity on cell transplantation models should be

  8. CD36/fatty acid translocase in rats: distribution, isolation from hepatocytes, and comparison with the scavenger receptor SR-B1.

    PubMed

    Zhang, Xingqi; Fitzsimmons, Rebecca L; Cleland, Leslie G; Ey, Peter L; Zannettino, Andrew C W; Farmer, Elizabeth-Anne; Sincock, Paul; Mayrhofer, Graham

    2003-03-01

    The new mAb UA009 recognizes an antigen expressed by microvascular endothelium, by lymphatic endothelium, and by some epithelia in a number of organs, including the small intestine, lactating mammary gland, kidney, lung, sebaceous glands, and circumvallate papillae of the tongue. This antigen is also expressed abundantly in the splenic red pulp and marginal zone and by monocytes, macrophages, and erythrocytes (but not by platelets). Among tissues that store or metabolize fatty acids, the antigen is expressed by adipocytes, cardiomyocytes, and red skeletal muscle. Importantly, it is expressed by steroidogenic cells in the adrenal gland, testis, and ovary, whereas in the liver it is expressed by hepatocytes in a pattern that is dependent on gender and genetic background. mAb UA009 immunoprecipitated a mol wt 85-kDa surface protein from detergent extracts of hepatocytes from Dark Agouti female rats. The N-terminal amino acid sequence of this protein was identical to fatty acid translocase (FAT), the rat cluster of differentiation 36 (CD36) ortholog. The mAb also reacted with COS-7 cells transfected with cDNA encoding FAT. cDNAs encoding a CD36/FAT-like polypeptide were prepared from both liver and heart RNA by RT-PCR. The nucleotide sequences obtained from these cDNAs (Dark Agouti rats) revealed identity and 99% similarity, respectively, with the published sequences of Cd36/Fat in rats of the Wistar and Sprague-Dawley strains. The absence of the UA009 antigen in CD36/FAT-deficient SHR/N rats confirmed the identity of the UA009 antigen and CD36/FAT. We suggest that CD36/FAT might function in the liver as a sex-regulated accessory molecule, either in reverse cholesterol transport and/or in fatty acid uptake.

  9. Metabolic changes in lipids of rat plasma and hepatocytes induced by 17 alpha-ethynylestradiol treatment.

    PubMed

    Hayashi, K; Koide, K; Hirata, Y; Ohtani, H; Yamada, K; Kajiyama, G

    1986-11-14

    Cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats were used to investigate the change of lipid metabolism induced by administration of 17 alpha-ethynylestradiol. Treatment with 17 alpha-ethynylestradiol caused a decrease of rat plasma lipids (free cholesterol, cholesterol ester, triacylglycerol and phosphatidylcholine). No difference in the ability of urea nitrogen synthesis could be demonstrated between cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats and propylene glycol-treated rats (control). Total cholesterol and cholesterol ester contents of cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats were increased in comparison with those of the control. Triacylglycerol content of cultured hepatocytes was not affected by 17 alpha-ethynylestradiol treatment. There was no difference in the composition of lipid content between liver tissues and cultured hepatocytes. These results suggest that hepatocytes isolated from livers maintain the character of livers treated with 17 alpha-ethynylestradiol or livers treated with propylene glycol. Free cholesterol and cholesterol ester synthesis from [14C]acetic acid by cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats were decreased to about 30% of the control. Triacylglycerol and polar lipid (phospholipid) synthesis from [14C]acetic acid were not affected by 17 alpha-ethynylestradiol treatment. Microsomal hydroxymethylglutaryl-CoA reductase activity of rat liver treated with 17 alpha-ethynylestradiol was decreased to about 50% of control. The secretions of free cholesterol, cholesterol ester, triacylglycerol, phosphatidylcholine, apolipoprotein BL and BS by cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol treated rats were not decreased when compared with the control. Because lipid and apolipoprotein secretions from cultured hepatocytes treated with 17 alpha

  10. Effect of growth hormone and estrogen administration on hepatocyte alterations in old ovariectomized female wistar rats.

    PubMed

    Castillo, Carmen; Salazar, Veronica; Ariznavarreta, Carmen; Vara, Elena; Tresguerres, Jesus A F

    2005-02-01

    Aging could be due to the accumulation of oxidative damage. On the other hand, growth hormone (GH) and estrogen deficiency induce deleterious effects on different tissues, and hormonal replacement could counteract these effects. We have investigated whether GH and estrogen administration modify some parameters related to oxidative stress and inflammation in hepatocytes isolated from old ovariectomized female rats. Twenty-two month-old ovariectomized animals were divided into control rats, rats treated with GH, rats treated with estradiol, and rats treated with GH+estradiol. Two-month-old intact female rats were used as young reference group. Hepatocytes were isolated, cultured, and CO and NO release, ATP, cyclic-guanosyl monophosphate (cGMP), and lipid peroxide (LPO) content of cells, as well as phosphatidylcholine (PC)synthesis, were measured. Hepatocytes isolated from old ovariectomized rats showed a decrease in ATP content and PC synthesis compared to young rats. Age also induced an increase in LPO, NO, CO, and cGMP. Treating old rats with GH significantly increased ATP and reduced CO and cGMP levels. Estradiol administration improved all the parameters that were altered. Co-administration of GH and estrogens induced a more marked effect than estrogens alone only in cGMP content. In conclusion, administration of estrogens to old ovariectomized females seemed to prevent oxidative changes in hepatocytes, whereas the effect of GH is not so evident.

  11. Epidermal growth factor-stimulated protein phosphorylation in rat hepatocytes

    SciTech Connect

    Connelly, P.A.; Sisk, R.B.; Johnson, R.M.; Garrison, J.C.

    1987-05-01

    Epidermal growth factor (EGF) causes a 6-fold increase in the phosphorylation state of a cytosolic protein (pp36, M/sub r/ = 36,000, pI = 5.5) in hepatocytes isolated from fasted, male, Wistar rats. Stimulation of /sup 32/P incorporation is observed as early as 1 min following treatment of hepatocytes with EGF and is still present at 30 min after exposure to the growth factor. The phosphate incorporated into pp36 in response to EGF is located predominantly in serine but not tyrosine residues. Phosphorylation of pp36 does not occur in response to insulin or to agents which specifically activate the cAMP-dependent protein kinase (S/sub p/ -cAMPS), protein kinase C (PMA) or Ca/sup 2 +//calmodulin-dependent protein kinases (A23187) in these cells. Prior treatment of hepatocytes with the cAMP analog, S/sub p/-cAMPS, or ADP-ribosylation of N/sub i/, the inhibitory GTP-binding protein of the adenylate cyclase complex, does not prevent EGF-stimulated phosphorylation of pp36. However, as seen in other cell types, pretreatment of hepatocytes with PMA abolishes all EGF-mediated responses including phosphorylation of pp36. These results suggest that EGP specifically activates an uncharacterized, serine protein kinase in hepatocytes that is distal to the intrinsic EGF receptor tyrosine protein kinase. The rapid activation of this kinase suggests that it may play an important role in the early response of the cell to EGF.

  12. Allicin Modulates the Antioxidation and Detoxification Capabilities of Primary Rat Hepatocytes

    PubMed Central

    Wu, Chih-Chung; Chu, Yung-Lin; Sheen, Lee-Yan

    2012-01-01

    The effect of allicin, an active ingredient of garlic, on lactate dehydrogenase (LDH) leakage, lipid peroxidation, glutathione (GSH) content, and GSH-related enzyme activity was investigated in primary hepatocytes. In this study, allicin was synthesized in our laboratory as an experimental material, and primary hepatocytes isolated from Sprague-Dawley rats were used as an experimental model. According to the results, hepatocytes treated with 10 μM allicin did not differ from the control on LDH leakage during various incubation times. When the hepatocytes were treated with 10 μM allicin, their levels of thiobarbituric acid reactive-substances (TBARS) did not differ significantly from that of the control within the 8-h incubation. However, the TBARS values of hepatocytes treated with 30 and 50 μM allicin were higher compared to the control after incubation for 4 h and 8 h, respectively. The hepatocyte intracellular GSH content was significantly higher than that of the control after 30 μM allicin treatment, but treatment with 50 μM allicin caused a significant GSH depletion after incubation for 4 h or longer. In addition, when hepatocytes were treated for 24 h with 10 or 30 μM allicin, glutathione peroxidase (GPx) activity was significantly increased compared to that of the control, whereas 50 μM allicin treatment for 24 h or longer significantly decreased the GPx activity. Glutathione reductase (GRd) activity was significantly increased when the hepatocytes were treated with 10 μM allicin for 24 h, but GRd activity significantly decreased when the hepatocytes were treated with 50 μM allicin. However, hepatocytes treated for 24 h with 10 or 30 μM allicin showed significantly increased glutathione S-transferase (GST) activity compared to the control. These results suggest that 10 μM allicin potentially enhances the antioxidation and detoxification capabilities of primary rat hepatocytes. PMID:24716147

  13. Preincubation of rat and human hepatocytes with cytoprotectants prior to cryopreservation can improve viability and function upon thawing.

    PubMed

    Terry, Claire; Dhawan, Anil; Mitry, Ragai R; Lehec, Sharon C; Hughes, Robin D

    2005-12-01

    Cryopreservation of human hepatocytes is important for the treatment of liver disease by hepatocyte transplantation and also for the use of hepatocytes as an in vitro model of the liver. One factor in the success of cryopreservation is the quality of cells before freezing. Preincubation of hepatocytes with cytoprotective compounds to allow recovery from the isolation process prior to cryopreservation, such as those that will boost cellular adenosine triphosphate (ATP) content or antioxidants, may improve the viability and function of cells upon thawing. Rat hepatocytes were used to investigate the effects of preincubation with 10 compounds: precursors (glucose, fructose, glutathione, and S-adenosyl-L-methionine), antioxidants (ascorbic acid and alpha-lipoic acid), and compounds with multiple effects (N-acetylcysteine, pentoxifylline, prostaglandin E(1), and tauroursodeoxycholic acid). Human hepatocytes were then used to investigate 5 of the original 10 compounds (glucose, fructose, alpha-lipoic acid, S-adenosyl-L-methionine, and pentoxifylline). Glucose preincubation (100-300 mM) improved the viability and attachment efficiency of rat hepatocytes and improved the viability and reduced lactate dehydrogenase (LDH) leakage of human hepatocytes. Fructose preincubation (100-300 mM) improved the viability and attachment efficiency of rat hepatocytes and improved the attachment efficiency of human hepatocytes. alpha-lipoic acid preincubation (0.5-5 mM) improved the viability and attachment efficiency of both rat and human hepatocytes. At a concentration of 2.5 mM alpha-lipoic acid also improved the albumin production of human hepatocytes. In conclusion, preincubation of hepatocytes prior to cryopreservation can improve the viability and function of thawed cells and may provide a method of obtaining better-quality cryopreserved hepatocytes for transplantation.

  14. Isolation of centrolobular and perilobular hepatocytes after phenobarbital treatment

    PubMed Central

    1975-01-01

    Daily phenobarbital (PB) injections, on 3-7 consecutive days, induce an intense proliferation of smooth endoplasmic reticulum (ER) associated with a decrease of the glucose-6-phosphatase activity. This situation first affects the centrolobular hepatocytes, enhancing the degree of liver lobule heterogeneity. This experimental model was used for isolation and further subfractionation of hepatocytes on Ficoll density gradients, as described in the preceding paper. Profiles of protein, DNA, RNA, glycogen, phosphorylase, and glucose-6-phosphatase were determined all along the gradient. Two liver cell populations were distinguished: (a) light hepatocytes (mean density 1.10) present the same morphological characteristics as centrolobular cells, i.e., an abundant smooth ER composed of tubular elements, numerous small mitochondria, and few glycogen particles; (b) heavy hepatocytes (mean density 1.14) are characterized by large and compact glycogen areas and prominent rough endoplasmic cisternae, as are the perilobular cells. After incubation in the Wachstein-Meisel medium, Centrolobular hepatocytes exhibit dispersed reaction sites of glucose-6-phosphatase activity, whereas perilobular cells present a continuous and intense reaction. Morphometric determinations were carried out for both cell populations. Centrolobular PB hepatocytes are considerably enlarged (mean diameter: 23.7 mum); perilobular hepatocytes have a significantly smaller mean diameter of 19.2 mum, which is close to the values of control liver cells. PMID:167029

  15. Role of fructose 2,6-bisphosphate in the control by glucagon of gluconeogenesis from various precursors in isolated rat hepatocytes.

    PubMed Central

    Hue, L; Bartrons, R

    1984-01-01

    Hepatocytes from overnight-starved rats were incubated with 1-20 mM-fructose, -dihydroxyacetone, -glycerol, -alanine or -lactate and -pyruvate with or without 0.1 microM-glucagon. The production of glucose and lactate was measured, as was the content of fructose 2,6-bisphosphate. The concentrations of fructose (below 5 mM) and dihydroxyacetone (above 1 mM) that gave rise to an increase in fructose 2,6-bisphosphate were those at which a glucagon effect on the production of glucose and lactate could be observed. Glycerol had no effect on fructose 2,6-bisphosphate content or on production of lactate, and glucagon did not stimulate the production of glucose from this precursor. With alanine or lactate/pyruvate as substrates, glucagon stimulated glucose production whether the concentration of fructose 2,6-bisphosphate was increased or not. The extent of inactivation of pyruvate kinase by glucagon was not affected by the presence of the various gluconeogenic precursors. The role of fructose 2,6-bisphosphate in the effect of glucagon on gluconeogenesis from precursors entering the pathway at the level of triose phosphates or pyruvate is discussed. PMID:6546872

  16. Toxicity assessments of nonsteroidal anti-inflammatory drugs in isolated mitochondria, rat hepatocytes, and zebrafish show good concordance across chemical classes

    SciTech Connect

    Nadanaciva, Sashi; Aleo, Michael D.; Strock, Christopher J.; Stedman, Donald B.; Wang, Huijun; Will, Yvonne

    2013-10-15

    To reduce costly late-stage compound attrition, there has been an increased focus on assessing compounds in in vitro assays that predict attributes of human safety liabilities, before preclinical in vivo studies are done. Relevant questions when choosing a panel of assays for predicting toxicity are (a) whether there is general concordance in the data among the assays, and (b) whether, in a retrospective analysis, the rank order of toxicity of compounds in the assays correlates with the known safety profile of the drugs in humans. The aim of our study was to answer these questions using nonsteroidal anti-inflammatory drugs (NSAIDs) as a test set since NSAIDs are generally associated with gastrointestinal injury, hepatotoxicity, and/or cardiovascular risk, with mitochondrial impairment and endoplasmic reticulum stress being possible contributing factors. Eleven NSAIDs, flufenamic acid, tolfenamic acid, mefenamic acid, diclofenac, meloxicam, sudoxicam, piroxicam, diflunisal, acetylsalicylic acid, nimesulide, and sulindac (and its two metabolites, sulindac sulfide and sulindac sulfone), were tested for their effects on (a) the respiration of rat liver mitochondria, (b) a panel of mechanistic endpoints in rat hepatocytes, and (c) the viability and organ morphology of zebrafish. We show good concordance for distinguishing among/between NSAID chemical classes in the observations among the three approaches. Furthermore, the assays were complementary and able to correctly identify “toxic” and “non-toxic” drugs in accordance with their human safety profile, with emphasis on hepatic and gastrointestinal safety. We recommend implementing our multi-assay approach in the drug discovery process to reduce compound attrition. - Highlights: • NSAIDS cause liver and GI toxicity. • Mitochondrial uncoupling contributes to NSAID liver toxicity. • ER stress is a mechanism that contributes to liver toxicity. • Zebrafish and cell based assays are complimentary.

  17. Machine Perfusion Enhances Hepatocyte Isolation Yields From Ischemic Livers

    PubMed Central

    Izamis, Maria-Louisa; Perk, Sinem; Calhoun, Candice; Uygun, Korkut; Yarmush, Martin L.; Berthiaume, François

    2015-01-01

    Background High-quality human hepatocytes form the basis of drug safety and efficacy tests, cell-based therapies, and bridge-to-transplantation devices. Presently the only supply of cells derives from an inadequate pool of suboptimal disqualified donor livers. Here we evaluated whether machine perfusion could ameliorate ischemic injury that many of these livers experience prior to hepatocyte isolation. Methods Non-heparinized female Lewis rat livers were exposed to an hour of warm ischemia (34°C) and then perfused for 3 hours. Five different perfusion conditions that utilized the cell isolation apparatus were investigated, namely: (1) modified Williams Medium E and (2) Lifor, both with active oxygenation (95%O2/5%CO2), as well as (3) Lifor passively oxygenated with ambient air (21%O2/0.04%CO2), all at ambient temperatures (20±2°C). At hypothermic temperatures (5±1°C) and under passive oxygenation were (4) University of Wisconsin solution (UW) and (5) Vasosol. Negative and positive control groups comprised livers that had ischemia (WI) and livers that did not (Fresh) prior to cell isolation, respectively. Results Fresh livers yielded 32±9 million cells/g liver while an hour of ischemia reduced the cell yield to 1.6±0.6 million cells/g liver. Oxygenated Williams medium E and Lifor recovered yields of 39±11 and 31±2.3 million cells/g liver, respectively. The passively oxygenated groups produced 15±7 (Lifor), 13±7 (Vasosol), and 10±6 (UW) million cells/g liver. Oxygenated Williams Medium E was most effective at sustaining pH values, avoiding the accumulation of lactate, minimizing edematous weight gain and producing bile during perfusion. Conclusions Machine perfusion results in a dramatic increase in cell yields from livers that have had up to an hour of warm ischemia, but perfusate choice significantly impacts the extent of recovery. Oxygenated Williams Medium E at room temperature is superior to Lifor, UW and Vasosol, largely facilitated by its high

  18. DIFFERENTIATING MECHANISMS OF REACTIVE CHEMICAL TOXICITY IN ISOLATED TROUT HEPATOCYTES

    EPA Science Inventory

    The toxicity of four quinones, 2,3-dimethoxy-1,4-naphthoquinone (DMONQ), 2-methyl 1,4-naphthoquinone (MNQ ),1,4-naphthoquinone (NQ), and 1,4-benzoquinone (BQ), which redox cycle or arlyate in mammalian cells, was determined in isolated trout (Oncorhynchus mykiss) hepatocytes. Mor...

  19. Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes

    SciTech Connect

    Richert, L. Tuschl, G.; Pekthong, D.; Mantion, G.; Weber, J.-C.; Mueller, S.O.

    2009-02-15

    It is important to investigate the induction of cytochrome P450 (CYP) enzymes by drugs. The most relevant end point is enzyme activity; however, this requires many cells and is low throughput. We have compared the CYP1A, CYP2B and CYP3A induction response to eight inducers in rat and human hepatocytes using enzyme activities (CYP1A2 (ethoxyresorufin), 2B (benzoxyresorufin for rat and bupropion for human) and CYP3A (testosterone)) and Taqman{sup TM} Low Density Array (TLDA) analysis. There was a good correlation between the induction of CYP1A2, CYP2B6 and CYP3A4 enzyme activities and mRNA expression in human hepatocytes. In contrast, BROD activities and mRNA expression in rat hepatocytes correlated poorly. However, bupropion hydroxylation correlated well with Cyp2b1 expression in rat hepatocytes. TLDA analysis of a panel of mRNAs encoding for CYPs, phase 2 enzymes, nuclear receptors and transporters revealed that the main genes induced by the 8 compounds tested were the CYPs. AhR ligands also induced UDP-glucuronosyltransferases and glutathione S-transferases in rat and human hepatocytes. The transporters, MDR1, MDR3 and OATPA were the only transporter genes significantly up-regulated in human hepatocytes. In rat hepatocytes Bsep, Mdr2, Mrp2, Mrp3 and Oatp2 were up-regulated. We could then show a good in vivo:in vitro correlation in the induction response of isolated rat hepatocytes and ex-vivo hepatic microsomes for the drug development candidate, EMD392949. In conclusion, application of TLDA methodology to investigate the potential of compounds to induce enzymes in rat and human hepatocytes increases the throughput and information gained from one assay, without reducing the predictive capacity.

  20. Impaired mitochondrial metabolism and protein synthesis in streptozotocin diabetic rat hepatocytes

    SciTech Connect

    Memon, R.A.; Bessman, S.P.; Mohan, C. )

    1990-02-26

    Isolated hepatocytes prepared from control, streptozotocin diabetic rats were incubated at 30{degrees}C in Krebs-Henseleit bicarbonate buffer, pH 7.4, containing 0.5 mM concentration of each of the 20 natural amino acids. Effect of insulin on the oxidation of 2,3-{sup 14}C and 1,4-{sup 14}C succinate (suc) carbons and their incorporation into hepatocyte protein, lipid and various metabolic intermediates was studied. Mitochondrial oxidation of suc carbons and their incorporation into protein and lipid was significantly lower in diabetic and insulin treated diabetic rats. Diabetic rats failed to exhibit any significant insulin effect on the oxidation of either 2,3 or 1,4-{sup 14}C suc carbons. Amphibolic channeling of 2,3-{sup 14}C suc carbons into amino acids was significantly reduced in hepatocytes of diabetic rats, however, more of these carbons were diverted into the gluconeogenesis pathway. Diabetes caused a far greater decrease in the oxidation of 2,3-{sup 14}C suc carbons as compared to 1,4-{sup 14}C suc. Based on an earlier report that insulin stimulates only the intramitochondrial Krebs cycle reactions, the authors conclude that the diminished level of anabolic activities in the diabetic rat hepatocytes is due to the subsequent reduction in amphibolic channeling of metabolic intermediates.

  1. Evidence for two Ca2(+)-mobilizing purinoceptors on rat hepatocytes.

    PubMed Central

    Dixon, C J; Woods, N M; Cuthbertson, K S; Cobbold, P H

    1990-01-01

    Aequorin measurements of cytosolic free Ca2+ in single rat hepatocytes show that ADP and ATP, thought to act through the same P2Y purinoceptor, elicited very different responses in the majority of cells tested. ADP invariably induced transients of short duration (approx. 9 s), whereas ATP induced either similar transients or transients with a much longer duration (approx. 49 s). We explain this variability in terms of two separate purinoceptors on rat hepatocytes, one of which responds to either ATP or ADP to generate free-Ca2+ transients of short duration, and the other responds to ATP only, with transients of longer duration. PMID:2386488

  2. Evidence for expression of the facilitated glucose transporter in rat hepatocytes.

    PubMed Central

    Rhoads, D B; Takano, M; Gattoni-Celli, S; Chen, C C; Isselbacher, K J

    1988-01-01

    The eukaryotic facilitated glucose transporter (GT) is expressed by many cell types, with the notable exception of hepatocytes; however, GT is expressed by several hepatoma cell lines, including the well-differentiated lines Fao, Hep3B, and HepG2. We report on studies carried out to determine the aspect(s) of the transformed phenotype that might be responsible for activating GT expression. Using RNA blot analysis with probes derived from rat GT cDNA, we found that GT was expressed by rat hepatocytes under two conditions (i) in vitro, when isolated hepatocytes were placed in cell culture, and (ii) in vivo, when rats were subjected to starvation for greater than or equal to 2 days. However, GT expression was not an obligatory feature of hepatomas, since two primary hepatocellular carcinomas did not express any GT mRNA. GT expression in hepatocytes was reduced by addition of dimethyl sulfoxide or sodium butyrate to the culture medium. Since these reagents are known to promote differentiation in some cell culture systems, their effect on hepatocytes may be to maintain the GT repression normally observed in vivo. Inclusion or exclusion in the culture medium of several other agents that enhance hepatocyte viability (serum, insulin, corticosteroids, epidermal growth factor, or triiodothyronine) did not affect GT expression. It is unclear whether the two conditions that led to GT expression in hepatocytes are related by a common signaling mechanism. Possibly, both cases involve a "stress" response: in vivo, a normal physiological response to starvation; in vitro, a response to a major alteration in the cellular environment. Images PMID:3194405

  3. Lignans from Opuntia ficus-indica seeds protect rat primary hepatocytes and HepG2 cells against ethanol-induced oxidative stress.

    PubMed

    Kim, Jung Wha; Yang, Heejung; Kim, Hyeon Woo; Kim, Hong Pyo; Sung, Sang Hyun

    2017-01-01

    Bioactivity-guided isolation of Opuntia ficus-indica (Cactaceae) seeds against ethanol-treated primary rat hepatocytes yielded six lignan compounds. Among the isolates, furofuran lignans 4-6, significantly protected rat hepatocytes against ethanol-induced oxidative stress by reducing intracellular reactive oxygen species levels, preserving antioxidative defense enzyme activities, and maintaining the glutathione content. Moreover, 4 dose-dependently induced the heme oxygenase-1 expression in HepG2 cells.

  4. Effect of apolipoprotein a-I complex with tetrahydrocortisone on protein biosynthesis and glucose absorption by rat hepatocytes.

    PubMed

    Sumenkova, D V; Knyazev, R A; Guschya, R S; Polyakov, L M; Panin, L E

    2009-08-01

    We studied the effect of apolipoprotein A-I-tetrahydrocortisone complex on (14)C glucose absorption and lactate accumulation and on the rate of protein biosynthesis in isolated rat hepatocytes. The presence of apolipoprotein A-I-tetrahydrocortisone complex in the incubation medium increased absorption of labeled glucose by hepatocytes by 52%, while lactate content in the conditioning medium increased 4-fold. The rate of protein biosynthesis increased by 80% in comparison with control cells. It is hypothesized that the increase in protein biosynthesis rate in hepatocytes under the effect of apolipoprotein A-I-tetrahydrocortisone complex is due to stimulation of energy metabolism, specifically, of its glycolytic component.

  5. Comparative effects of UW and SLS solutions on concentrative proline uptake in cold preserved rat hepatocytes.

    PubMed

    Serrar, H; Musallam, L; Haddad, P

    1999-01-01

    In previous studies, we have shown that the rate of cell swelling induced by concentrative proline uptake in isolated rat hepatocytes decreased by 50 per cent after only 24 h of cold storage in University of Wisconsin (UW) solution, thereby representing a sensitive marker of alterations in hepatocyte functions after cold preservation and rewarming. We have thus used concentrative proline uptake to compare the capacity of UW and sodium-lactobionate-sucrose (SLS) solutions to maintain such differentiated hepatocyte functions. Isolated rat hepatocytes were kept at 4 degrees C for 4, 10, 24 and 48 h in UW or SLS solutions, and subsequently cultured at 37 degrees C for 1-2 h. Viability was measured by Trypan blue exclusion. After rewarming, cells were subjected to a 10 min administration of 10 mM proline and accumulation of the amino acid was assessed by changes in cell volume as measured by digital analysis of single-cell images obtained under bright-field illumination. Cell viability was reduced gradually and significantly after 0 to 48 h of preservation, and rewarming amplified this effect. However, loss of viability was similar in UW- and SLS-stored cells, as were initial steady-state cell volumes. Proline-induced swelling rate was reduced significantly by 13, 46 and by 57 per cent after 10, 24 and 48 h of preservation in UW solution, respectively. There is no significant difference between SLS- and UW-preserved hepatocyte swelling rates after 10 h and 48 h of cold preservation. However, the decline in the swelling rate of SLS-preserved hepatocytes incubated for 24 h is significantly lower than that of their UW-preserved counterparts. These results show that the SLS solution can preserve differentiated hepatic functions as well as the UW solution does.

  6. Molybdate and tungstate act like vanadate on glucose metabolism in isolated hepatocytes.

    PubMed Central

    Fillat, C; Rodríguez-Gil, J E; Guinovart, J J

    1992-01-01

    In rat hepatocytes, molybdate and tungstate inactivate glycogen synthase by a mechanism independent of Ca2+ and activate glycogen phosphorylase by a Ca(2+)-dependent mechanism. On the other hand, both molybdate and tungstate increase fructose 2,6-bisphosphate levels and counteract the decrease in this metabolite induced by glucagon. These effectors do not directly modify 6-phosphofructo-2-kinase activity, even though they partially counteract the inactivation of this enzyme induced by glucagon. These effects are related to an increase on the glycolytic flux, as indicated by the increase in L-lactate and CO2 production and the decrease in glucose 6-phosphate levels in the presence of glucose. All these effects are similar to those previously reported for vanadate, although molybdate and tungstate are less effective than vanadate. These results could indicate that molybdate, tungstate and vanadate act on glucose metabolism in isolated hepatocytes by a similar mechanism of action. PMID:1313228

  7. Regulation of activity and apical targeting of the Cl-/HCO3- exchanger in rat hepatocytes.

    PubMed Central

    Benedetti, A; Strazzabosco, M; Ng, O C; Boyer, J L

    1994-01-01

    To test the hypothesis that rat hepatocyte canalicular Cl-/HCO3- exchange activity might be regulated by HCO3- or protein kinase-induced changes in the apical targeting of vesicles, isolated rat hepatocytes were cultured in the presence or absence of HCO3-/CO2.Cl-/HCO3- exchange activity increased in cells cultured in the presence of HCO3-/CO2 or when stimulated by dibutyryl cAMP. Both of these effects were blocked by either colchicine or the protein kinase C agonist phorbol 12,13-dibutyrate. Fluorescence and confocal microscopy, respectively, revealed increased pericanalicular-apical membrane localization of two canalicular markers, peanut agglutinin and a 110-kDa canalicular ecto-ATPase, when hepatocyte couplets were preincubated in HCO3-/CO2-containing medium, an effect that was again blocked by colchicine. Dibutyryl cAMP also stimulated canalicular localization of the 110-kDa protein. These findings suggest that hepatocyte Cl-/HCO3- exchange activity is regulated by HCO3-/CO2 and by protein kinase A and protein kinase C agonists through microtubule-dependent targeting of vesicles containing this exchanger to the canalicular domain. Images Fig. 3 PMID:8290601

  8. Effects of phenobarbital on metabolism and toxicity of diclofenac sodium in rat hepatocytes in vitro.

    PubMed

    Wang, A G; Xia, T; Yuan, J; Yu, R A; Yang, K D; Chen, X M; Qu, W; Waalkes, M P

    2004-10-01

    Diclofenac sodium (DF-Na) was a nonsteroidal anti-inflammatory drug used in various aspects of inflammatory disease. The purpose of this study was to examine the effects of phenobarbital (PB) on metabolism and toxicity of DF-Na in vitro and explore the potential mechanism of DF-Na induced hepatotoxicity. Rat hepatocytes were isolated by a modification of the two-step in situ collagenase perfusion technique and the harvested rat hepatocytes were cultured with sandwich method. Control or PB (2 mM) pre-treated hepatocytes were incubated with DF-Na (0.1, 0.05 or 0.01 mM) in vitro and cytosolic enzyme leakage levels, cytochrome P450 (CYP) 3A activity, and metabolite content of DF-Na in cell culture medium were measured. The results showed that without any treatment hepatocyte CYP 3A activity gradually decreased with culture time. On day four, CYP 3A activity was 53% of the initial value. The decline of CYP 3A was partially reversed by CYP inducer PB, and the maximum induction of CYP 3A was 2.2-fold over control after continuous exposure of hepatocytes to 2 mM PB for 48 h. Lactic dehydrogenase (LDH), aspartate transaminase (AST), and alanine transamine (ALT) activity and the contents of the DF-Na metabolites 4'-hydroxydiclofenac (4'-OH-DF) and 5-hydroxydiclofenac (5-OH-DF) in media appeared to increase with increasing DF-Na concentrations, though there were no significant differences between DF-Na exposed and control hepatocytes. However, if the hepatocytes first were pre-treated with 2 mM PB for 2 days and then exposed to DF-Na, the concentrations of DF-Na metabolites and the activity of LDH in the media were significantly higher than that of control group. These findings suggest that the hepatotoxicity and metabolism of DF-Na in rat hepatocytes are increased when hepatic CYP 3A activity is increased.

  9. The effect of cysteine oxidation on isolated hepatocytes.

    PubMed Central

    Viña, J; Saez, G T; Wiggins, D; Roberts, A F; Hems, R; Krebs, H A

    1983-01-01

    Isolated hepatocytes incubated with 4mM-cysteine lose reduced glutathione, adenine nucleotides and intracellular enzymes, thus showing extensive membrane damage. The toxic effects of cysteine are enhanced by NH4Cl. Lactate, ethanol and unsaturated fatty acids afford significant protection against cysteine-induced cytoxicity. Addition of catalase to the incubation medium also protected against cysteine toxicity, indicating that H2O2 formed during the oxidation of cysteine is involved in the toxic effects observed. Under anaerobic conditions cysteine did not cause leakage of lactate dehydrogenase from cells, confirming that rapid autoxidation is an essential condition for development of the toxic effects of cysteine. PMID:6870855

  10. Metabolism of cysteine by cyteinesulfinate-independent pathway(s) in rat hepatocytes

    SciTech Connect

    Stipanuk, M.H.; De La Rosa, J.; Drake, M.R.

    1986-05-01

    The metabolism of cysteine (CYS) and that of cysteinesulfinate (CSA) were studied in freshly isolated hepatocytes from fed rats. In incubations of rat hepatocytes with either 1 or 25 mM CSA, over 90% of the /sup 14/CO/sub 2/ formed from (1-/sup 14/C)CSA could be accounted for by production of hypotaurine plus taurine. In similar incubations with 1 or 25 mM CYS, only 4% of /sup 14/CO/sub 2/ evolution from (1-/sup 14/C)CYS could be accounted for by production of hypotaurine plus taurine. Addition of unlabeled CSA inhibited recovery of label from (1-/sup 14/C)CYS as /sup 14/CO/sub 2/ by 33%. Metabolism of CYS and of CSA were affected differently by addition of ..cap alpha..-ketoglutarate, a cosubstrate for transamination, or of propargylglycine, an inhibitor of cystathionase activity. These data suggest that a substantial proportion of CYS is catabolized by CSA-independent pathways in the rat hepatocyte. Although addition of ..cap alpha..-ketoglutarate to incubations of hepatocytes with CSA resulted in a marked increase in CSA catabolism via the transamination pathway, addition of keto acids to incubation systems had little or no effect on production of any metabolite from CYS. Thus, CYS transamination does not appear to be a major pathway of CYS metabolism in the hepatocyte. Inhibition of cystathionase with propargylglycine reduced both /sup 14/CO/sub 2/ production from (1-/sup 14/C)CYS and ammonia plus urea nitrogen production from CYS by about 50%; CSA catabolism was not affected. Thus, cleavage of cyst(e)ine by cystathionase may be an important physiological pathway for CYS catabolism in the liver.

  11. Comparison of rat and hamster hepatocyte primary culture/DNA repair assays

    SciTech Connect

    Kornbrust, D.J.; Barfknect, T.R.

    1984-01-01

    Previous studies have demonstrated marked differences in the capacity of hepatocytes from rats or hamsters to mediate the metabolic activation of chemical carcinogens to genotoxic (i.e., mutagenic) products. Thus far, very few investigations of species differences in DNA repair have been performed. Therefore, a comparison of the relative extent of DNA repair elicited by various genotoxic chemicals in rat and hamster hepatocyes was conducted, using the hepatocyte primary culture/DNA repair (HPC/DR) assay. Of the ll chemicals tested, eight were more potent in inducing DNA repair in hamster hepatocytes than in rat hepatocytes. Dimethylnitrosamine, diethylnitrosamine, 2-acetylaminofluorene, 9-aminoacridine, pararosaniline hydrochloride, 1-naphthylamine, benzidine and 1,2,3,4-diepoxybutane were all active in hamster hepatocytes at a concentration at least ten times less than the lowest effective concentration in rat hepatocytes. The direct-acting alkylating agent, methylmethane sulfonate, was equipotent inducing DNA repair in both rat and hamster hepatocytes, indicating that the differences in DNA repair observed for the other chemicals were probably not a result of species differences in DNA repair capacities. In contrast, 1-nitropyrene produced a greater DNA repair response in rat hepatocyes than hamster hepatocytes, while the bacterial mutagen 3-(chloromethyl)pyridine hydrochloride was inactive in both hepatocyte systems. These studies demonstrate the feasibility of using hamster hepatocytes in the HPC/DR assay and illustrate the utility of performing the assay with hepatocytes from more than one species.

  12. Cryopreservation of isolated human hepatocytes for transplantation: State of the art.

    PubMed

    Terry, Claire; Dhawan, Anil; Mitry, Ragai R; Hughes, Robin D

    2006-10-01

    Hepatocytes isolated from unused donor livers are being used for transplantation in patients with acute liver failure and liver-based metabolic defects. As large numbers of hepatocytes can be prepared from a single liver and hepatocytes need to be available for emergency and repeated treatment of patients it is essential to be able to cryopreserve and store cells with good thawed cell function. This review considers the current status of cryopreservation of human hepatocytes discussing the different stages involved in the process. These include pre-treatment of cells, freezing solution, cryoprotectants and freezing and thawing protocols. There are detrimental effects of cryopreservation on hepatocyte structure and metabolic function, including cell attachment, which is important to the engraftment of transplanted cells in the liver. Cryopreserved human hepatocytes have been successfully used in clinical transplantation, with evidence of replacement of missing function. Further optimisation of hepatocyte cryopreservation protocols is important for their use in hepatocyte transplantation.

  13. Caffeine induces CYP1A2 expression in rat hepatocytes but not in human hepatocytes.

    PubMed

    Vaynshteyn, David; Jeong, Hyunyoung

    2012-06-01

    Caffeine is the active constituent in coffee. Continual consumption of caffeine can lead to an attenuated response also known as tolerance. Results from rat studies have shown that caffeine is an inducer of CYP1A2, the enzyme responsible for caffeine's metabolism. This suggests that CYP1A2 induction by caffeine may be in part responsible for caffeine tolerance. However, whether caffeine induces CYP1A2 expression in humans remains unknown. Our results from luciferase assays performed in HepG2 cells showed that caffeine is not an activator of the aromatic hydrocarbon receptor (AhR), a major transcription factor involved in upregulation of CYP1A2. Furthermore, caffeine did not induce CYP1A2 expression in primary human hepatocytes at a concentration attained by ordinary coffee drinking. On the other hand, caffeine enhanced CYP1A2 expression by 9-fold in rat hepatocytes. Our results suggest that caffeine from ordinary coffee drinking does not induce CYP1A2 expression in humans and that factors other than CYP1A2 induction by caffeine likely contribute to development of caffeine tolerance in humans.

  14. Impaired mitochondrial functions contribute to 3-bromopyruvate toxicity in primary rat and mouse hepatocytes.

    PubMed

    Sobotka, Ondřej; Endlicher, René; Drahota, Zdeněk; Kučera, Otto; Rychtrmoc, David; Raad, Marjan; Hakeem, Khurum; Červinková, Zuzana

    2016-08-01

    A compound with promising anticancer properties, 3-bromopyruvate (3-BP) is a synthetic derivative of a pyruvate molecule; however, its toxicity in non-malignant cells has not yet been fully elucidated. Therefore, we elected to study the effects of 3-BP on primary hepatocytes in monolayer cultures, permeabilized hepatocytes and isolated mitochondria. After a 1-h treatment with 100 μM 3-BP cell viability of rat hepatocytes was decreased by 30 % as measured by the WST-1 test (p < 0.001); after 3-h exposure to ≥200 μM 3-BP lactate dehydrogenase leakage was increased (p < 0.001). Reactive oxygen species production was increased in the cell cultures after a 1-h treatment at concentrations ≥100 μmol/l (p < 0.01), and caspase 3 activity was increased after a 20-h incubation with 150 μM and 200 μM 3-BP (p < 0.001). This toxic effect of 3-BP was also proved using primary mouse hepatocytes. In isolated mitochondria, 3-BP induced a dose- and time-dependent decrease of mitochondrial membrane potential during a 10-min incubation both with Complex I substrates glutamate + malate or Complex II substrate succinate, although this decrease was more pronounced with the latter. We also measured the effect of 3-BP on respiration of isolated mitochondria. ADP-activated respiration was inhibited by 20 μM 3-BP within 10 min. Similar effects were also found in permeabilized hepatocytes of both species.

  15. Hepatocytic parental progenitor cells of rat small hepatocytes maintain self-renewal capability after long-term culture

    PubMed Central

    Ishii, Masayuki; Kino, Junichi; Ichinohe, Norihisa; Tanimizu, Naoki; Ninomiya, Takafumi; Suzuki, Hiromu; Mizuguchi, Toru; Hirata, Koichi; Mitaka, Toshihiro

    2017-01-01

    The liver has a variety of functions for maintaining homeostasis, and hepatocytes play a major role. In contrast with the high regenerative capacity of mature hepatocytes (MHs) in vivo, they have not been successfully expanded ex vivo. Here we demonstrate that CD44-positive cells sorted from small hepatocyte (SH) colonies derived from a healthy adult rat liver can proliferate on a Matrigel-coated dish in serum-free chemically defined medium; in addition, a subpopulation of the cells can divide more than 50 times in a period of 17 weeks every 4-week-passage. The passage cells retained the capability to recover highly differentiated functions, such as glycogen storage, CYP activity and bile secretion. When Matrigel-treated cells from the third passage were transplanted into retrorsine/partial hepatectomy-treated rat livers, the cells engrafted to differentiate into MHs and cholangiocytes. These results suggest that long-term cultured CD44+ SHs retain hepatocytic characteristics in vitro and the capability to differentiate into hepatocytes and cholangiocytes in vivo. Thus, a newly identified subpopulation of MHs possessing the attributes of hepatocytic stem/progenitor cells can be passaged several times without losing hepatocytic characteristics.

  16. In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration.

    PubMed

    Hu, Chenxia; Li, Lanjuan

    2015-08-01

    Various liver diseases result in terminal hepatic failure, and liver transplantation, cell transplantation and artificial liver support systems are emerging as effective therapies for severe hepatic disease. However, all of these treatments are limited by organ or cell resources, so developing a sufficient number of functional hepatocytes for liver regeneration is a priority. Liver regeneration is a complex process regulated by growth factors (GFs), cytokines, transcription factors (TFs), hormones, oxidative stress products, metabolic networks, and microRNA. It is well-known that the function of isolated primary hepatocytes is hard to maintain; when cultured in vitro, these cells readily undergo dedifferentiation, causing them to lose hepatocyte function. For this reason, most studies focus on inducing stem cells, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), hepatic progenitor cells (HPCs), and mesenchymal stem cells (MSCs), to differentiate into hepatocyte-like cells (HLCs) in vitro. In this review, we mainly focus on the nature of the liver regeneration process and discuss how to maintain and enhance in vitro hepatic function of isolated primary hepatocytes or stem cell-derived HLCs for liver regeneration. In this way, hepatocytes or HLCs may be applied for clinical use for the treatment of terminal liver diseases and may prolong the survival time of patients in the near future.

  17. High concentrations of stavudine impair fatty acid oxidation without depleting mitochondrial DNA in cultured rat hepatocytes.

    PubMed

    Igoudjil, Anissa; Massart, Julie; Begriche, Karima; Descatoire, Véronique; Robin, Marie-Anne; Fromenty, Bernard

    2008-06-01

    The antiretroviral nucleoside reverse-transcriptase inhibitor (NRTI) stavudine (d4T) can induce mild to severe liver injuries such as steatosis (i.e. triglyceride accumulation), steatohepatitis and liver failure. NRTI-induced toxicity has been ascribed to the inhibition of mitochondrial DNA (mtDNA) replication causing mtDNA depletion and respiratory chain dysfunction. This can secondarily impair the tricarboxylic acid cycle and fatty acid oxidation (FAO), thus leading to lactic acidosis and hepatic steatosis. However, NRTIs could also impair mitochondrial function and induce hepatic steatosis through other mechanisms. In this study, we sought to determine whether d4T could inhibit mitochondrial FAO and induce triglyceride accumulation through a mtDNA-independent mechanism. Since human tumoral and non-tumoral hepatic cell lines were unable to efficiently oxidize palmitic acid, the effects of d4T on mitochondrial FAO were assessed on cultured rat hepatocytes. Our results showed that 750 microM of d4T significantly inhibited palmitic acid oxidation after 48 or 72 h of culture, without inducing cell death. Importantly, high concentrations of zidovudine and zalcitabine (two other NRTIs that can induce hepatic steatosis), or beta-aminoisobutyric acid (a d4T metabolite), did not impair FAO in rat hepatocytes. D4T-induced FAO inhibition was observed without mtDNA depletion and lactate production, and was fully prevented with l-carnitine or clofibrate coincubation. l-carnitine also prevented the accretion of neutral lipids within rat hepatocytes. High concentrations of d4T were unable to inhibit FAO on freshly isolated liver mitochondria. Moreover, a microarray analysis was performed to clarify the mechanism whereby d4T can inhibit mitochondrial FAO and induce triglyceride accumulation in rat hepatocytes. The microarray data, confirmed by quantitative real-time PCR analysis, showed that d4T increased the expression of sterol regulatory element-binding protein-1c (SREBP1c

  18. Insulin-Regulated Srebp-1c and Pck1 mRNA Expression in Primary Hepatocytes from Zucker Fatty but Not Lean Rats Is Affected by Feeding Conditions

    PubMed Central

    Zhang, Yan; Chen, Wei; Li, Rui; Li, Yang; Ge, Yuebin; Chen, Guoxun

    2011-01-01

    Insulin regulates the transcription of genes for hepatic glucose and lipid metabolism. We hypothesized that this action may be impaired in hepatocytes from insulin resistant animals. Primary hepatocytes from insulin sensitive Zucker lean (ZL) and insulin resistant Zucker fatty (ZF) rats in ad libitum or after an overnight fasting were isolated, cultured and treated with insulin and other compounds for analysis of gene expression using real-time PCR. The mRNA levels of one insulin-induced (Srebp-1c) and one insulin-suppressed (Pck1) genes in response to insulin, glucagon, and compactin treatments in hepatocytes from ad libitum ZL and ZF rats were analyzed. Additionally, the effects of insulin and T1317 on their levels in hepatocytes from ad libitum or fasted ZL or ZF rats were compared. The mRNA levels of Srebp-1c, Fas, and Scd1, but not that of Insr, Gck and Pck1, were higher in freshly isolated hepatocytes from ad libitum ZF than that from ZL rats. These patterns of Srebp-1c and Pck1 mRNA levels remained in primary hepatocyte cultured in vitro. Insulin's ability to regulate Srebp-1c and Pck1 expression was diminished in hepatocytes from ad libitum ZF, but not ZL rats. Glucagon or compactin suppressed Srebp-1c mRNA expression in lean, but not fatty hepatocytes. However, glucagon induced Pck1 mRNA expression similarly in hepatocytes from ad libitum ZL and ZF rats. Insulin caused the same dose-dependent increase of Akt phosphorylation in hepatocytes from ad libitum ZL and ZF rats. It synergized with T1317 to induce Srebp-1c, and suppressed Pck1 mRNA levels in hepatocytes from fasted, but not that from ad libitum ZF rats. We demonstrated that insulin was unable to regulate its downstream genes' mRNA expression in hepatocytes from ad libitum ZF rats. This impairment can be partially restored in hepatocytes from ZF rats after an overnight fasting, a phenomenon that deserves further investigation. PMID:21731709

  19. Cytotoxic mechanisms of hydrosulfide anion and cyanide anion in primary rat hepatocyte cultures.

    PubMed

    Thompson, Rodney W; Valentine, Holly L; Valentine, William M

    2003-06-30

    Hydrogen sulfide and hydrogen cyanide are known to compromise mitochondrial respiration through inhibition of cytochrome c oxidase and this is generally considered to be their primary mechanism of toxicity. Experimental studies and the efficiency of current treatment protocols suggest that H(2)S may exert adverse physiological effects through additional mechanisms. To evaluate the role of alternative mechanisms in H(2)S toxicity, the relative contributions of electron transport inhibition, uncoupling of mitochondrial respiration, and opening of the mitochondrial permeability transition pore (MPTP) to hydrosulfide and cyanide anion cytotoxicity in primary hepatocyte cultures were examined. Supplementation of hepatocytes with the glycolytic substrate, fructose, rescued hepatocytes from cyanide anion induced toxicity, whereas fructose supplementation increased hydrosulfide anion toxicity suggesting that hydrosulfide anion may compromise glycolysis in hepatocytes. Although inhibitors of the MPTP opening were protective for hydrosulfide anion, they had no effect on cyanide anion toxicity, consistent with an involvement of the permeability transition pore in hydrosulfide anion toxicity but not cyanide anion toxicity. Exposure of isolated rat liver mitochondria to hydrosulfide did not result in large amplitude swelling suggesting that if H(2)S induces the permeability transition it does so indirectly through a mechanism requiring other cellular components. Hydrosulfide anion did not appear to be an uncoupler of mitochondrial respiration in hepatocytes based upon the inability of oligomycin and fructose to protect hepatocytes from hydrosulfide anion toxicity. These findings support mechanisms additional to inhibition of cytochrome c oxidase in hydrogen sulfide toxicity. Further investigations are required to assess the role of the permeability transition in H(2)S toxicity, determine whether similar affects occur in other cell types or in vivo and evaluate whether this may

  20. Effect of Dimethyl Sulfoxide and Melatonin on the Isolation of Human Primary Hepatocytes.

    PubMed

    Solanas, Estela; Sostres, Carlos; Serrablo, Alejandro; García-Gil, Agustín; García, Joaquín J; Aranguren, Francisco J; Jiménez, Pilar; Hughes, Robin D; Serrano, María T

    2015-01-01

    The availability of fully functional human hepatocytes is critical for progress in human hepatocyte transplantation and the development of bioartificial livers and in vitro liver systems. However, the cell isolation process impairs the hepatocyte status and determines the number of viable cells that can be obtained. This study aimed to evaluate the effects of using dimethyl sulfoxide (DMSO) and melatonin in the human hepatocyte isolation protocol. Human hepatocytes were isolated from liver pieces resected from 10 patients undergoing partial hepatectomy. Each piece was dissected into 2 equally sized pieces and randomized, in 5 of 10 isolations, to perfusion with 1% DMSO-containing perfusion buffer or buffer also containing 5 mM melatonin using the 2-step collagenase perfusion technique (experiment 1), and in the other 5 isolations to standard perfusion or perfusion including 1% DMSO (experiment 2). Tissues perfused with DMSO yielded 70.6% more viable hepatocytes per gram of tissue (p = 0.076), with a 26.1% greater albumin production (p < 0.05) than those perfused with control buffer. Melatonin did not significantly affect (p > 0.05) any of the studied parameters, but cell viability, dehydrogenase activity, albumin production, urea secretion, and 7-ethoxycoumarin O-deethylase activity were slightly higher in cells isolated with melatonin-containing perfusion buffer compared to those isolated with DMSO. In conclusion, addition of 1% DMSO to the hepatocyte isolation protocol could improve the availability and functionality of hepatocytes for transplantation, but further studies are needed to clarify the mechanisms involved.

  1. Prostaglandins E2 and F2 alpha increase fructose 2,6-bisphosphate levels in isolated hepatocytes.

    PubMed Central

    Gómez-Foix, A M; Rodríguez-Gil, J E; Guinovart, J J; Bosch, F

    1991-01-01

    In hepatocytes isolated from fed rats, prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) increased, in a time- and dose-dependent manner, fructose 2,6-bisphosphate [Fru(2,6)P2] levels and stimulated the glycolytic flux. The rise in Fru(2,6)P2 was related to an increase in glucose 6-phosphate levels which resulted from the stimulation of glycogenolysis. In cells obtained from 24 h-starved rats, no effects of either PGE2 or PGF2 alpha could be observed. In addition, when the stimulation of glycogenolysis was abolished by incubation of fed-rat hepatocytes in a Ca2(+)-depleted medium, Fru(2,6)P2 levels did not increase. Furthermore, no effects of PGs on 6-phosphofructo-2-kinase activity could be observed. These results indicate that PGE2 and PGF2 alpha show similar actions to Ca2(+)-dependent hormones on hepatic glucose metabolism. PMID:2001249

  2. Free fatty acid inhibition of the insulin induction of glucose-6-phosphate dehydrogenase in rat hepatocyte monolayers.

    PubMed

    Salati, L M; Adkins-Finke, B; Clarke, S D

    1988-01-01

    Rat hepatocytes in monolayer culture were utilized to determine if the decrease in glucose-6-phosphate dehydrogenase (G6PD) activity resulting from the ingestion of fat can be mimicked by the addition of fatty acids to a chemically, hormonally defined medium. G6PD activity in cultured hepatocytes was induced several-fold by insulin. Dexamethasone or T3 did not amplify the insulin induction of G6PD. Glucose alone increased G6PD activity in cultured hepatocytes from fasted donors by nearly 500%. Insulin in combination with glucose induced G6PD an additional two-fold. The increase in G6PD activity caused by glucose was greater in hepatocytes isolated from 72 hr-fasted rats as compared to fed donor rats. Such a response was reminiscent of the "overshoot" phenomenon in which G6PD activity is induced well above the normal level by fasting-refeeding rats a high glucose diet. Addition of linoleate to the medium resulted in a significant suppression of insulin's ability to induce G6PD, but linoleate had no effect on the induction of G6PD activity by glucose alone. A shift to the right in the insulin-response curve for the induction of G6PD also was detected for the induction of malic enzyme and acetyl-CoA carboxylase. Arachidonate (0.25 mM) was a significantly more effective inhibitor of the insulin action than linoleate was. Apparently rat hepatocytes in monolayer culture can be utilized as a model to investigate the molecular mechanism by which fatty acids inhibit the production of lipogenic enzymes. In part, this mechanism of fatty acid inhibition involves desensitization of hepatocytes to the lipogenic action of insulin.

  3. Elevated level of. beta. -adrenergic receptors in hepatocytes from regenerating rat liver

    SciTech Connect

    Sandnes, D.; Sand, T.E.; Sager, G.; Broenstad, G.O.; Refsnes, M.R.; Gladhaug, I.P.; Jacobsen, S.; Christoffersen, T.

    1986-01-01

    Hepatocytes from regenerating rat liver show an enhanced epinephrine-sensitive adenylate cyclase activity and cAMP response, which may be involved in triggering of the cell proliferation. We have determined adrenergic receptors and adenylate cyclase activity in hepatocytes isolated at various time points after partial hepatectomy. The number of ..beta..-adrenergic receptors, measured by binding of (/sup 125/I)iodocyanopindolol ((/sup 125/I)CYP) to a particulate fraction prepared from isolated hepatocytes, increased rapidly after partial hepatectomy as compared with sham-operated or untreated controls. The maximal increase, which was observed at 48 h, was between 5- and 6-fold (from approx.1800 to approx.10,500 sites per cell). Thereafter, the number of ..beta..- adrenergic receptors decreased gradually. Competition experiments indicated ..beta../sub 2/-type receptors. Parallelism was found between the change in the number of ..beta../sub 2/-adrenergic receptors and the isoproterenol-responsive adenylate cyclase activity. The number of ..cap alpha../sub 1/-adrenergic receptors, determined by binding of (/sup 3/H)prazosin, was transiently lowered by about 35% at 18-24 h. with no significant change in K/sub d/. Although the results of this study do not exclude the possibility of post-receptor events, they suggest that the increased number of..beta../sub 2/-adrenergic receptors is a major factor responsible for the enhanced catecholamine-responsive adenylate cyclase activity in regenerating liver.

  4. Species-specific toxicity of troglitazone on rats and human by gel entrapped hepatocytes

    SciTech Connect

    Shen, Chong; Meng, Qin; Zhang, Guoliang

    2012-01-01

    Troglitazone, despite passing preclinical trials on animals, was shortly withdrawn from market due to its severe hepatotoxicity in clinic. As rat hepatocyte monolayer consistently showed sensitive troglitazone toxicity as human hepatocyte monolayer in contrast to the species-specific toxicity in vivo, this paper utilized both hepatocytes in three-dimensional culture of gel entrapment to reflect the species difference on hepatotoxicity. Rat hepatocytes in gel entrapment did not show obvious cellular damage even under a long-term exposure for 21 days while gel entrapped human hepatocytes significantly displayed oxidative stress, steatosis, mitochondrial damage and cell death at a short exposure for 4 days. As a result, the detected species-specific toxicity of troglitazone between gel entrapped rat and human hepatocytes consisted well with the situation in vivo but was in a sharp contrast to the performance of two hepatocytes by monolayer culture. Such contradictory toxicity of rat hepatocytes between monolayer and gel entrapment culture could be explained by the fact that troglitazone was cleared more rapidly in gel entrapment than in monolayer culture. Similarly, the differential clearance of troglitazone in rat and human might also explain its species-specific toxicity. Therefore, gel entrapment of hepatocytes might serve as a platform for evaluation of drug toxicity at early stage of drug development by reducing costs, increasing the likelihood of clinical success and limiting human exposure to unsafe drugs. -- Highlights: ► Species-specific toxicity of troglitazone reflected by rat/human hepatocytes ► 3D hepatocytes in 21 days’ long-term culture used for drug hepatotoxicity ► Oversensitive toxicity in hepatocyte monolayer by slow troglitazone clearance.

  5. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

    SciTech Connect

    Dever, Joseph T.; Elfarra, Adnan A.

    2009-05-01

    L-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 {sup o}C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increases in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-D-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-DL-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

  6. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: potential roles in toxicity.

    PubMed

    Dever, Joseph T; Elfarra, Adnan A

    2009-05-01

    L-methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 degrees C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increases in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-d-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-dl-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

  7. Toxicity of microcystins in the isolated hepatocytes of common carp (Cyprinus carpio L.).

    PubMed

    Li, Xiao-Yu; Wang, Jie; Liang, Jun-Bo; Liu, Yong-Ding

    2007-07-01

    The toxicity of hepatotoxic microcystins produced mainly by Microcystis aeruginosa in mammals and fishes was well studied in recent years. However, there were scarcely reports in toxic effects of microcystins on isolated hepatocytes of fishes, especially investigation of microcystin-induced apoptosis and/or necrosis in carp hepatocytes. In the present study, the isolated hepatocytes of common carp were exposed to various concentrations of microcystins (0.01, 0.1, 1, 10, 100, 1000 microg L(-1)) for 2, 4, 8, 16 and 24h, respectively, and cytotoxicity of microcystins in the toxin-treated cells was determined. Results of this study showed that cytotoxicity of microcystins on carp hepatocytes was time and dose-dependent, and the approximate LC(50) of microcystins in carp hepatocytes was 169.2 microg L(-1). The morphological changes typical of apoptosis, such as blebbing of cell membrane, condensation and fragmentation of cell nucleus were observed in the hepatocytes exposed to microcystins (1, 10 and 100 microg L(-1)) using fluorescence and differential interference contrast microscopy. Agarose gel electrophoresis of DNA demonstrated a typical apoptotic "ladder pattern" in microcystin-treated hepatocytes after 16 h of exposure. Results of the present study indicated that the form of cell death in microcystin-treated hepatocytes depend on the exposure dose of toxin. When lower concentration of microcystins (10 and 100 microg L(-1)) was used for exposure, carp hepatocytes died in apoptosis while, when higher one used (1000 microg L(-1)), they died in the form of necrosis.

  8. Investigation of functional and morphological integrity of freshly isolated and cryopreserved human hepatocytes.

    PubMed

    Ostrowska, A; Bode, D C; Pruss, J; Bilir, B; Smith, G D; Zeisloft, S

    2000-01-01

    There is a pressing need for alternative therapeutic methods effective in the treatment of patients with liver insufficiency. Isolated human hepatocytes may be a viable alternative or adjunct to orthotopic liver transplantation in such patients. The purpose of this study was to evaluate the viability and functional integrity of freshly isolated and cryopreserved human hepatocytes, in preparation for a multi-center human hepatocyte transplantation trial. We are currently processing transplant-grade human parenchymal liver cells from nondiseased human livers that are obtained through a network of organ procurement organizations (OPOs). Thus far, sixteen hepatocyte transplants have been performed using hepatocytes processed by our methods. At the time of referral all specimens were deemed unsuitable for transplantation due to anatomical anomalies, high fat content, medical history, etc. Hepatocytes were isolated from encapsulated liver sections by a modified two-step perfusion technique. Isolated cells were cryopreserved and stored in liquid nitrogen for one to twelve months. The total yield of freshly isolated hepatocytes averaged 3.7x10(7) cells per gram of wet tissue. Based on trypan blue exclusion, fresh preparations contained an average of 85% viable hepatocytes vs. 70% in cryopreserved samples. The plating efficiencies of cells seeded immediately after isolation ranged from 87% to 98%, while those of cryopreserved/thawed cells were markedly lower. Flow cytometry analysis of cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that there was no significant difference in viability compared with trypan blue staining. Both freshly isolated hepatocytes and those recovered from cryopreservation showed typical and intact morphology as demonstrated by light and electron microscopy. The product of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reaction was always expressed more intensely in cultures of freshly

  9. Metabolism and Toxicity of Thioacetamide and Thioacetamide S-Oxide in Rat Hepatocytes

    PubMed Central

    Hajovsky, Liz; Hu, Gang; Koen, Yakov; Sarma, Diganta; Cui, Wenqi; Moore, David S.; Staudinger, Jeff L.; Hanzlik, Robert P.

    2012-01-01

    The hepatotoxicity of thioacetamide (TA) has been known since 1948. In rats, single doses cause centrilobular necrosis accompanied by increases in plasma transaminases and bilirubin. To elicit these effects TA requires oxidative bioactivation leading first to its S-oxide (TASO) and then to its chemically reactive S,S-dioxide (TASO2) which ultimately modifies amine-lipids and proteins. To generate a suite of liver proteins adducted by TA metabolites for proteomic analysis, and to reduce the need for both animals and labeled compounds, we treated isolated hepatocytes directly with TA. Surprisingly, TA was not toxic at concentrations up to 50 mM for 40 hr. On the other hand, TASO was highly toxic to isolated hepatocytes as indicated by LDH release, cellular morphology and vital staining with Hoechst 33342/propidium iodide. TASO toxicity was partially blocked by the CYP2E1 inhibitors diallyl sulfide and 4-methylpyrazole, and was strongly inhibited by TA. Significantly, we found that hepatocytes produce TA from TASO relatively efficiently by back-reduction. The covalent binding of [14C]-TASO is inhibited by unlabeled TA which acts as a “cold-trap” for [14C]-TA and prevents its re-oxidation to [14C]-TASO. This in turn increases the net consumption of [14C]-TASO despite the fact that its oxidation to TASO2 is inhibited. The potent inhibition of TASO oxidation by TA, coupled with the back-reduction of TASO and its futile redox cycling with TA may help explain phenomena previously interpreted as “saturation toxicokinetics” in the in vivo metabolism and toxicity of TA and TASO. The improved understanding of the metabolism and covalent binding of TA and TASO facilitates the use of hepatocytes to prepare protein adducts for target protein identification. PMID:22867114

  10. Curcumin inhibits activation of TRPM2 channels in rat hepatocytes

    PubMed Central

    Kheradpezhouh, E.; Barritt, G.J.; Rychkov, G.Y.

    2015-01-01

    Oxidative stress is a hallmark of many liver diseases including viral and drug-induced hepatitis, ischemia-reperfusion injury, and non-alcoholic steatohepatitis. One of the consequences of oxidative stress in the liver is deregulation of Ca2+ homeostasis, resulting in a sustained elevation of the free cytosolic Ca2+ concentration ([Ca2+]c) in hepatocytes, which leads to irreversible cellular damage. Recently it has been shown that liver damage induced by paracetamol and subsequent oxidative stress is, in large part, mediated by Ca2+ entry through Transient Receptor Potential Melastatin 2 (TRPM2) channels. Involvement of TRPM2 channels in hepatocellular damage induced by oxidative stress makes TRPM2 a potential therapeutic target for treatment of a range of oxidative stress-related liver diseases. We report here the identification of curcumin ((1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), a natural plant-derived polyphenol in turmeric spice, as a novel inhibitor of TRPM2 channel. Presence of 5 µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca2+]c rise in rat hepatocytes. Furthermore, in patch clamping experiments incubation of hepatocytes with curcumin inhibited activation of TRPM2 current by intracellular ADPR with IC50 of approximately 50 nM. These findings enhance understanding of the actions of curcumin and suggest that the known hepatoprotective properties of curcumin are, at least in part, mediated through inhibition of TRPM2 channels. PMID:26609559

  11. Curcumin inhibits activation of TRPM2 channels in rat hepatocytes.

    PubMed

    Kheradpezhouh, E; Barritt, G J; Rychkov, G Y

    2016-04-01

    Oxidative stress is a hallmark of many liver diseases including viral and drug-induced hepatitis, ischemia-reperfusion injury, and non-alcoholic steatohepatitis. One of the consequences of oxidative stress in the liver is deregulation of Ca(2+) homeostasis, resulting in a sustained elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)]c) in hepatocytes, which leads to irreversible cellular damage. Recently it has been shown that liver damage induced by paracetamol and subsequent oxidative stress is, in large part, mediated by Ca(2+) entry through Transient Receptor Potential Melastatin 2 (TRPM2) channels. Involvement of TRPM2 channels in hepatocellular damage induced by oxidative stress makes TRPM2 a potential therapeutic target for treatment of a range of oxidative stress-related liver diseases. We report here the identification of curcumin ((1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), a natural plant-derived polyphenol in turmeric spice, as a novel inhibitor of TRPM2 channel. Presence of 5µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca(2+)]c rise in rat hepatocytes. Furthermore, in patch clamping experiments incubation of hepatocytes with curcumin inhibited activation of TRPM2 current by intracellular ADPR with IC50 of approximately 50nM. These findings enhance understanding of the actions of curcumin and suggest that the known hepatoprotective properties of curcumin are, at least in part, mediated through inhibition of TRPM2 channels.

  12. Glutathione (GSH) production is increased in copper deficient isolated hepatocytes and inhibition of GSH synthesis decreases intracellular cholesterol

    SciTech Connect

    Pi Yu Chao; Allen, K.G.D )

    1991-03-15

    Thirty male weanling Sprague Dawley rats were assigned to three groups of 10 animals each and fed AIN based copper deficient ad libitum, copper control meal fed and copper deficient meal fed diets. 12h meals were provided. Beginning on day 54 animals were used to prepare isolated hepatocytes, following a 4h meal, by portal vein collagenase infusion. Washed isolated hepatocytes were incubated in Krebs Henseleit bicarbonate buffer pH 7.4 under 95/5 O{sub 2}/CO{sub 2} at 37{degree} with 2.5 mM Ca, 0.75% gelatin and 10 mM glucose, either with or without 1 mM buthionine sulfoximine (BSO) a specific inhibitor of GSH synthesis. Hepatocyte viability was assessed by trypan blue exclusion. Copper deficiency significantly increased GSH production by 30-50% at 1.5 and 3 h, with no changes in GSSG. GSH production was inhibited by BSO which also significantly inhibited intracellular cholesterol appearance at 3 h. The intracellular (GHS){sup 2}/GSSG at 3 hr showed a significant correlation with cholesterol appearance rate at 3 h. Copper deficiency increases hepatocyte GSH production. Inhibition of GSH production is correlated with decreased cholesterol appearance.

  13. Persistence and accumulation of micronucleated hepatocytes in liver of rats after repeated administration of diethylnitrosamine.

    PubMed

    Narumi, Kazunori; Ashizawa, Koji; Fujiishi, Yohei; Tochinai, Ryota; Okada, Emiko; Tsuzuki, Yasuhiro; Tatemoto, Hideki; Hamada, Shuichi; Kaneko, Kimiyuki; Ohyama, Wakako

    2013-08-15

    A repeat-dose micronucleus assay in adult rat liver was recently developed [Mutat. Res. 747 (2012) 234-239]. This assay demonstrated a high detectability of hepatocarcinogens at relatively low doses, as indicated by dose-dependent micronucleus induction. Because the adult rat liver is known to have a long life-span, this desirable property of the assay will be an advantage in detecting micronucleated hepatocytes (MNHEPs) that have persisted for long periods in the liver following repeated dosing. However, no data directly supporting the underlying mechanisms have been published to date. In the present study, we verified the mechanisms by means of pulse-labeling of micronucleated hepatocytes with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU). The rodent hepatocarcinogen diethylnitrosamine (DEN) was repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old) for up to 2 weeks, and EdU was injected intraperitoneally on days 1, 7, or 14. Hepatocytes were isolated by use of a non-perfusion technique at 24h, 1 week, or 2 weeks after EdU injection and analyzed for EdU incorporation and micronucleus formation. The results of our study confirmed that MNHEPs labeled with EdU on the first day of DEN administration persisted until 2 weeks post-administration in the rat livers. However, the frequency of MHNEPs among EdU-labeled hepatocytes decreased over time. In addition, the number of terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive cells in the liver tissue increased, suggesting selective removal of micronucleated cells. Theoretical calculation of the cumulative MNHEP frequency on each of the days on which DEN was administered, taking into account the rate of loss, came out closer to the actual value observed in the liver micronucleus test. Taken together, these results indicate that although micronucleated cells induced in rat livers by administration of the genotoxic hepatocarcinogen DEN undergo selective removal, they

  14. AMP-activated protein kinase counteracted the inhibitory effect of glucose on the phosphoenolpyruvate carboxykinase gene expression in rat hepatocytes.

    PubMed

    Hubert, A; Husson, A; Chédeville, A; Lavoinne, A

    2000-09-22

    The effect of AMP-activated protein kinase (AMPK) in the regulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene expression was studied in isolated rat hepatocytes. Activation of AMPK by AICAR counteracted the inhibitory effect of glucose on the PEPCK gene expression, both at the mRNA and the transcriptional levels. It is proposed that a target for AMPK is involved in the inhibitory effect of glucose on PEPCK gene transcription.

  15. Gel entrapment culture of rat hepatocytes for investigation of tetracycline-induced toxicity

    SciTech Connect

    Shen Chong; Meng Qin Schmelzer, Eva; Bader, Augustinus

    2009-07-15

    This paper aimed to explore three-dimensionally cultured hepatocytes for testing drug-induced nonalcoholic steatohepatitis. Gel entrapped rat hepatocytes were applied for investigation of the tetracycline-induced steatohepatitis, while hepatocyte monolayer was set as a control. The toxic responses of hepatocytes were systematically evaluated by measuring cell viability, liver-specific function, lipid accumulation, oxidative stress, adenosine triphosphate content and mitochondrial membrane potential. The results suggested that gel entrapped hepatocytes showed cell death after 96 h of tetracycline treatment at 25 {mu}M which is equivalent to toxic serum concentration in rats, while hepatocyte monolayer showed cell death at a high dose of 200 {mu}M. The concentration-dependent accumulation of lipid as well as mitochondrial damage were regarded as two early events for tetracycline hepatotoxicity in gel entrapment culture due to their detectability ahead of subsequent increase of oxidative stress and a final cell death. Furthermore, the potent protection of fenofibrate and fructose-1,6-diphosphate were evidenced in only gel entrapment culture with higher expressions on the genes related to {beta}-oxidation than hepatocyte monolayer, suggesting the mediation of lipid metabolism and mitochondrial damage in tetracycline toxicity. Overall, gel entrapped hepatocytes in three-dimension reflected more of the tetracycline toxicity in vivo than hepatocyte monolayer and thus was suggested as a more relevant system for evaluating steatogenic drugs.

  16. COVALENT BINDING OF TRICHLOROETHYLENE TO PROTEINS IN HUMAN AND RAT HEPATOCYTES. (R826409)

    EPA Science Inventory

    The environmental contaminant and occupational solvent trichloroethylene is metabolized to a reactive intermediate that covalently binds to specific hepatic proteins in exposed mice and rats. In order to compare covalent binding between humans and rodents, primary hepatocyte c...

  17. Rat-derived amniotic epithelial cells differentiate into mature hepatocytes in vivo with no evidence of cell fusion.

    PubMed

    Marongiu, Michela; Serra, Maria Paola; Contini, Antonella; Sini, Marcella; Strom, Stephen C; Laconi, Ezio; Marongiu, Fabio

    2015-06-15

    Amniotic epithelial cells (AEC) derived from human placenta represent a useful and noncontroversial source for liver-based regenerative medicine. Previous studies suggested that human- and rat-derived AEC differentiate into hepatocyte-like cells upon transplantation. In the retrorsine (RS) model of liver repopulation, clusters of donor-derived cells engrafted in the recipient liver and, importantly, showed characteristics of mature hepatocytes. The aim of the current study was to investigate the possible involvement of cell fusion in the emergence of hepatocyte clusters displaying a donor-specific phenotype. To this end, 4-week-old GFP(+)/DPP-IV(-) rats were treated with RS and then transplanted with undifferentiated AEC isolated from the placenta of DPP-IV(+) pregnant rats at 16-19 days of gestational age. Results indicated that clusters of donor-derived cells were dipeptidyl peptidase type IV (DPP-IV) positive, but did not express the green fluorescent protein (GFP), suggesting that rat amniotic epithelial cells (rAEC) did not fuse within the host parenchyma, as no colocalization of the two tags was observed. Moreover, rAEC-derived clusters expressed markers of mature hepatocytes (eg, albumin, cytochrome P450), but were negative for the expression of biliary/progenitor markers (eg, epithelial cell adhesion molecule [EpCAM]) and did not express the marker of preneoplastic hepatic nodules glutathione S-transferase P (GST-P). These results extend our previous findings on the potential of AEC to differentiate into mature hepatocytes and suggest that this process can occur in the absence of cell fusion with host-derived cells. These studies support the hypothesis that amnion-derived epithelial cells can be an effective cell source for the correction of liver disease.

  18. Detection of DNA single-strand breaks induced by procarcinogens in Chinese hamster ovary cells cocultured with rat hepatocytes

    SciTech Connect

    Yang, K.H.; Shin, C.G.; Choe, S.Y.; Kim, D.H.

    1984-01-01

    DNA single-strand breaks induced by procarcinogens were detected in Chinese hamster overy (CHO) cell cocultured with adult rat hepatocytes. Freshly isolated adult rat hepatocytes were added to the CHO cell culture prelabeled with (/sup 3/H) thymidine. After allowing the hepatocytes to attach on or near the CHO cells, aflatoxin B/sub 1/ or benzo(a)pyrene was added to the culture and incubated for the desired time. DNA single-strand breaks in CHO cells were measured by the alkaline elution technique. Aflatoxin B/sub 1/ induced some DNA single-strand breaks in CHO cells cultured alone, but in coculture system with hepatocytes the number of DNA single-strand breaks increased greatly. The magnitude of the increase was related to the dose and the time of exposure to aflatoxin B/sub 1/. Addition of proteinase-K to the cell lysates increased the elution of DNA compared to that of samples without proteinase-K. Benzo(a)pyrene did not induce any DNA single-strand breaks in CHO cells in the absence of liver cells, but a significant number of single-strand breaks were detected in the coculture system.

  19. Latency is the major determinant of UDP-glucuronosyltransferase activity in isolated hepatocytes.

    PubMed

    Bánhegyi, G; Garzó, T; Fulceri, R; Benedetti, A; Mandl, J

    1993-08-09

    The glucuronidation of p-nitrophenol was measured in intact, saponin- and alamethicin-treated isolated mouse hepatocytes. In saponin-permeabilized cells the elevation of extrareticular UDP-glucuronic acid concentration enhanced the rate of glucuronidation threefold. When intracellular membranes were also permeabilized by alamethicin, a further tenfold increase in the glucuronidation of p-nitrophenol was present. Parallel measurements of the ER mannose 6-phosphatase activity revealed that saponin selectively permeabilized the plasma membrane, whereas alamethicin permeabilized both plasma membrane and ER membranes. The inhibition of p-nitrophenol glucuronidation by dbcAMP in intact hepatocytes was still present in saponin-treated cells and disappeared in alamethicin-permeabilized hepatocytes. It is suggested that the permeability of the endoplasmic reticulum membrane is a major determinant of glucuronidation not only in microsomes but in isolated hepatocytes as well.

  20. Induction of unscheduled DNA synthesis in suspensions of rat hepatocytes by an environmental toxicant, 3,3'4,4'-tetrachloroazobenzene.

    PubMed

    Hsia, M T; Kreamer, B L

    1979-04-01

    Unscheduled DNA synthesis was induced by 3,3'4,4'-tetrachloroazobenzene (TCAB)) in freshly isolated suspensions of rat hepatocytes. A dose-dependent response was demonstrated. Hepatocellular DNA was obtained after the chloroform-isoamyl alchohol-phenol extraction of the isolated nuclei. The induction of unscheduled DNA synthesis was measured by the incorporation of [3H]-thymidine in the presence of hydroxyurea as determined by the scintillation counting assay. DNA repair data obtained in this study on benzo[a]pyrene and methyl methanesulfonate are comparable to a previous report using primary cultures of hepatocytes and cesium chloride gradients. Hence, the present method offers promise as a rapid and sensitive screen for chemical carcinogens.

  1. Pancreastatin increases free cytosolic Ca2+ in rat hepatocytes, involving both pertussis-toxin-sensitive and -insensitive mechanisms.

    PubMed Central

    Sánchez-Margalet, V; Lucas, M; Goberna, R

    1993-01-01

    Freshly isolated rat hepatocytes, loaded with the Ca2+ probe Fluo-3, responded to homologous pancreastatin with a sudden increase in free cytosolic Ca2+ ([Ca2+]i) as well as glucose release. Addition of rat pancreastatin (0.1 microM) to hepatocytes resulted in an increase in [Ca2+]i from 150 nM to 700 nM, which declined back to nearly basal values within 2-3 min. Half-maximal and maximal effects were observed at 0.3 and 100 nM pancreastatin respectively. The increase in [Ca2+]i induced by vasopressin and noradrenaline was very similar in extent (from 150 to 800 nM) to that produced by pancreastatin. Neither the alpha 1-adrenergic blocker prazosin nor the vasopressin antagonist V1 modified the increase in [Ca2+]i induced by pancreastatin. Pig pancreastatin and its 33-49 C-terminal fragment produced about 65 and 75% of the effect of homologous pancreastatin respectively. Glucose production correlated with changes in [Ca2+]i in the same order of potency: vasopressin > rat pancreastatin > pig 33-49 pancreastatin > pig 1-49 pancreastatin. The effect of pancreastatin on [Ca2+]i was decreased by 50% when Ca2+ was omitted from the medium, and totally abolished when hepatocytes were depleted of internal Ca2+ stores by preincubation without Ca2+ and with 2 mM EGTA. When hepatocytes were preincubated for 5 min with PMA, the effects of ATP and noradrenaline were prevented, and those of vasopressin and pancreastatin remained unchanged. The pretreatment of hepatocytes with pertussis toxin diminished the response to pancreastatin and vasopressin. These results suggest that pancreastatin is a new Ca(2+)-mobilizing glycogenolytic hormone acting through a specific receptor which may involve both pertussis-toxin-sensitive and -insensitive GTP-binding regulatory proteins. PMID:8373359

  2. Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes.

    PubMed Central

    Bassuk, J A; Tsichlis, P N; Sorof, S

    1987-01-01

    Hepatocytes in normal rat liver were found previously to contain a cytoplasmic 14,000-dalton polypeptide (p14) that is associated with mitosis and is the principal early covalent target of activated metabolites of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene). The level of immunohistochemically detected p14 was low when growth activity of hepatocytes was low, was markedly elevated during mitosis in normal and regenerating livers, but was very high throughout interphase during proliferation of hyperplastic and malignant hepatocytes induced in rat liver by a carcinogen (N-2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene). We report here that p14 is the liver fatty acid binding protein. The nucleotide sequence of p14 cDNA clones, isolated by screening a rat liver cDNA library in bacteriophage lambda gt11 using p14 antiserum, was completely identical to part of the sequence reported for liver fatty acid binding protein. Furthermore, the two proteins shared the following properties: size of mRNA, amino acid composition, molecular size according to NaDodSO4 gel electrophoresis, and electrophoretic mobilities in a Triton X-100/acetic acid/urea gel. Their pI values overlapped in 2-dimensional isoelectric focusing/NaDodSO4 gel electrophoresis and showed the same response to delipidation. Either polypeptide reacted with and blocked the antiserum raised against the other polypeptide. The two polypeptides bound oleic acid similarly. Finally, identical elevations of cytoplasmic immunostain were detected specifically in mitotic hepatocytes with either antiserum. The collected findings are suggestive that liver fatty acid binding protein may carry ligands that promote hepatocyte division and may transport certain activated chemical carcinogens. Images PMID:3478711

  3. Equine hepatocytes: isolation, cryopreservation, and applications to in vitro drug metabolism studies.

    PubMed

    Shibany, Khaled A; Tötemeyer, Sabine; Pratt, Stefanie L; Paine, Stuart W

    2016-10-01

    Despite reports of the successful isolation of primary equine hepatocytes, there are no published data regarding the successful cryopreservation of these isolated cells. In this study, a detailed description of the procedures for isolation, cryopreservation, and recovery of equine hepatocytes are presented. Furthermore, the intrinsic clearance (Clint) and production of metabolites for three drugs were compared between freshly isolated and recovered cryopreserved hepatocytes. Primary equine hepatocytes were isolated using a two-step collagenase perfusion method, with an average cell yield of 2.47 ± 2.62 × 10(6) cells/g of perfused liver tissue and viability of 84.1 ± 2.62%. These cells were cryopreserved with William's medium E containing 10% fetal bovine serum with 10% DMSO. The viability of recovered cells, after a 30% Percoll gradient, was 77 ± 11% and estimated recovery rate was approximately 27%. These purified cells were used to determine the in vitro Clint of three drugs used in equine medicine; omeprazole, flunixin, and phenylbutazone, via the substrate depletion method. Cryopreserved suspensions gave a comparable estimation of Clint compared to fresh cells for these three drugs as well as producing the same metabolites. This work paves the way for establishing a bank of cryopreserved equine hepatocytes that can be used for estimating pharmacokinetic parameters such as the hepatic metabolic in vivo clearance of a drug as well as producing horse-specific drug metabolites.

  4. Ultrastructural and biochemical alterations induced in human, rat and mouse hepatocytes in primary culture exposed to selected carcinogens

    SciTech Connect

    Cole, K.H.

    1987-01-01

    Aflatoxin B1 (AFB{sub 1}), dimethylnitrosamine (DMN), 2-acetylaminofluorene (2-AAF) and actinomycin D are all potential human liver carcinogens. In order to investigate carcinogenic susceptibility of human liver to these agents, primary cultures of normal human hepatocytes were exposed to the four carcinogens. In the first series of experiments, human, rat, and mouse hepatocytes in primary culture were exposed to actinomycin D, AFB{sub 1}, and DMN for 24 h and examined for ultrastructural alterations. In an effort to relate the ultrastructural effects with total covalent binding of the carcinogen to DNA, human, rat and mouse hepatocytes were exposed to 2.0 {times} 10{sup {minus}7} M ({sup 3}H)AFB{sub 1} for 24 h. Hepatocytes from male rats had the highest degree of ({sup 3}H)AFB{sub 1}-DNA binding. Human hepatocytes contained the next highest binding level, while hepatocytes from female rats bound 38 pmoles/mg DNA. The AFB{sub 1}-DNA binding level in mouse hepatocytes was 1.4 pmoles/mg DNA. In similar experiments, human, and male and female rat hepatocytes in primary culture were exposed to the carcinogen 2-acetylamino (9-{sup 14}C)fluorene for 24 h. It was determined that male rat hepatocytes had the highest amount of radiolabeled 2-AAF bound to their DNA, female rats contained 0.57 nmoles/mg DNA, while human hepatocytes contained 0.29 nmoles/mg DNA.

  5. Differential entry of ricin into malignant and normal rat hepatocytes

    SciTech Connect

    Decastel, M.; Haentjens, G.; Aubery, M.; Goussault, Y. )

    1989-02-01

    The authors have compared the mechanisms of ricin binding to and entry into Zajdela hepatoma cells (ZHC) and normal rat hepatocytes (HyC). Lactose but not mannan was found to inhibit ricin binding to and toxicity on ZHC and HyC. This finding suggests that ricin binding, entry, and toxicity are expressed only through the galactose binding sites on ZHC and HyC. Nevertheless, the characteristics of ricin binding and its entry pathway appeared to be different in several respects in ZHC and HyC. Scatchard analysis of equilibrium data determined over a wide range of {sup 125}I-labeled ricin concentrations yielded a curvilinear plot for ZHC, while a straight line was obtained for HyC. These results indicate that only ZHC possess high-affinity receptors for ricin. Analysis of ricin toxicity of ZHC and HyC, in the presence of ammonium chloride or after K{sup +}-depletion in both cell types, suggests that the ricin bound to galactose receptors entered through neutral vesicles in ZHC, and through both neutral and acidic vesicles in HyC. The qualitative and quantitative differences found between the process of receptor-mediated endocytosis of ricin in ZHC and HyC might explain the differential sensitivity of the two cell types toward the toxin.

  6. Differential induction of the expression of GST subunits by geniposide in rat hepatocytes.

    PubMed

    Kuo, Wu-Hsien; Wang, Chau-Jong; Young, Shun-Chieh; Sun, Yuan-Chang; Chen, Yi-Jun; Chou, Fen-Pi

    2004-01-01

    Geniposide, an iridoid glycoside isolated from the fruit of Gardenia jasminoides Ellis, has the biological capabilities of detoxication, antioxidation, and anticarcinogenesis. In this study, the mechanism of geniposide affecting the GST (glutathione S-transferase) system was investigated. Primary cultured rat hepatocytes were treated with geniposide and examined for total GST activity and expression of GST subunits. The results showed that the geniposide-induced GST activity was dose and time dependent. Western blotting data demonstrated that geniposide induced increased protein levels of GSTM1 and GSTM2 (approximately 1.7- and 1.8-fold of control, respectively), but did not increase those of GSTA1. The corresponding transcripts levels were confirmed by RT-PCR. Using PD98059, the effect of geniposide was verified to be via the MEK pathway. The results suggest that geniposide possesses a potential for detoxication by inducing GST activity via increasing the transcription of GSTM1 and GSTM2.

  7. Biochemical characterization of domain-specific glycoproteins of the rat hepatocyte plasma membrane

    SciTech Connect

    Bartles, J.R.; Braiterman, L.T.; Hubbard, A.L.

    1985-10-15

    Seven integral proteins (CE 9, HA 21, HA 116, HA 16, HA 4, HA 201, and HA 301) were isolated from rat hepatocyte plasma membranes by immunoaffinity chromatography on monoclonal antibody-Sepharose. Six of the proteins (all but HA 16) exhibit domain-specific localizations (either bile canalicular or sinusoidal/lateral) about the hepatocyte surface. The authors identified three of these protein antigens as leucine aminopeptidase (HA 201), dipeptidyl peptidase IV (HA 301), and the asialoglycoprotein receptor (HA 116). They also developed SVI-lectin blotting procedures that, when used in conjunction with chemical and glycosidase treatments, permitted a comparison of the types of oligosaccharides present on the seven proteins. All seven are sialoglycoproteins, based upon the effects of prior neuraminidase and periodate-aniline-cyanoborohydride treatments of blots on labeling by SVI-wheat germ agglutinin. Depending upon the protein, they estimated the presence of 2-26 N-linked oligosaccharides/polypeptide chain from the Mr reductions accompanying chemical or enzymatic deglycosylation. Three of these mature plasma membrane proteins (HA 21, HA 116, and HA 4) have both high mannose-type and complex-type oligosaccharides on every copy of their polypeptide chains.

  8. Alcohol-induced suppression of gluconeogenesis is greater in ethanol fed female rat hepatocytes than males.

    PubMed

    Sumida, Ken D; Cogger, Alma A; Matveyenko, Aleksey V

    2007-03-01

    The impact of alcohol-induced suppression on hepatic gluconeogenesis (HGN) after chronic ethanol consumption between males and females is unknown. To determine the effects of chronic alcohol consumption (8 weeks) on HGN, the isolated hepatocyte technique was used on 24 h fasted male and female Wistar rats. Livers were initially perfused with collagenase and the hepatocytes were isolated. Aliquots of the cell suspension were placed in Krebs-Henseleit buffer and incubated for 30 min with lactate, [U -14C]lactate, and nine different concentrations of ethanol (EtOH). Dose-effect curves were generated for the determination of maximal and half-maximal alcohol-induced inhibition on HGN. There was no significant difference in HGN (lactate only and no EtOH) between males and females fed the control diet (88.5 +/- 5.1 nmol/mg protein/30 min). Similarly, the HGN (lactate only and no EtOH) in males fed the ethanol diet (ME) were not significantly different (82.8 +/- 3.5 nmol/mg protein/30 min) compared to controls. In contrast, the females chronically fed the ethanol diet (FE) had significantly (P < .05) lower HGN (67.8 +/- 4.6 nmol/mg protein/30 min) compared to both ME and controls. With alcohol in the incubation medium, the HGN significantly (P<.05) declined in all groups. While alcohol suppressed HGN to a larger (P < .05) extent in ME (45.8 +/- 3.7 nmol/mg protein/30 min) compared to controls (64.0 +/- 3.8 nmol/mg protein/30 min), the inhibition was even greater (P < .05) in FE (32.7 +/- 3.2 nmol/mg protein/30 min). The more pronounced effect of chronic alcohol consumption on HGN in the presence of ethanol in female hepatocytes was supported by the concomitant decreases (P < .05) in 14C-lactate incorporation into 14C-glucose, lactate uptake, and 14C-lactate uptake. The results suggest that chronic alcohol consumption elicits a greater reduction on HGN in the presence of ethanol in the hepatocytes of females compared to males.

  9. A comparison of adrenergic receptors of rat ascites hepatoma AH130 cells with those of normal rat hepatocytes.

    PubMed

    Sanae, F; Miyamoto, K; Koshiura, R

    1988-04-01

    The pharmacological specificity of adrenergic receptors in the plasma membrane of rat ascites hepatoma AH130 cells was compared with that in normal rat hepatocytes. The number of [125I]iodocyanopindolol-binding sites was much greater in AH130 cells than in the hepatocytes. We characterized the alpha-adrenergic receptor subtypes using the alpha 1-selective ligand [3H]prazosin and the alpha 2-selective ligand [3H]clonidine. AH130 cells had fewer prazosin-binding sites than the hepatocytes and about 8 times as many clonidine-binding sites of high affinity. The results showed that the adrenergic receptors in AH130 cells have pharmacological properties that are very different from those of the receptors in normal rat hepatocytes.

  10. Changes in the content of short, medium and long-chain fatty acids in isolated hepatocytes incubated in the presence of magnesium ions and/or ethanol.

    PubMed

    Grochowska-Niedworok, E; Calyniuk, B; Nowakowska-Zajdel, E; Muc-Wierzgon', M

    2013-01-01

    Magnesium is one of the commonly used dietary supplements. Therefore, this study was to evaluate the content of short, medium and long-chain fatty acids and their esters in isolated rat hepatocytes induced by magnesium and/or ethanol. Isolation of hepatocytes was carried out by the Seglen's enzymatic method using collagenase. To thus prepared samples ethanol and/or MgCl2 solution were added, respectively, so that their concentrations were as follows: 150 mM/dm3 ethanol and/or 2 mM/dm3 MgCl2, 4 mM/dm3 MgCl2. The contents of short, medium and long-chain fatty acids and those of ester-bound acids were determined. The statistical evaluation of the experiment was made by comparing the area normalized for the analysed fatty acids in hepatocytes incubated for 5 h in the presence of the test substances. The effect of magnesium ions on the content of fatty acids and their esters in isolated hepatocytes incubated for 5 h depended on their concentration in the medium. A normalizing effect of magnesium ions on ethanol-induced changes in the content of C14-C17, C18-C20 and C21-C24 fatty acids was demonstrated. A normalizing effect of magnesium on ethanol-induced changes in the content of ester-bound fatty acids in hepatocytes was not confirmed.

  11. Depression of alcohol dehydrogenase activity in rat hepatocyte culture by dihydrotestosterone.

    PubMed

    Mezey, E; Potter, J J; Diehl, A M

    1986-01-15

    Hepatocytes harvested from castrated rats retained a higher alcohol dehydrogenase (EC 1.1.1.1) activity than hepatocytes harvested from normal rats during 7 days of culture. Dihydrotestosterone (1 microM) decreased the enzyme activity, after 2 and 5 days of culture, in hepatocytes from castrated and control animals respectively. Dihydrotestosterone decreased the enzyme activity to similar values in both groups of hepatocytes by the end of 7 days of culture. Testosterone (1 microM) had no effect on the enzyme activity in normal hepatocytes and only a transitory effect in decreasing the enzyme activity in hepatocytes from castrated animals. The increases in alcohol dehydrogenase activity after castration and their suppression by dihydrotestosterone were associated with parallel changes in the rate of ethanol elimination. Additions of substrates of the malate-aspartate shuttle or dinitrophenol did not modify ethanol elimination. These observations indicate that dihydrotestosterone has a direct suppressant effect on hepatocyte alcohol dehydrogenase and that the enzyme activity is a major determinant of the rate of ethanol elimination.

  12. U. v. -enhanced reactivation of u. v. -irradiated herpes virus by primary cultures of rat hepatocytes

    SciTech Connect

    Zurlo, J.; Yager, J.D. )

    1984-04-01

    Carcinogen treatment of cultured mammalian cells prior to infection with u.v.-irradiated virus results in enhanced virus survival and mutagenesis suggesting the induction of SOS-type processes. The development of a primary rat hepatocyte culture system is reported to investigate cellular responses to DNA damage which may be relevant to hepatocarcinogenesis in vivo. Enhanced reactivation of u.v.-irradiated Herpes simplex virus type 1 (HSV-1) occurred in hepatocytes irradiated with u.v. Cultured hepatocytes were pretreated with u.v. at the time of enhanced DNA synthesis. These treatments caused an inhibition followed by a recovery of DNA synthesis. At various times after pretreatment, the hepatocytes were infected with control or u.v.-irradiated HSV-1 at low multiplicity, and virus survival was measured. U.v.-irradiated HSV-1 exhibited the expected two-component survival curve in control or u.v. pretreated hepatocytes. The magnitude of enhanced reactivation of HSV-1 was dependent on the u.v. dose to the hepatocytes, the time of infection following u.v. pretreatment, and the level of DNA synthesis at the time of pretreatment. These results suggest that u.v. treatment of rat hepatocytes causes the induction of SOS-type functions tht may have a role in the initiation of hepatocarcinogenesis.

  13. Nanofabricated Collagen-Inspired Synthetic Elastomers for Primary Rat Hepatocyte Culture

    PubMed Central

    Bettinger, Christopher J.; Kulig, Katherine M.; Vacanti, Joseph P.

    2009-01-01

    Synthetic substrates that mimic the properties of extracellular matrix proteins hold significant promise for use in systems designed for tissue engineering applications. In this report, we designed a synthetic polymeric substrate that is intended to mimic chemical, mechanical, and topological characteristics of collagen. We found that elastomeric poly(ester amide) substrates modified with replica-molded nanotopographic features enhanced initial attachment, spreading, and adhesion of primary rat hepatocytes. Further, hepatocytes cultured on nanotopographic substrates also demonstrated reduced albumin secretion and urea synthesis, which is indicative of strongly adherent hepatocytes. These results suggest that these engineered substrates can function as synthetic collagen analogs for in vitro cell culture. PMID:18847357

  14. Toxin-Induced Activation of Rat Hepatocyte Prostaglandin Synthesis and Phospholipid Metabolism

    DTIC Science & Technology

    1989-12-22

    SUBJECT TERMS (Continue on reverse if necesay and identify by block number) FIELD GROUP SUB-GROUP - microcystin -LR, arachidonic acid, phospholipid...pool was reduced to 47% (p ɘ.025) by l’ 3’ microcystin -LR. Changes in phospholipid classes indicated that prostaglandin formation induced by microcystin ... microcystin -LR has important effects on the regulation of inflammatory mediator synthesis in hepatocytes. 7.,1 ,- TOXIN-INDUCED ACTIVATION OF RAT HEPATOCYTE

  15. [An experimental study on the protective effect of hepatocyte growth factor (HGF) for the primary cultured hepatocytes obtained from iron-loaded rats].

    PubMed

    Yoshida, J

    1995-01-01

    Pathological iron deposition in liver is often found in various liver diseases. The deposited iron is thought to be one of the most important factor of liver cell injury, not only in hemochromotosis but also in cirrhosis following hepatitis virus B or C infection. To investigate the influence of the deposited iron on damage and regeneration of hepatocyte, primary cultured hepatocytes obtained from carbonyl iron-loaded rats were treated with carbon tetrachloride (CCl4) in the presence or absence of hepatocyte growth factor (HGF). Although the section of liver from carbonyl iron-loaded rats showed no necrosis and fibrosis, iron-loaded hepatocytes contained about twofold more iron than control. The damage of iron-loaded hepatocytes induced by CCl4 was more serious than that of control, and HGF decreased this injury only in iron-loaded hepatocytes but not in control. There is no difference in DNA synthesis stimulated by HGF between iron-loaded hepatocytes and control. These findings suggest that the pathological iron deposition induces the fragility of hepatocyte and that cytoprotective effect of HGF is induced by this pathological iron.

  16. Deduced primary structure of rat hepatocyte growth factor and expression of the mRNA in rat tissues.

    PubMed Central

    Tashiro, K; Hagiya, M; Nishizawa, T; Seki, T; Shimonishi, M; Shimizu, S; Nakamura, T

    1990-01-01

    The primary structure of rat hepatocyte growth factor (HGF) was elucidated by determining the base sequence of the complementary DNA (cDNA) of HGF. The cDNA for rat HGF was isolated by screening a liver cDNA library with oligonucleotides based on the partial N-terminal amino acid sequence of the beta subunit of purified rat HGF. HGF is encoded in an mRNA of about 6 kilobases. Both alpha and beta subunits of HGF are specified in a single open reading frame for a 728-amino acid protein with a calculated molecular weight of 82,904. The N-terminal part of HGF has a signal sequence and a prosequence with 30 and 25 amino acid residues, respectively. The mature heterodimer structure is derived proteolytically from this single pre-pro precursor polypeptide. The calculated molecular weights of the alpha and beta subunits are 50,664 and 25,883, respectively, and each subunit has two potential N-linked glycosylation sites. The amino acid sequence of HGF is 38% identical with that of plasminogen. The alpha subunit of HGF contains four "kringle" structures, and the beta subunit has 37% amino acid identity with the serine protease domain of plasmin. Northern blot analysis revealed that HGF mRNA was expressed in rat various tissues, including the liver, kidney, lung, and brain. Images PMID:2139229

  17. In vitro drug metabolism of green tea catechins in human, monkey, dog, rat and mouse hepatocytes.

    PubMed

    Chen, Wendy W; Qin, Geng-Yao; Zhang, Ting; Feng, Wan-Yong

    2012-06-01

    The metabolic fate of green tea catechins [(-)-epicatechin ((-)-EC), (-)-epicatechin-3-gallate (ECG) (-)- epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG)] in cryopreserved human, monkey, dog, rat and mouse hepatocytes was studied. Methylation, glucuronidation, sulfation and isomerization pathways of (-)-EC in all five species were found. Methylation, glucuronidation, sulfation, hydrolysis, isomerization and glucosidation pathways of ECG were found. Species differences in metabolism of (-)-EC or ECG were observed. Surprisingly, no metabolites of EGC or EGCG were detected, but chemical oxidation and polymerization were observed under these experimental conditions. It appeared that enzymatic reactions and chemical reactions were differentiated by an additional hydroxyl group on the B-ring between (-)-EC/ECG and EGC/EGCG. For (-)-EC, thirty-five metabolites including isomerized (M6. M10 and M25), glucuronidated (M3, M5 and M11), sulfated (M7, M15, M16, M18, M20, M23, M26), methylated (M2, M9, M12, M17, M19, M21, M27, M30, M32), glucuronated/methylated (M4, M8, M13, M14), sulfated/methylated (M22, M24, M28, M29, M31, M33, M34, M35) and diglucuronidate (M1), were detected and characterized. M11, M18, M19 and M23 were major metabolites in human hepatocytes; M11, M26 and M31 were major metabolites in monkey hepatocytes; M10, M20, M22, M26 and M31 were major metabolites in dog hepatocytes; M5, M6 and M10 were major metabolites in rat hepatocytes; and M5, M6 and M13 were major metabolites in mouse hepatocytes. For ECG, twelve metabolites including isomerized (M1), hydrolyzed (M3), glucosidated (M2), glucuronidated (M4 and M6), sulfated (M9, M11 and M12), methylated (M7), sulfated/glucuronidated/methylated (M8 and M10) and diglucuronidated (M5), were detected and characterized. M4, M11 and M12 were major metabolites in human hepatocytes; M11 and M12 were major metabolites in monkey hepatocytes; M3 and M11 were major metabolites in dog hepatocytes; M4, M6 and

  18. Effect of dexamethasone treatment on the expression and function of transport proteins in sandwich-cultured rat hepatocytes.

    PubMed

    Turncliff, Ryan Z; Meier, Peter J; Brouwer, Kim L R

    2004-08-01

    Dexamethasone (DEX) is a well established inducer of CYP3A. These studies examined the influence of DEX treatment on transport protein expression and function in sandwich-cultured (SC) rat hepatocytes. Freshly isolated hepatocytes were cultured between two layers of gelled collagen and maintained in Dulbecco's modified Eagle's medium supplemented with DEX (0.1 microM, 0-48 h and 0.1-100 microM, 48-96 h). The expression of sinusoidal [(organic anion transporting polypeptide 1a1 (Oatp1a1), Oatp1a4, multidrug resistance-associated protein 3 (Mrp3), and Na(+)-dependent taurocholate cotransporting polypeptide (Ntcp)] and canalicular [bile salt export pump (Bsep), multidrug resistance protein 1a/b (Mdr1a/b), and Mrp2] transport proteins was determined by Western blot analysis. The accumulation and biliary excretion index (BEI; percentage of accumulated substrate in canalicular networks) of the probe substrates taurocholate (TC; 1 microM, 10 min), rhodamine 123 (Rh123; 10 microM, 30 min), and carboxy-2',7'-dichlorofluorescein (CDF; 10 microM, 10 min) were employed as measures of canalicular transport protein function in SC rat hepatocytes. DEX treatment increased CYP3A1/2, Oatp1a4, and Mrp2 expression, decreased the expression of Ntcp, and did not seem to alter the expression of Oatp1a1, Mrp3, Mdr1a/b, or Bsep. The BEI of CDF, an Mrp2 substrate, increased from 18 to 37% after DEX treatment (100 microM). The accumulation of TC, an Ntcp substrate, was reduced (<50% of control), whereas the BEI of TC, also a Bsep substrate, was unchanged. Treatment of SC rat hepatocytes with DEX resulted in alterations in the expression of CYP3A1/2 and some hepatic transport proteins. Modest alterations in hepatic transport protein function were consistent with changes in protein expression.

  19. Effect of recombinant human growth hormone on age-related hepatocyte changes in old male and female Wistar rats.

    PubMed

    Castillo, Carmen; Salazar, Veronica; Ariznavarreta, Carmen; Vara, Elena; Tresguerres, Jesus A F

    2004-10-01

    Aging induces changes in several organs, such as the liver, and this process might be due to damage caused by free radicals and inflammatory mediators. The growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis shows a reduction with age, and this fact could be associated with some age-related changes. The aim of this study was to investigate the effect of GH administration on age-induced alterations in hepatocytes. Two and twenty two month-old male and female Wistar rats were used. Old rats were treated with human recombinant GH for 10 wk. At the end of the treatment, hepatocytes were isolated from the liver and cultured, and different parameters were measured in cells and medium. Plasma IGF-1 was also measured. Aging significantly decreased plasma IGF-1 in males. In females, plasma IGF-1 was also reduced, but not significantly. GH treatment restored plasma IGF-1 levels to values similar to young males. Aging was associated with a significant increase in lipid peroxidation (LPO), nitric oxide (NO), carbon monoxide (CO) and cyclic guanosyl-monophosphate (cGMP), as well as a reduction in adenosyl triphosphate (ATP) and phosphatidylcholine (PC) synthesis. GH administration partially prevented all these changes in males. In females, some of the parameters were significantly improved by GH (ATP, CO, cGMP), while others showed a tendency to improvement, although differences did not reach significance. In conclusion, GH administration could exert beneficial effects against age-related changes in hepatocytes, mainly in males.

  20. Screening of herbal components for attenuating amiodarone-induced hepatotoxicity on gel-entrapped rat hepatocytes.

    PubMed

    Deng, Xudong; Shen, Chong; Meng, Qin

    2014-01-01

    Amiodarone (AMD) is a hepatotoxic drug that has been widely used as a class III antiarrhythmic drug. Because, to date, only a few kinds of protectants are able to reduce AMD hepatotoxicity, this article utilized gel-entrapped rat hepatocytes to screen effective protectants from a series of herbal compounds for their effects against AMD-induced toxicity. Herbal compounds, including matrine, silibinin, glycyrrhizic acid, schisandrin B, epigallocatechin gallate and anisodamine, were cotreated with AMD to assess their protective effect, whereas vitamin E, which has been shown to be protective in rats, was selected as a control. It was found that vitamin E, as with its function in rats, provided the best protection in gel-entrapped rat hepatocytes, whereas silibinin, a major component of silymarin, could largely reduce AMD-induced hepatotoxicity, performing a similar function as silymarin in rats. The results illustrated that gel-entrapped hepatocytes may reflect the protective effects of drugs and serve as a reliable model for screening hepatoprotectants. Moreover, matrine, a widely used monomer of the traditional Chinese medicine, Sophora flavescens, for treatment of arrhythmia, was evidenced to show some effective protections against AMD hepatotoxicity. Taken together, gel-entrapped rat hepatocytes may provide a platform for screening effective candidates from the herbal component library.

  1. Demonstration of direct lineage between hepatocytes and hepatocellular carcinoma in diethylnitrosamine-treated rats.

    PubMed

    Bralet, Marie-Pierre; Pichard, Virginie; Ferry, Nicolas

    2002-09-01

    The question whether hepatocellular carcinoma (HCC) arises from dedifferentiation of mature hepatocytes or from proliferation of liver stem cells is still debated. In the present study, we used retroviral-mediated genetic labeling to investigate the fate of mature hepatocytes in rats after administration of diethylnitrosamine (DEN). Mature hepatocytes were genetically labeled by intravenous injection of retroviral vectors containing the Escherichia coli beta-galactosidase gene coupled to a nuclear localization signal (nls-LacZ) 1 day after partial hepatectomy. Liver biopsies performed after completion of hepatic regeneration showed that 18.3% of hepatocytes expressed the nls-LacZ transgene. Rats were then treated with DEN in drinking water for 12 weeks and sacrificed between 98 and 151 days after the onset of DEN administration. Clones of beta-galactosidase positive cells were observed, half of which (53%) also expressed the placental form of glutathione-S-transferase (GSTp), a marker of preneoplastic cells. HCCs of various sizes expressing GSTp were present in all animals. Careful examination of 90 HCCs revealed that 16 (17.7%) also expressed nls-LacZ. This figure precisely matched the proportion of labeled hepatocytes before DEN treatment (18.3%). In conclusion, a random clonal origin of HCC from mature hepatocytes is seen in the DEN model of hepatocarcinogenesis.

  2. Quantitative assessment of canalicular bile formation in isolated hepatocyte couplets using microscopic optical planimetry.

    PubMed Central

    Gautam, A; Ng, O C; Strazzabosco, M; Boyer, J L

    1989-01-01

    Isolated rat hepatocyte couplets (IRHC) are primary units of bile secretion that accumulate fluid in an enclosed canalicular space with time in culture. We have quantitated the rate of canalicular secretion in IRHC cultured for 4-8 h by measuring the change in canalicular space volume by video-microscopic optical planimetry using high resolution Nomarski optics. Electron microscopic morphometric studies revealed significant increases in canalicular membrane area after 4-6 h in culture. Canalicular secretion in basal L-15 medium (3.8 +/- 1.3 fl/min) increased significantly with the choleretic bile salts (10 microM), taurocholate, and ursodeoxycholate (14 +/- 7 fl/min each). Secretion rates after exposure to bile acids correlated directly with the canalicular surface area before stimulation. In contrast, expansion times after stimulation varied inversely with initial canalicular volumes. Ursodeoxycholic acid failed to produce a hypercholeresis at 10-, 100-, or 200-microM concentrations compared with taurocholate, either in normal or taurine-depleted IRHC. The present findings establish that rates of canalicular bile secretion can be quantitated in IRHC by serial optical planimetry, both in the basal state and after stimulation with bile acids. Furthermore, ursodeoxycholate does not acutely induce hypercholeresis at the canalicular level in this model. Rather, both taurocholic and ursodeoxycholic acids induced secretion in proportion to the surface area of the canalicular membrane. The IRHC are a useful model to identify canalicular choleretics and for studies of canalicular bile formation. Images PMID:2913052

  3. Comparison of acetaminophen toxicity in primary hepatocytes isolated from transgenic mice with different appolipoprotein E alleles.

    PubMed

    Mezera, V; Kucera, O; Moravcova, A; Peterova, E; Rousar, T; Rychtrmoc, D; Sobotka, O; Cervinkova, Z

    2015-12-01

    The nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor, important for combating electrophilic and oxidative stress in the liver and other organs. This encompasses detoxification of hepatotoxic drugs, including acetaminophen (APAP). Recently, an association between apolipoprotein E (ApoE) genotype and Nrf2 expression was described. We compared the toxicity of APAP on primary culture hepatocytes isolated from transgenic mice carrying two different human ApoE alleles and wild-type controls. The cells were exposed to APAP in concentrations from 0.5 to 4 mM for up to 24 hours. APAP led to a dose-dependent hepatotoxicity from 1 mM after 16 h exposure in all mice tested. The toxicity was higher in hepatocytes isolated from both transgenic strains than in wild-type controls and most pronounced in ApoE3 mice. Concurrently, there was a decline in mitochondrial membrane potential, especially in ApoE3 hepatocytes. The formation of reactive oxygen species was increased after 24 hours with 2.5 mM APAP in hepatocytes of all strains tested, with the highest increase being in the ApoE3 genotype. The activity of caspases 3 and 7 did not differ among groups and was minimal after 24 hour incubation with 4 mM APAP. We observed higher lipid accumulation in hepatocytes isolated from both transgenic strains than in wild-type controls. The expression of Nrf2-dependent genes was higher in ApoE3 than in ApoE4 hepatocytes and some of these genes were induced by APAP treatment. In conclusion, transgenic mice with ApoE4 and ApoE3 alleles displayed higher susceptibility to acute APAP toxicity in vitro than wild-type mice. Of the two transgenic genotypes tested, ApoE3 allele carriers were more prone to injury.

  4. A transgenic rat hepatocyte - Kupffer cell co-culture model for evaluation of direct and macrophage-related effect of poly(amidoamine) dendrimers.

    PubMed

    Jemnitz, Katalin; Bátai-Konczos, Attila; Szabó, Mónika; Ioja, Enikő; Kolacsek, Orsolya; Orbán, Tamás I; Török, György; Homolya, László; Kovács, Eszter; Jablonkai, István; Veres, Zsuzsa

    2017-02-01

    Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in the development of drug induced liver injury (DILI). Furthermore, elevated intracellular Ca(2+) level of hepatocytes is considered as a common marker of DILI. Here we applied an in vitro model based on hepatocyte mono- and hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing the GCaMP2 fluorescent Ca(2+) sensor protein to investigate the effects of polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5 Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity assay and by Ca(2+) imaging, while hepatocyte functions were studied by CYP2B1/2 inducibility, and bilirubin and taurocholate transport. G5 was significantly more cytotoxic than G4.5 for hepatocytes and induced Ca(2+) oscillation and sustained Ca(2+) signals at 1μM and10 μM, respectively both in hepatocytes and KCs. Dendrimer-induced Ca(2+) signals in hepatocytes were attenuated by macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the inducibility of CYP2B1/2, which was restored by depleting the KCs with gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In conclusion, H/KC provides a good model for the prediction of hepatotoxic potential of drugs, especially of nanomaterials known to be trapped by macrophages, activation of which presumably contributes to DILI.

  5. N-acetylcysteine protects against cadmium-induced oxidative stress in rat hepatocytes

    PubMed Central

    Wang, Jicang; Zhu, Huali; Liu, Xuezhong

    2014-01-01

    Cadmium (Cd) is a well-known hepatotoxic environmental pollutant. We used rat hepatocytes as a model to study oxidative damage induced by Cd, effects on the antioxidant systems, and the role of N-acetylcysteine (NAC) in protecting cells against Cd toxicity. Hepatocytes were incubated for 12 and 24 h with Cd (2.5, 5, 10 µM). Results showed that Cd can induce cytotoxicity: 10 µM resulted in 36.2% mortality after 12 h and 47.8% after 24 h. Lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase activities increased. Additionally, reactive oxygen species (ROS) generation increased in Cd-treated hepatocytes along with malondialdehyde levels. Glutathione concentrations significantly decreased after treatment with Cd for 12 h but increased after 24 h of Cd exposure. In contrast, glutathione peroxidase activity significantly increased after treatment with Cd for 12 h but decreased after 24 h. superoxide dismutase and catalase activities increased at 12 h and 24 h. glutathione S-transferase and glutathione reductase activities decreased, but not significantly. Rat hepatocytes incubated with NAC and Cd simultaneously had significantly increased viability and decreased Cd-induced ROS generation. Our results suggested that Cd induces ROS generation that leads to oxidative stress. Moreover, NAC protects rat hepatocytes from cytotoxicity associated with Cd. PMID:25234327

  6. Significant improvement of survival by intrasplenic hepatocyte transplantation in totally hepatectomized rats.

    PubMed

    Vogels, B A; Maas, M A; Bosma, A; Chamuleau, R A

    1996-01-01

    The effect of intrasplenic hepatocyte transplantation (HTX) was studied in an experimental model of acute liver failure in rats with chronic liver atrophy. Rats underwent a portacaval shunt operation on Day -14 to induce liver atrophy, and underwent total hepatectomy on Day 0 as a start of acute liver failure. Intrasplenic hepatocyte or sham transplantation was performed on Day -7,-3, or -1 (n = 4 to 6 per group). During the period following hepatectomy, mean arterial blood pressure was maintained above 80 mm Hg and hypoglycaemia was prevented. Severity of hepatic encephalopathy was assessed by clinical grading and EEG spectral analysis, together with determination of blood ammonia and plasma amino acid concentrations, and "survival" time. Histological examination of the spleen and lungs was performed after sacrifice. Intrasplenic hepatocyte transplantation resulted in a significant improvement in clinical grading in all transplanted groups (p < 0.05), whereas a significant improvement in EEG left index was seen only in the group with transplantation on Day -1 (p < 0.05). In contrast to hepatocyte transplantation 1 day before total hepatectomy, rats with hepatocyte transplantation 3 and 7 days before total hepatectomy showed a significant 3- and 2-fold increase in "survival" time compared to sham transplanted controls: HTX at Day -1: 7.5 +/- 0.3 h vs. 5.9 +/- 0.6 h (p > 0.05), HTX at Day -3: 19.7 +/- 3.7 h vs. 6.5 +/- 0.3 h (p < 0.05), and HTX at Day -7: 13.8 +/- 3.2 h vs. 6.3 +/- 0.3 h (p < 0.05). Furthermore, rats with hepatocyte transplantation on Day -3 and -7 showed significantly lower blood ammonia concentrations after total hepatectomy (p < 0.0001). Histological examination of the spleens after sacrifice showed clusters of hepatocytes in the red pulp. Hepatocytes present in the spleen for 3 and 7 days showed bile accumulation and spots of beginning necrosis. The present data show that in a hard model of complete liver failure in portacaval shunted rats

  7. Hydroxylation, conjugation and sulfation of bile acids in primary monolayer cultures of rat hepatocytes

    SciTech Connect

    Princen, H.M.; Meijer, P.

    1988-08-15

    Hydroxylation of lithocholic, chenodeoxycholic, deoxycholic and cholic acids was studied in monolayers of rat hepatocytes cultured for 76 h. The majority of added lithocholic and chenodeoxycholic acids was metabolized to beta-muricholic acid (56-76%). A small part of these bile acids (9%), however, and a considerable amount of deoxycholic and cholic acids (21%) were converted into metabolites more polar than cholic acid in the first culture period. Formation of these compounds decreased during the last day of culture. Bile acids synthesized after addition of (4-/sup 14/C)-cholesterol were almost entirely (97%) sulfated and/or conjugated, predominantly with taurine (54-66%), during culture. Sulfated bile acids were mainly composed of free bile acids. The ability of hepatocytes to sulfurylate bile acids declined with culture age. Thus, rat hepatocytes in primary monolayer culture are capable to sulfurylate bile acids and to hydroxylate trihydroxylated bile acids, suggesting formation of polyhydroxylated metabolites.

  8. Decellularization and Recellularization of Rat Livers With Hepatocytes and Endothelial Progenitor Cells.

    PubMed

    Zhou, Pengcheng; Huang, Yan; Guo, Yibing; Wang, Lei; Ling, Changchun; Guo, Qingsong; Wang, Yao; Zhu, Shajun; Fan, Xiangjun; Zhu, Mingyan; Huang, Hua; Lu, Yuhua; Wang, Zhiwei

    2016-03-01

    Whole-organ decellularization has been identified as a promising choice for tissue engineering. The aim of the present study was to engineer intact whole rat liver scaffolds and repopulate them with hepatocytes and endothelial progenitor cells (EPCs) in a bioreactor. Decellularized liver scaffolds were obtained by perfusing Triton X-100 with ammonium hydroxide. The architecture and composition of the original extracellular matrix were preserved, as confirmed by morphologic, histological, and immunolabeling methods. To determine biocompatibility, the scaffold was embedded in the subcutaneous adipose layer of the back of a heterologous animal to observe the infiltration of inflammatory cells. Hepatocytes were reseeded using a parenchymal injection method and cultured by continuous perfusion. EPCs were reseeded using a portal vein infusion method. Morphologic and functional examination showed that the hepatocytes and EPCs grew well in the scaffold. The present study describes an effective method of decellularization and recellularization of rat livers, providing the foundation for liver engineering and the development of bioartificial livers.

  9. Methanethiol and dimethylsulfide formation from 3-methylthiopropionate in human and rat hepatocytes.

    PubMed

    Blom, H J; van den Elzen, J P; Yap, S H; Tangerman, A

    1988-11-18

    This study was designed to investigate the metabolism of methanethiol, and the involvement of methanethiol and its metabolites in the transamination pathway of methionine. Gaseous methanethiol, methanethiol-mixed disulfides and dimethylsulfide were formed from 3-methylthiopropionate, a metabolite in the transamination pathway of methionine, during incubation with human and rat hepatocytes. An increase of the 3-methylthiopropionate concentration resulted in an increased formation of the products, up to a substrate concentration of 4.4 mM. Higher substrate levels resulted in a decreased methanethiol formation, probably due to poisoning of the system. However, in human hepatocytes the formation of dimethylsulfide increased up to a 3-methylthiopropionate concentration of 12.5 mM. The formation of methanethiol, dimethylsulfide and methanethiol-mixed disulfides from 3-methylthiopropionate in hepatocytes of both human and rat support the hypothesis that methanethiol can be formed from methionine via the transamination pathway.

  10. Human hepatocyte and kidney cell metabolism of 2-acetylaminofluorene and comparison to the respective rat cells.

    PubMed

    Langenbach, R; Rudo, K

    1988-12-01

    The metabolism and mutagenic activation of 2-acetylaminofluorene by human and rat hepatocytes and kidney cells were measured. High performance liquid chromatography was used to separate the 2-acetylaminofluorene metabolites, and a cell-mediated Salmonella typhimurium mutagenesis assay was used to detect mutagenic intermediates. Rat and human differences were observed with cells from both organs and levels of metabolism and mutagenesis were higher in human cells. Within a species, liver and kidney cell differences were also evident, with levels of hepatocyte-mediated metabolism and mutagenesis being greater than kidney cells. Human inter-individual variation was apparent with cells from both organs, but the variation observed was significantly greater in hepatocytes than kidney cells. A knowledge of such differences, including an understanding that they may vary with the chemical being studied, should be useful in the extrapolation of rodent carcinogenesis data to humans.

  11. Ginkgolide A contributes to the potentiation of acetaminophen toxicity by Ginkgo biloba extract in primary cultures of rat hepatocytes

    SciTech Connect

    Rajaraman, Ganesh; Chen, Jie; Chang, Thomas K.H. . E-mail: tchang@interchange.ubc.ca

    2006-12-01

    The present cell culture study investigated the effect of Ginkgo biloba extract pretreatment on acetaminophen toxicity and assessed the role of ginkgolide A and cytochrome P450 3A (CYP3A) in hepatocytes isolated from adult male Long-Evans rats provided ad libitum with a standard diet. Acetaminophen (7.5-25 mM for 24 h) conferred hepatocyte toxicity, as determined by the lactate dehydrogenase (LDH) assay. G. biloba extract alone increased LDH leakage in hepatocytes at concentrations {>=} 75 {mu}g/ml and {>=} 750 {mu}g/ml after a 72 h and 24 h treatment period, respectively. G. biloba extract (25 or 50 {mu}g/ml once every 24 h for 72 h) potentiated LDH leakage by acetaminophen (10 mM for 24 h; added at 48 h after initiation of extract pretreatment). The effect was confirmed by a decrease in [{sup 14}C]-leucine incorporation. At the level present in a modulating concentration (50 {mu}g/ml) of the extract, ginkgolide A (0.55 {mu}g/ml), which increased CYP3A23 mRNA levels and CYP3A-mediated enzyme activity, accounted for part but not all of the potentiating effect of the extract on acetaminophen toxicity. This occurred as a result of CYP3A induction by ginkgolide A because triacetyloleandomycin (TAO), a specific inhibitor of CYP3A catalytic activity, completely blocked the effect of ginkgolide A. Ginkgolide B, ginkgolide C, ginkgolide J, quercetin, kaempferol, isorhamnetin, and isorhamnetin-3-O-rutinoside did not alter the extent of LDH leakage by acetaminophen. In summary, G. biloba pretreatment potentiated acetaminophen toxicity in cultured rat hepatocytes and ginkgolide A contributed to this novel effect of the extract by inducing CYP3A.

  12. SIMPLE MACHINE PERFUSION SIGNIFICANTLY ENHANCES HEPATOCYTE YIELDS OF ISCHEMIC AND FRESH RAT LIVERS.

    PubMed

    Izamis, Maria-Louisa; Calhoun, Candice; Uygun, Basak E; Guzzardi, Maria Angela; Price, Gavrielle; Luitje, Martha; Saeidi, Nima; Yarmush, Martin L; Uygun, Korkut

    2013-01-01

    The scarcity of viable hepatocytes is a significant bottleneck in cell transplantation, drug discovery, toxicology, tissue engineering, and bioartificial assist devices, where trillions of high-functioning hepatocytes are needed annually. We took the novel approach of using machine perfusion to maximize cell recovery, specifically from uncontrolled cardiac death donors, the largest source of disqualified donor organs. In a rat model, we developed a simple 3 hour room temperature (20±2°C) machine perfusion protocol to treat non-premedicated livers exposed to 1 hour of warm (34°C) ischemia. Treated ischemic livers were compared to fresh, fresh-treated and untreated ischemic livers using viable hepatocyte yields and in vitro performance as quantitative endpoints. Perfusion treatment resulted in both a 25-fold increase in viable hepatocytes from ischemic livers, and a 40% increase from fresh livers. While cell morphology and function in suspension and plate cultures of untreated warm ischemic cells was significantly impaired, treated warm ischemic cells were indistinguishable from fresh hepatocytes. Further, a strong linear correlation between tissue ATP and cell yield enabled accurate evaluation of the extent of perfusion recovery. Maximal recovery of warm ischemic liver ATP content appears to be correlated with optimal flow through the microvasculature. These data demonstrate that the inclusion of a simple perfusion-preconditioning step can significantly increase the efficiency of functional hepatocyte yields and the number of donor livers that can be gainfully utilized.

  13. Simple Machine Perfusion Significantly Enhances Hepatocyte Yields of Ischemic and Fresh Rat Livers

    PubMed Central

    Izamis, Maria-Louisa; Calhoun, Candice; Uygun, Basak E.; Guzzardi, Maria Angela; Price, Gavrielle; Luitje, Martha; Saeidi, Nima; Yarmush, Martin L.; Uygun, Korkut

    2013-01-01

    The scarcity of viable hepatocytes is a significant bottleneck in cell transplantation, drug discovery, toxicology, tissue engineering, and bioartificial assist devices, where trillions of high-functioning hepatocytes are needed annually. We took the novel approach of using machine perfusion to maximize cell recovery, specifically from uncontrolled cardiac death donors, the largest source of disqualified donor organs. In a rat model, we developed a simple 3-h room temperature (20 ± 2°C) machine perfusion protocol to treat nonpremedicated livers exposed to 1 h of warm (34°C) ischemia. Treated ischemic livers were compared to fresh, fresh-treated, and untreated ischemic livers using viable hepatocyte yields and in vitro performance as quantitative endpoints. Perfusion treatment resulted in both a 25-fold increase in viable hepatocytes from ischemic livers and a 40% increase from fresh livers. While cell morphology and function in suspension and plate cultures of untreated warm ischemic cells was significantly impaired, treated warm ischemic cells were indistinguishable from fresh hepatocytes. Furthermore, a strong linear correlation between tissue ATP and cell yield enabled accurate evaluation of the extent of perfusion recovery. Maximal recovery of warm ischemic liver ATP content appears to be correlated with optimal flow through the microvasculature. These data demonstrate that the inclusion of a simple perfusion-preconditioning step can significantly increase the efficiency of functional hepatocyte yields and the number of donor livers that can be gainfully utilized. PMID:25431743

  14. DICHLOROACETIC ACID (DCA) INHIBITS PROLIFERATION AND APOPTOSIS IN NORMAL HEPATOCYTES OF MALE F344 RATS

    EPA Science Inventory

    Dichloroacetic acid (DCA} inhibits proliferation and apoptosis in nonnal hepatocytes of
    male F344 rats.

    Large segments of the population are chronically exposed to dichloroacetic acid (DCA}: DCA is a by product of the chlorine disinfection of drinking water, a metab...

  15. Enhancement of proliferation in a rat hepatocyte co-culture model after mitogenic stimulation.

    EPA Science Inventory

    Primary mouse and rat hepatocyte cultures have long been the gold standard for assessment of cellular changes following chemical exposure. While helpful for assessing proliferative and responses in vitro, these cultures are limited to 1 or 2 days of incubation. Our motivation was...

  16. Pyrrolizidine alkaloid clivorine induced oxidative injury on primary cultured rat hepatocytes.

    PubMed

    Ji, LiLi; Liu, TianYu; Wang, ZhengTao

    2010-04-01

    Clivorine is an otonecine-type hepatotoxic pyrrolizidine alkaloid (HPAs), to which humans are exposed when consuming herbs containing such components. In the present study, we investigated clivorine-induced oxidative stress injury on primary cultured rat hepatocytes. Rat hepatocytes were treated with various concentrations of clivorine (1-100 microM) for 48 hours, and then cell viability was detected by 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay, while lipid peroxidation (LPO) level, glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD) activities were determined to evaluate the oxidative injury. The results of MTT assay showed that clivorine decreased cell viability in a concentration-dependent manner. Clivorine also increased LPO amounts in rat hepatocytes at the concentrations of 50 microM and 100 microM. Further results showed that clivorine decreased GPx, GST and GR activities, which are all reduced glutathione (GSH)-related antioxidant enzymes. CAT and SOD are both important antioxidant enzymes, and the results showed that clivorine increased CAT activity at the low concentration of 5 muM and decreased cellular SOD activity at all concentrations. Taken together, our results demonstrated that clivorine induced toxicity on primary cultured rat hepatocytes by causing the damage on cellular redox balance.

  17. Oxidative stress is involved in Dasatinib-induced apoptosis in rat primary hepatocytes

    SciTech Connect

    Xue, Tao; Luo, Peihua; Zhu, Hong; Zhao, Yuqin; Wu, Honghai; Gai, Renhua; Wu, Youping; Yang, Bo; Yang, Xiaochun; He, Qiaojun

    2012-06-15

    Dasatinib, a multitargeted inhibitor of BCR–ABL and SRC kinases, exhibits antitumor activity and extends the survival of patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL). However, some patients suffer from hepatotoxicity, which occurs through an unknown mechanism. In the present study, we found that Dasatinib could induce hepatotoxicity both in vitro and in vivo. Dasatinib reduced the cell viability of rat primary hepatocytes, induced the release of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in vitro, and triggered the ballooning degeneration of hepatocytes in Sprague–Dawley rats in vivo. Apoptotic markers (chromatin condensation, cleaved caspase-3 and cleaved PARP) were detected to indicate that the injury induced by Dasatinib in hepatocytes in vitro was mediated by apoptosis. This result was further validated in vivo using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Here we found that Dasatinib dramatically increased the level of reactive oxygen species (ROS) in hepatocytes, reduced the intracellular glutathione (GSH) content, attenuated the activity of superoxide dismutase (SOD), generated malondialdehyde (MDA), a product of lipid peroxidation, decreased the mitochondrial membrane potential, and activated nuclear factor erythroid 2-related factor 2 (Nrf2) and mitogen-activated protein kinases (MAPK) related to oxidative stress and survival. These results confirm that oxidative stress plays a pivotal role in Dasatinib-mediated hepatotoxicity. N-acetylcysteine (NAC), a typical antioxidant, can scavenge free radicals, attenuate oxidative stress, and protect hepatocytes against Dasatinib-induced injury. Thus, relieving oxidative stress is a viable strategy for reducing Dasatinib-induced hepatotoxicity. -- Highlights: ►Dasatinib shows potential hepatotoxicity both in vitro and in vivo. ►Apoptosis plays a vital role in Dasatinib

  18. Polyploidization delay in rat hepatocytes under liver growth inhibition by hypokinesia

    NASA Technical Reports Server (NTRS)

    Faktor, V. M.; Malyutin, V. F.; Li, S. Y.; Brodskiy, V. Y.

    1981-01-01

    A study of young rats, weighing 55 to 59 g, after being for 10 days in conditions of limited mobility, shows a retardation of body growth as well as that of liver growth. The decrease in the rate of growth is accompanied by a reduction of cell proliferation and by delay polyploidization of hepatocytes in the liver of experimental rats. The materials, methods, and results of research are discussed.

  19. Autophagic sequestration of (/sup 14/C)sucrose, introduced into rat hepatocytes by reversible electro-permeabilization

    SciTech Connect

    Gordon, P.B.; Seglen, P.O.

    1982-11-01

    Isolated rat hepatocytes could be made permeable to small molecules such as (/sup 14/C)sucrose (but not to proteins) but subjecting the cells to repeated discharges in a high-voltage field. During subsequent incubation at 37/sup 0/C, the permeability changes were reversed within 15 min, the electron-injected (/sup 14/C)sucros remaining trapped inside the re-sealed plasma membrane. Autophagic sequestration of (/sup 14/C)sucrose, i.e., the transfer of radioactivity from cytosol to sedimentable vesicles (autophagosomes and lysosomes), could be followed by incubating the (/sup 14/C)sucrose-loaded hepatocytes for up to 2 h at 37/sup 0/C. After incubation, the cells were disrupted by a single high-voltage discharge in electrolyte-free medium (sucrose), and sedimentable cell components were separated from the cytosol by centrifugation through metrizamide. By the use of these methods, which are particularly suitable for the analysis of many small cell samples, it could be shown that (/sup 14/C)sucros was autophagically sequestered in the hepatocytes at a rate of 4-5%/h. The sequestration was nearly completely inhibited by the specific autophagy inhibitor 3-methyladenine.

  20. Exposure of primary rat hepatocytes in long-term DMSO culture to selected transition metals induces hepatocyte proliferation and formation of duct-like structures.

    PubMed

    Cable, E E; Isom, H C

    1997-12-01

    We previously showed that primary rat hepatocytes plated on a rat-tail collagen coated dish and fed a chemically-defined medium supplemented with 2% dimethylsulfoxide (DMSO) can be maintained in a well-differentiated, non-replicating state for periods of several months. In this study, we show that the addition of copper, iron, and zinc to the DMSO-containing chemically defined medium induced DNA synthesis and cell replication during the first two months in culture without loss of hepatic differentiation. DNA synthesis occurred throughout the hepatocyte population without regard to cellular size. No changes were observed in properties indicative of well-differentiated hepatocytes, including cellular morphology, ultrastructure, albumin, or cytokeratin-8 expression. During the third month in culture, after the hepatocytes had become confluent, pseudoduct structures became apparent. Examination of cells lining the ducts by immunohistochemistry showed that these cells lost the ability to express albumin and stained more intensely for cytokeratin 19 and laminin. The ultrastructure of the cells lining the ducts was altered and became more characteristic of bile duct cells. Immunoelectron microscopy revealed that connexin 43, a marker of bile-duct proliferation, was expressed in the duct-like cells. We conclude that under these specific nutritive conditions, primary rat hepatocytes proliferate and, with time, begin to form duct-like structures with altered gene expression and ultrastructural properties.

  1. Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract.

    PubMed

    Housley, R M; Morris, C F; Boyle, W; Ring, B; Biltz, R; Tarpley, J E; Aukerman, S L; Devine, P L; Whitehead, R H; Pierce, G F

    1994-11-01

    Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, was identified as a specific keratinocyte mitogen after isolation from a lung fibroblast line. Recently, recombinant (r)KGF was found to influence proliferation and differentiation patterns of multiple epithelial cell lineages within skin, lung, and the reproductive tract. In the present study, we designed experiments to identify additional target tissues, and focused on the rat gastrointestinal (GI) system, since a putative receptor, K-sam, was originally identified in a gastric carcinoma. Expression of KGF receptor and KGF mRNA was detected within the entire GI tract, suggesting the gut both synthesized and responded to KGF. Therefore, rKGF was administered to adult rats and was found to induce markedly increased proliferation of epithelial cells from the foregut to the colon, and of hepatocytes, one day after systemic treatment. Daily treatment resulted in the marked selective induction of mucin-producing cell lineages throughout the GI tract in a dose-dependent fashion. Other cell lineages were either unaffected (e.g., Paneth cells), or relatively decreased (e.g., parietal cells, enterocytes) in rKGF-treated rats. The direct effect of rKGF was confirmed by demonstrating markedly increased carcinoembryonic antigen production in a human colon carcinoma cell line, LIM1899. Serum levels of albumin were specifically and significantly elevated after daily treatment. These results demonstrate rKGF can induce epithelial cell activation throughout the GI tract and liver. Further, endogenous KGF may be a normal paracrine mediator of growth within the gut.

  2. Effects of model inducers on thyroxine UDP-glucuronosyl-transferase activity in vitro in rat and mouse hepatocyte cultures.

    PubMed

    Viollon-Abadie, C; Bigot-Lasserre, D; Nicod, L; Carmichael, N; Richert, L

    2000-12-01

    Thyroxine (T(4))-UDP-glucuronosyltransferase (UGT) activity was measured directly in cultured male Sprague-Dawley rat and OF-1 mouse hepatocyte monolayers. The activity of T(4)-UGT (pmol/min/g liver) in vitro in hepatocyte cultures was, after 24 hr in culture, equivalent to that previously measured in vivo in rat and mouse liver microsomes (Viollon-Abadie et al., 1999). A progressive decline in T(4)-UGT activity occurred over time in both rat and mouse hepatocyte cultures. Treatment of cultures with various model inducers such as phenobarbital (PB), beta-naphthoflavone (NF) and clofibric acid (CLO) induced a strong increase in T(4)-UGT activity in rat hepatocyte monolayers. In addition, and as expected from available in vivo data, treatment of rat hepatocyte cultures with NF also increased p-nitrophenol (PNP)-UGT activity and treatment with PB or CLO increased bilirubin (Bili)-UGT activity. In contrast, T(4)-UGT activity in mouse hepatocyte monolayers was not affected by the treatments, neither were PNP- and Bili- UGT activities. These in vitro data confirm our previous in vivo observations that these inducers increase rat but not mouse liver T(4)-UGT activities (Viollon-Abadie et al., 1999). The present study thus demonstrates that hepatocyte monolayers are appropriated for the evaluation and inter-species comparison of the effects of xenobiotics on T(4)-UGT activities.

  3. Formation of glutathione conjugates by reactive metabolites of vinylidene chloride in microsomes and isolated hepatocytes

    SciTech Connect

    Liebler, D.C.; Meredith, M.J.; Guengerich, F.P.

    1985-01-01

    Oxidation of the vinyl halide carcinogen and hepatotoxin vinylidene chloride (VDC) by microsomal cytochrome P-450 yields 2,2-dichloroacetaldehyde, 2-chloroacetyl chloride, 2-chloroacetic acid, and 1,1-dichloroethylene oxide. The roles of these metabolites in covalent modification of proteins and reduced glutathione (GSH) were examined. 2-Chloroacetyl chloride reacted with model thiols at least 10(3)-fold faster than did 1,1-dichloroethylene oxide and at least 10(5)-fold faster than did 2,2-dichloroacetaldehyde or 2-chloroacetic acid. Microsomal covalent binding of (/sup 14/C)VDC was inhibited by GSH but not by lysine, suggesting that protein thiols, rather than amino groups, are major targets. Liver microsomes catalyzed the formation of three GSH:VDC metabolite conjugates, identified as S-(2,2-dichloro-1-hydroxy)ethylglutathione, 2-(S-glutathionyl)acetate, and S-(2-glutathionyl)acetylglutathione, a novel conjugate containing both stable (thioether) and labile (thioester) linkages. The latter two conjugates also were formed in isolated rat hepatocytes and measurable amounts of 2-(S-glutathionyl)acetate were released into the incubation medium. Both 2-(S-glutathionyl)acetate and S-(2-glutathionyl)acetylglutathione were formed with (/sup 35/S)GSH added to the hepatic medium, indicating that reactive VDC metabolites are capable of crossing the plasma membrane to react with extracellular targets. Unlabeled S-(2-glutathionyl)-acetylglutathione underwent carbonyl substitution with added (/sup 35/S)GSH, suggesting that this conjugate may participate in modification of protein thiols. This conjugate also underwent hydrolysis with a half-life of approximately 3 hr. GSH:VDC metabolite conjugates may serve as accessible models for labile covalent adducts formed between VDC metabolites and protein thiols.

  4. Role of elevated S-adenosylhomocysteine in rat hepatocyte apoptosis: protection by betaine.

    PubMed

    Kharbanda, Kusum K; Rogers, David D; Mailliard, Mark E; Siford, Gerri L; Barak, Anthony J; Beckenhauer, Harriet C; Sorrell, Michael F; Tuma, Dean J

    2005-12-05

    Previous studies from our laboratory have shown that ethanol consumption results in an increase in hepatocellular S-adenosylhomocysteine levels. Because S-adenosylhomocysteine is a potent inhibitor of methylation reactions, we propose that increased intracellular S-adenosylhomocysteine levels could be a major contributor to ethanol-induced pathologies. To test this hypothesis, hepatocytes isolated from rat livers were grown on collagen-coated plates in Williams' medium E containing 5% FCS and exposed to varying concentrations of adenosine in order to increase intracellular S-adenosylhomocysteine levels. We observed increases in caspase-3 activity following exposure to adenosine. This increase in caspase activity correlated with increases in intracellular S-adenosylhomocysteine levels and DNA hypoploidy. The adenosine-induced changes could be significantly attenuated by betaine administration. The mechanism of betaine action appeared to be via the methylation reaction catalyzed by betaine-homocysteine-methyltransferase. To conclude, our results indicate that the elevation of S-adenosylhomocysteine levels in the liver by ethanol is a major factor in altering methylation reactions and in increasing apoptosis in the liver. We conclude that ethanol-induced alteration in methionine metabolic pathways may play a crucial role in the pathologies associated with alcoholic liver injury and that betaine administration may have beneficial therapeutic effects.

  5. Effect of hepatocyte growth factor and angiotensin II on rat cardiomyocyte hypertrophy

    PubMed Central

    Chen, Ai-Lan; Ou, Cai-Wen; He, Zhao-Chu; Liu, Qi-Cai; Dong, Qi; Chen, Min-Sheng

    2012-01-01

    Angiotensin II (Ang II) plays an important role in cardiomyocyte hypertrophy. The combined effect of hepatocyte growth factor (HGF) and Ang II on cardiomyocytes is unknown. The present study was designed to determine the effect of HGF on cardiomyocyte hypertrophy and to explore the combined effect of HGF and Ang II on cardiomyocyte hypertrophy. Primary cardiomyocytes were isolated from neonatal rat hearts and cultured in vitro. Cells were treated with Ang II (1 µM) alone, HGF (10 ng/mL) alone, and Ang II (1 µM) plus HGF (10 ng/mL) for 24, 48, and 72 h. The amount of [3H]-leucine incorporation was then measured to evaluate protein synthesis. The mRNA levels of β-myosin heavy chain and atrial natriuretic factor were determined by real-time PCR to evaluate the presence of fetal phenotypes of gene expression. The cell size of cardiomyocytes was also studied. Ang II (1 µM) increased cardiomyocyte hypertrophy. Similar to Ang II, treatment with 1 µM HGF promoted cardiomyocyte hypertrophy. Moreover, the combination of 1 µM Ang II and 10 ng/mL HGF clearly induced a combined pro-hypertrophy effect on cardiomyocytes. The present study demonstrates for the first time a novel, combined effect of HGF and Ang II in promoting cardiomyocyte hypertrophy. PMID:23044624

  6. Arsenic trioxide induced indirect and direct inhibition of glutathione reductase leads to apoptosis in rat hepatocytes.

    PubMed

    Ray, Atish; Chatterjee, Sarmishtha; Mukherjee, Sandip; Bhattacharya, Shelley

    2014-06-01

    Glutathione reductase (GR) is an essential enzyme which maintains the reduced state of a cell. Therefore GR malfunction is closely associated with several disorders related to oxidative damage. The present study reports toxic manifestation of arsenic trioxide in respect of GR leading to apoptosis. Isolated rat hepatocytes exposed to arsenic trioxide were analyzed for GR expression and activity. Arsenic resulted in a time dependent inhibition of GR mediated by the superoxide anion. The cellular demand of functional enzyme is achieved by concomitant rise in gene expression. However, direct inhibition of GR by arsenic trioxide was also evident. Furthermore, arsenic induced free radical mediated inhibition of GR was found to be partially uncompetitive and associated with time dependent decrease in the substrate binding rate. Externalization of phosphatidylserine, nuclear degradation, apoptosis inducing factor leakage, apoptosome formation, caspase activation, DNA damage and break down of PARP suggest consequential induction of apoptosis due to inhibition of GR. The implication of GR was further established from the reduced rate of caspase activation in the arsenic trioxide treated cell, supplemented with complete and incomplete enzyme systems.

  7. Proliferation of rat small hepatocytes requires follistatin expression.

    PubMed

    Ooe, Hidekazu; Chen, Qijie; Kon, Junko; Sasaki, Kazunori; Miyoshi, Hiroyuki; Ichinohe, Norihisa; Tanimizu, Naoki; Mitaka, Toshihiro

    2012-06-01

    Small hepatocytes (SHs) are a subpopulation of hepatocytes that have high growth potential in culture and can differentiate into mature hepatocytes (MHs). The activin (Act)/follistatin (Fst) system critically contributes to homeostasis of cell growth in the normal liver. ActA and ActB consist of two disulfide-linked Inhibin (Inh)β subunits, InhβA and InhβB, respectively. Fst binds to Act and blocks its bioactivity. In the present study we carried out the experiments to clarify how Fst regulates the proliferation of SHs. The gene expression was analyzed using DNA microarray analysis, reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR, and protein expression was examined by western blots, immunocytochemistry, and enzyme-linked immunosorbent assay. RT-PCR showed that Fst expression was high in SHs and low in MHs. Although the ActA expression was opposite to that of Fst, ActB expression was high in SHs and low in MHs and increased with time in culture. Fst protein was detected in the cytoplasm of SHs and secreted into the culture medium. ActB protein was also secreted into the medium. Although the exogenous administration of ActA and ActB apparently suppressed the proliferation of SHs, apoptosis of SHs was not induced by treatment with ActA or ActB. On the other hand, Fst treatment did not affect the colony formation of SHs but prevented the inhibitory effect of ActA. Neutralization by the anti-Fst antibody resulted in the suppression of DNA synthesis in SHs, and small hairpin RNA against Fst suppressed the expansion of SH colonies. In conclusion, Fst expression is necessary for the proliferation of SHs.

  8. Effect of endogenous nitric oxide on mitochondrial respiration of rat hepatocytes in vitro and in vivo

    SciTech Connect

    Stadler, J.; Curran, R.D.; Ochoa, J.B.; Harbrecht, B.G.; Hoffman, R.A.; Simmons, R.L.; Billiar, T.R. )

    1991-02-01

    Nitric oxide, a highly reactive radical, was recently identified as an intermediate of L-arginine metabolism in mammalian cells. We have shown that nitric oxide synthesis is induced in vitro in cultured hepatocytes by supernatants from activated Kupffer cells or in vivo by injecting rats with nonviable Corynebacterium parvum. In both cases, nitric oxide biosynthesis in hepatocytes was associated with suppression of total protein synthesis. This study attempts to determine the effect of nitric oxide biosynthesis on the activity of specific hepatocytic mitochondrial enzymes and to determine whether inhibition of protein synthesis is caused by suppression of energy metabolism. Exposure of hepatocytes to supernatants from activated Kupffer cells led to a 30% decrease of aconitase (Krebs cycle) and complex I (mitochondrial electron transport chain) activity. Using NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthesis, we demonstrated that the inhibition of mitochondrial aconitase activity was due, in part, to the action of nitric oxide. In contrast, in vivo nitric oxide synthesis of hepatocytes from Corynebacterium parvum-treated animals had no effect on mitochondrial respiration. This suggests that inhibition of protein synthesis by nitric oxide is not likely to be mediated by inhibition of energy metabolism.

  9. A light- and electron-microscope study of hepatocytes of rats fed different diets.

    PubMed

    Eagles, Douglas A; Chapman, George B

    2007-01-01

    Ketogenic diets are used in the treatment of epilepsy in children refractory to drug therapy. This study identifies changes in liver morphology in rats fed four different diets: a normal rodent chow diet, a calorie-restricted high-fat (ketogenic) diet and each diet supplemented with clofibric acid. Hepatocytes of rats fed the ketogenic diet show many lipid droplets and these are reduced to control levels when clofibrate is present in the diet. Mitochondria are enlarged in the livers of rats fed the ketogenic diet and further enlarged if clofibrate is present. Alterations in the appearance or numbers of other organelles are also found.

  10. Targeted gene transfer of human hepatocyte growth factor into rat skin by the use of laser-induced stress waves

    NASA Astrophysics Data System (ADS)

    Terakawa, Mitsuhiro; Sato, Shunichi; Saitoh, Daizoh; Ashida, Hiroshi; Okano, Hideyuki; Obara, Minoru

    2006-02-01

    We successfully delivered therapeutic gene, Hepatocyte Growth Factor (HGF), to rat skin in vivo. The level of hHGF protein increased by the application of LISWs when compared with that of control samples without LISW application.

  11. Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes.

    PubMed

    Liu, Cong; Sekine, Shuichi; Ito, Kousei

    2016-07-01

    Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and in hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity.

  12. Soyasaponin Bb Protects Rat Hepatocytes from Alcohol-Induced Oxidative Stress by Inducing Heme Oxygenase-1

    PubMed Central

    Lijie, Zhu; Ranran, Fu; Xiuying, Liu; Yutang, He; Bo, Wang; Tao, Ma

    2016-01-01

    Background: It has been known that oxidative stress induced by alcohol played a crucial role in the formation of alcoholic liver disease. Although the formation mechanisms underlying liver injury induced by alcohol still remained largely unknown, it has been considered that oxidative stress played a core role in the pathogenesis of hepatocyte damage. Objective: The aim of this study was to investigate the effects of soyasaponin Bb (Ss-Bb) on oxidative stress in alcohol-induced rat hepatocyte injury. Results: It has been shown that the administration of Ss-Bb could significantly restore antioxidant activity in BRL 3A cells. Moreover, the impaired liver function and morphology changes resulting from ethanol exposure were improved by Ss-Bb treatment. Treatment with a pharmacological inhibitor of haem oxygenase-1 (HO-1) indicated a critical role of HO-1 in mediating the protective role. Finally, we found that pretreatment with Ss-Bb to ethanol exposure cells increased the expression level of HO-1. Conclusion: It was suggested that Ss-Bb may protect against alcohol-induced hepatocyte injury through ameliorating oxidative stress, and the induction of HO-1 was an important protective mechanism. SUMMARY Effects of soyasaponin Bb was investigated on oxidative stress in rat hepatocytesCell viability and antioxidant capacities were evaluated to determine the effectsThe expression level of HO-1 was measured to reveal the proptective mechanisms PMID:27867273

  13. Metabolism of S-adenosylmethionine in rat hepatocytes: transfer of methyl group from S-adenosylmethionine by methyltransferase reactions

    SciTech Connect

    Tsukada, K.; Abe, T.; Kuwahata, T.; Mitsui, K.

    1985-08-19

    Treatment of rats with a methionine diet leads not only to a marked increase of S-adenosylmethionine synthetase in liver, but also to the increase of glycine, guanidoacetate and betaine-homocysteine methyltransferases. The activity of tRNA methyltransferase decreased with the increased amounts of methionine in the diets. However, the activities of phospholipids and S-adenosylmethionine-homocysteine methyltransferases did not show any significant change. When hepatocarcinogenesis induced by 2-fluorenylacetamide progresses, the activities of glycine and guanidoacetate methyltransferases in rat liver decreased, and could not be detected in tumorous areas 8 months after treatment. The levels of S-adenosylmethionine in the liver also decreased to levels of one-fifth of control animals at 8 months. The uptake and metabolism of (methyl-/sup 3/H)-methionine and -S-adenosylmethionine have been investigated by in vivo and isolated hepatocytes. The uptake of methionine and transfer of methyl group to phospholipid in the cells by methionine were remarkably higher than those by S-adenosylmethionine. These results indicate that phospholipids in hepatocytes accept methyl group from S-adenosylmethionine immediately, when it is synthesized from methionine, before mixing its pool in the cells. 39 references, 1 figure, 2 tables.

  14. Phenobarbital induces alterations in the proteome of hepatocytes and mesenchymal cells of rat livers.

    PubMed

    Klepeisz, Philip; Sagmeister, Sandra; Haudek-Prinz, Verena; Pichlbauer, Melanie; Grasl-Kraupp, Bettina; Gerner, Christopher

    2013-01-01

    Preceding studies on the mode of action of non-genotoxic hepatocarcinogens (NGCs) have concentrated on alterations induced in hepatocytes (HCs). A potential role of non-parenchymal liver cells (NPCs) in NGC-driven hepatocarcinogenesis has been largely neglected so far. The aim of this study is to characterize NGC-induced alterations in the proteome profiles of HCs as well as NPCs. We chose the prototypic NGC phenobarbital (PB) which was applied to male rats for a period of 14 days. The livers of PB-treated rats were perfused by collagenase and the cell suspensions obtained were subjected to density gradient centrifugation to separate HCs from NPCs. In addition, HCs and NPC isolated from untreated animals were treated with PB in vitro. Proteome profiling was done by CHIP-HPLC and ion trap mass spectrometry. Proteome analyses of the in vivo experiments showed many of the PB effects previously described in HCs by other methods, e.g. induction of phase I and phase II drug metabolising enzymes. In NPCs proteins related to inflammation and immune regulation such as PAI-1 and S100-A10, ADP-ribosyl cyclase 1 and to cell migration such as kinesin-1 heavy chain, myosin regulatory light chain RLC-A and dihydropyrimidinase-related protein 1 were found to be induced, indicating major PB effects on these cells. Remarkably, in vitro treatment of HCs and NPCs with PB hardly reproduced the proteome alterations observed in vivo, indicating differences of NGC induced responses of cells at culture conditions compared to the intact organism. To conclude, the present study clearly demonstrated that PB induces proteome alterations not only in HCs but also in NPCs. Thus, any profound molecular understanding on the mode of action of NGCs has to consider effects on cells of the hepatic mesenchyme.

  15. Kinetics of Arsenic Methylation by Freshly Isolated B6C3F1 Mouse Hepatocytes

    SciTech Connect

    Kedderis, Gregory L.; Elmore, Amy R.; Crecelius, Eric A.; Yager, Janice W.; Goldsworthy, Thomas L.

    2006-06-10

    The toxic and carcinogenic effects of arsenic may be mediated by both inorganic and methylated arsenic species. The methylation of arsenicIII takes place via sequential oxidative methylation and reduction steps to form monomethylarsenic (MMA) and dimethylarsenic (DMA) species. The kinetics of arsenic methylation were determined in freshly isolated hepatocytes from male B6C3F1 mice. Hepatocytes (>90% viability) were isolated by collagenase perfusion and suspended in Williams Medium E with various concentrations of arsenicIII (sodium m-arsenite). Aliquots of the cell suspension were lysed with 1.0% Triton X-100 and analyzed for arsenic species by hydride generation-atomic absorption spectrometry. The formation of MMAIII from sodium arsenite (1 ?M) was linear with respect to time for >90 min. DMAIII formation did not become significant until 60 min. MMAV and DMAV were not consistently observed in the incubations. These results suggest that the reduction of MMAV to MMAIII is rapid relative to the methylation rate since MMAV was not observed as a major product of arsenicIII metabolism in mouse hepatocytes. Metabolism of arsenicV was not observed in mouse hepatocytes, consistent with inhibition of arsenicV active cellular uptake by phosphate in the medium. The formation of MMAIII increased with increasing arsenicIII concentrations up to approximately 2 ?M and declined thereafter. The concentration dependence is consistent with a saturable methylation reaction accompanied by substrate inhibition of the reaction by arsenicIII. Kinetic analysis of the data suggested an apparent KM of approximately 3.6 ?M arsenicIII, an apparent Vmax of approximately 38.9 ?g MMAIII formed/L/hr/million cells, and an apparent KI of approximately 1.3 ?M arsenicIII. The results of this study can be used in the physiologically based pharmacokinetic model for arsenic disposition in mice to predict the concentration of MMAIII in liver and other tissues.

  16. Effect of aging on regulation of sdi-1 in rat hepatocytes.

    PubMed

    Kitano, S; Venable, S; Smith, J R; Reed, T D; Roth, G S

    1996-08-05

    We examined basal and EGF stimulated DNA synthesis as well as sdi-1 mRNA and protein in primary hepatocyte cultures, and basal levels of sdi-1 mRNA and protein in whole liver homogenates from 6 and 24 month old rats. Since EGF stimulated DNA synthesis decreases with age, it was hypothesized that basal and EGF stimulated levels of sdi-1 mRNA and protein, an inhibitor of DNA synthesis, might increase. Surprisingly, however both sdi-1 mRNA and protein actually decreased both in cells and homogenates of old rats. These results indicate that the age-related impairment in EGF stimulated DNA synthesis in hepatocytes appears to occur prior to or parallel with sdi-1 expression and cannot be explained on the basis of increased inhibition due to elevated levels of this protein.

  17. Differential regulation of the mitogen-activated protein and stress-activated protein kinase cascades by adrenergic agonists in quiescent and regenerating adult rat hepatocytes.

    PubMed Central

    Spector, M S; Auer, K L; Jarvis, W D; Ishac, E J; Gao, B; Kunos, G; Dent, P

    1997-01-01

    To study the mechanisms by which catecholamines regulate hepatocyte proliferation after partial hepatectomy (PHX), hepatocytes were isolated from adult male rats 24 h after sham operation or two-thirds PHX and treated with catecholamines and other agonists. In freshly isolated sham cells, p42 mitogen-activated protein (MAP) kinase activity was stimulated by the alpha1-adrenergic agonist phenylephrine (PHE). Activation of p42 MAP kinase by growth factors was blunted by pretreatment of sham hepatocytes with glucagon but not by that with the beta2-adrenergic agonist isoproterenol (ISO). In PHX cells, the ability of PHE to activate p42 MAP kinase was dramatically reduced, whereas ISO became competent to inhibit p42 MAP kinase activation. PHE treatment of sham but not PHX and ISO treatment of PHX but not sham hepatocytes also activated the stress-activated protein (SAP) kinases p46/54 SAP kinase and p38 SAP kinase. These data demonstrate that an alpha1- to beta2-adrenergic receptor switch occurs upon PHX and results in an increase in SAP kinase versus MAP kinase signaling by catecholamines. In primary cultures of hepatocytes, ISO treatment of PHX but not sham cells inhibited [3H]thymidine incorporation. In contrast, PHE treatment of sham but not PHX cells stimulated [3H]thymidine incorporation, which was reduced by approximately 25 and approximately 95% with specific inhibitors of p42 MAP kinase and p38 SAP kinase function, respectively. Inhibition of the p38 SAP kinase also dramatically reduced basal [3H]thymidine incorporation. These data suggest that p38 SAP kinase plays a permissive role in liver regeneration. Alterations in the abilities of catecholamines to modulate the activities of protein kinase A and the MAP and SAP kinase pathways may represent one physiological mechanism by which these agonists can regulate hepatocyte proliferation after PHX. PMID:9199291

  18. Effect of guava (Psidium guajava L.) leaf extract on glucose uptake in rat hepatocytes.

    PubMed

    Cheng, Fang-Chi; Shen, Szu-Chuan; Wu, James Swi-Bea

    2009-06-01

    People in oriental countries, including Japan and Taiwan, boil guava leaves (Psidium guajava L.) in water and drink the extract as a folk medicine for diabetes. The present study investigated the enhancement of aqueous guava leaf extract on glucose uptake in rat clone 9 hepatocytes and searched for the active compound. The extract was eluted with MeOH-H(2)O solutions through Diaion, Sephadex, and MCI-gel columns to separate into fractions with different polarities. The uptake test of 2-[1-(14)C] deoxy-D-glucose in rat clone 9 hepatocytes was performed to evaluate the hypoglycemic effect of these fractions. The active compound was identified by nuclear magnetic resonance analysis and high-performance liquid chromatography (HPLC). The results revealed that phenolics are the principal component of the extract, that high polarity fractions of the guava leaf extract are enhancers to glucose uptake in rat clone 9 hepatocytes, and that quercetin is the major active compound. We suggest that quercetin in the aqueous extract of guava leaves promotes glucose uptake in liver cells, and contributes to the alleviation of hypoglycemia in diabetes as a consequence.

  19. Hepatocyte differentiation.

    PubMed

    Olsavsky Goyak, Katy M; Laurenzana, Elizabeth M; Omiecinski, Curtis J

    2010-01-01

    Increasingly, research suggests that for certain systems, animal models are insufficient for human toxicology testing. The development of robust, in vitro models of human toxicity is required to decrease our dependence on potentially misleading in vivo animal studies. A critical development in human toxicology testing is the use of human primary hepatocytes to model processes that occur in the intact liver. However, in order to serve as an appropriate model, primary hepatocytes must be maintained in such a way that they persist in their differentiated state. While many hepatocyte culture methods exist, the two-dimensional collagen "sandwich" system combined with a serum-free medium, supplemented with physiological glucocorticoid concentrations, appears to robustly maintain hepatocyte character. Studies in rat and human hepatocytes have shown that when cultured under these conditions, hepatocytes maintain many markers of differentiation including morphology, expression of plasma proteins, hepatic nuclear factors, phase I and II metabolic enzymes. Functionally, these culture conditions also preserve hepatic stress response pathways, such as the SAPK and MAPK pathways, as well as prototypical xenobiotic induction responses. This chapter will briefly review culture methodologies but will primarily focus on hallmark hepatocyte structural, expression and functional markers that characterize the differentiation status of the hepatocyte.

  20. Metabolism of cyclosporin A. I. Study in freshly isolated rabbit hepatocytes

    SciTech Connect

    Fabre, G.; Bertault-Peres, P.; Fabre, I.; Maurel, P.; Just, S.; Cano, J.P.

    1987-05-01

    The metabolism of cyclosporin A (CsA), a widely used immunosuppressive agent, was evaluated in freshly isolated rabbit hepatocytes by HPLC which separated CsA from its major group of derivatives, e.g. first generation metabolites (monohydroxylated and N-demethylated) and second generation derivatives (dihydroxylated and dihydroxy-N-demethylated). After exposure of hepatocytes to radiolabeled CsA (0.5 mg/liter), CsA was rapidly accumulated inside the cells and metabolized. The dihydroxylated metabolites represent the major intracellular forms after 1 hr. CsA metabolites synthesized inside the cells are then rapidly detected in the extracellular compartment. Unchanged drug and the various metabolites are concentrated inside the cells with transmembrane chemical gradients ranging between 20:1 and 40:1. Transport and metabolic processes for CsA have been evaluated over the following CsA extracellular concentration range, 0.1-10 mg/liter. Metabolism appears to be the rate-limiting step. The apparent affinity constant of CsA for the enzyme system involved in its metabolism is approximately 15 microM. Besides the lipophilicity of the molecule, which is responsible for the retention of CsA and its metabolites in the intracellular compartment, the presence of a binding component(s) in the hepatocytes was also demonstrated. CsA and its metabolites seem to have similar affinities for this binding site. These studies demonstrate that CsA is rapidly transformed inside the hepatocytes to various metabolites which may play an important role in the pharmacological activity of the drug and/or in its clinical toxicity.

  1. Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes

    SciTech Connect

    Novicki, D.L.; Rosenberg, M.R.; Michalopoulos, G.

    1985-01-01

    Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats. The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of (3H)thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity.

  2. The impairment of hepatocytes and sinusoidal endothelial cells during cold preservation in rat fatty liver induced by alcohol and the beneficial effect of hepatocyte growth factor.

    PubMed

    Takeda, Yoshihisa; Arii, Shigeki; Kaido, Toshimi; Imamura, Masayuki

    2003-04-01

    A fatty liver resulting from alcohol intake is often unattractive for grafting. In this study, we investigated the impairment of hepatocytes and sinusoidal endothelial cells (SECs) during cold preservation of alcohol-induced fatty liver and examined the efficacy of human recombinant hepatocyte growth factor (hrHGF). Rats were fed an alcohol diet. We performed histological examinations of the hepatocytes and observed the ultrastructural alteration of the SECs. Additionally, we measured hepatic transaminase and peroxidative lipids for hepatocellular injury and the hyaluronic acid uptake rate (HUR) to determine SEC injury. We added hrHGF to University of Wisconsin (UW) solution to assess the protective effect of the agent. Numerous fatty deposits were observed in ethanol-induced fatty livers. These grew with the duration of cold storage. Hepatic transaminases of the effluents increased during cold preservation in the livers of alcohol-treated rats. Additionally, peroxidative lipids in the effluents increased during cold preservation in the livers of alcohol-treated rats, whereas they were undetectable in non-alcohol-treated rat livers. The sinusoidal endothelium had severely deteriorated in the livers of alcohol-treated rats. Further, the HUR decreased with ethanol treatment and/or cold preservation. The addition of hrHGF suppressed the increase of hepatic transaminase in the effluent of cold-preserved alcohol-treated livers. Peroxidative lipids in the same effluents were undetectable. In fatty livers, both hepatocytes and SECs received severe damage during cold preservation. Furthermore, we demonstrated that hepatocellular injury was significantly inhibited by hrHGF.

  3. Tanshinone I increases CYP1A2 protein expression and enzyme activity in primary rat hepatocytes.

    PubMed

    Lee, Wayne Y W; Zhou, Xuelin; Or, Penelope M Y; Kwan, Yiu Wa; Yeung, John H K

    2012-01-15

    This study investigated the effects of Danshen and its active ingredients on the protein expression and enzymatic activity of CYP1A2 in primary rat hepatocytes. The ethanolic extract of Danshen roots (containing mainly tanshinones) inhibited CYP1A2-catalyzed phenacetin O-deethylation (IC(50)=24.6 μg/ml) in primary rat hepatocytes while the water extract containing mainly salvianolic acid B and danshenshu had no effect. Individual tanshinones such as cryptotanshinone, dihydrotanshinone, tanshinone IIA inhibited the CYP1A2-mediated metabolism with IC(50) values at 12.9, 17.4 and 31.9 μM, respectively. After 4-day treatment of the rat hepatocytes, the ethanolic extract of Danshen and tanshinone I increased rat CYP1A2 activity by 6.8- and 5.2-fold, respectively, with a concomitant up-regulation of CYP1A2 protein level by 13.5- and 6.5-fold, respectively. CYP1A2 induction correlated with the up-regulation of mRNA level of aryl hydrocarbon receptor (AhR), which suggested a positive feedback mechanism of tanshinone I-mediated CYP1A2 induction. A formulated Danshen pill (containing mainly danshensu and salvianolic acid B and the tanshinones) up-regulated CYP1A2 protein expression and enzyme activity, but danshensu and salvianolic acid B, when used individually, did not affect CYP1A2 activity. This study was the first report on the Janus action of the tanshinones on rat CYP1A2 activity.

  4. Cytoprotective Effects of Hydrophilic and Lipophilic Extracts of Pistacia vera against Oxidative Versus Carbonyl Stress in Rat Hepatocytes

    PubMed Central

    Shahraki, Jafar; Zareh, Mona; Kamalinejad, Mohammad; Pourahmad, Jalal

    2014-01-01

    This study was conducted to evaluate the cytoprotection of various extracts and bioactive compounds found in Pistacia vera againts cytotoxicity, ROS formation, lipid peroxidation, protein carbonylation, mitochondrial and lysosomal membrane damages in cell toxicity models of diabetes related carbonyl (glyoxal) and oxidative stress (hydroperoxide). Methanol, water and ethyl acetate were used to prepare crude pistachios extracts, which were then used to screen for in-vitro cytoprotection of freshly isolated rat hepatocytes against these toxins. The order of protection by Pistacia vera extracts against both hydroperoxide induced oxidative stress (ROS formation) and glyoxal induced protein carbonylation was: pistachio methanolic extract >pistachio water extract, gallic acid, catechin> α-tochoferol and pistachio ethyl acetate extract. Finally due to higher protection achieved by methanolic extract even compared to sole pretreatment of gallic acid, catechin or α-tochoferol, we suggest that cytoprotection depends on the variety of polar and non-polar compounds found in methanolic extract, it is likely that multiple cytoprotective mechanisms are acting against oxidative and carbonyl induced cytotoxicity. To our knowledge, we are the first to report the cytoprotective activity of Pistacia vera extracts against oxidative and carbonyl stress seen in type 2 diabetes hepatocytes model. PMID:25587316

  5. Activation of Poly(ADP-Ribose)Polymerase in rat hepatocytes does not contribute to their cell death by oxidative stress.

    PubMed

    Latour, I; Leunda-Casi, A; Denef, J F; Buc Calderon, P

    2000-01-10

    Oxidative stress induced by tert-butyl hydroperoxide (tBOOH) in freshly isolated rat hepatocytes caused DNA damage and loss of membrane integrity. Such DNA lesions are likely to be single strand breaks since neither caryolysis nor chromatine condensation was seen in electron micrographs from tBOOH-treated cells. In addition, pulsed field gel electrophoresis of genomic DNA from both control and tBOOH-treated hepatocytes showed similar profiles, indicating the absence of internucleosomal DNA cleavage, a classical reflection of apoptotic endonuclease activity. The activation of the repair enzyme poly(ADP-ribose)polymerase (PARP) following DNA damage by tBOOH induced a dramatic drop in both NAD(+) and ATP. The inhibition of PARP by 3-aminobenzamide enhanced DNA damage by tBOOH, restored NAD(+) and ATP levels, but did not result in better survival against cell killing by tBOOH. The lack of the protective effect of PARP inhibitor, therefore, does not implicate PARP in the mechanism of tBOOH-induced cytotoxicity. Electron micrographs also show no mitochondrial swelling in cells under oxidative stress, but such organelles were mainly located around the nucleus, a picture already observed in autoschizis, a new suggested kind of cell death which shows both apoptotic and necrotic morphological characteristics.

  6. Isolation of rat adrenocortical mitochondria

    SciTech Connect

    Solinas, Paola; Fujioka, Hisashi; Tandler, Bernard; Hoppel, Charles L.

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer A method for isolation of adrenocortical mitochondria from the adrenal gland of rats is described. Black-Right-Pointing-Pointer The purified isolated mitochondria show excellent morphological integrity. Black-Right-Pointing-Pointer The properties of oxidative phosphorylation are excellent. Black-Right-Pointing-Pointer The method increases the opportunity of direct analysis of adrenal mitochondria from small animals. -- Abstract: This report describes a relatively simple and reliable method for isolating adrenocortical mitochondria from rats in good, reasonably pure yield. These organelles, which heretofore have been unobtainable in isolated form from small laboratory animals, are now readily accessible. A high degree of mitochondrial purity is shown by the electron micrographs, as well as the structural integrity of each mitochondrion. That these organelles have retained their functional integrity is shown by their high respiratory control ratios. In general, the biochemical performance of these adrenal cortical mitochondria closely mirrors that of typical hepatic or cardiac mitochondria.

  7. Induction of mitochondrial biogenesis and respiration is associated with mTOR regulation in hepatocytes of rats treated with the pan-PPAR activator tetradecylthioacetic acid (TTA)

    SciTech Connect

    Hagland, Hanne R.; Nilsson, Linn I.H.; Burri, Lena; Nikolaisen, Julie; Berge, Rolf K.; Tronstad, Karl J.

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We investigated mechanisms of mitochondrial regulation in rat hepatocytes. Black-Right-Pointing-Pointer Tetradecylthioacetic acid (TTA) was employed to activate mitochondrial oxidation. Black-Right-Pointing-Pointer Mitochondrial biogenesis and respiration were induced. Black-Right-Pointing-Pointer It was confirmed that PPAR target genes were induced. Black-Right-Pointing-Pointer The mechanism involved activation mTOR. -- Abstract: The hypolipidemic effect of peroxisome proliferator-activated receptor (PPAR) activators has been explained by increasing mitochondrial fatty acid oxidation, as observed in livers of rats treated with the pan-PPAR activator tetradecylthioacetic acid (TTA). PPAR-activation does, however, not fully explain the metabolic adaptations observed in hepatocytes after treatment with TTA. We therefore characterized the mitochondrial effects, and linked this to signalling by the metabolic sensor, the mammalian target of rapamycin (mTOR). In hepatocytes isolated from TTA-treated rats, the changes in cellular content and morphology were consistent with hypertrophy. This was associated with induction of multiple mitochondrial biomarkers, including mitochondrial DNA, citrate synthase and mRNAs of mitochondrial proteins. Transcription analysis further confirmed activation of PPAR{alpha}-associated genes, in addition to genes related to mitochondrial biogenesis and function. Analysis of mitochondrial respiration revealed that the capacity of both electron transport and oxidative phosphorylation were increased. These effects coincided with activation of the stress related factor, ERK1/2, and mTOR. The protein level and phosphorylation of the downstream mTOR actors eIF4G and 4E-BP1 were induced. In summary, TTA increases mitochondrial respiration by inducing hypertrophy and mitochondrial biogenesis in rat hepatocytes, via adaptive regulation of PPARs as well as mTOR.

  8. Uptake of Free Choline by Isolated Perfused Rat Liver

    NASA Astrophysics Data System (ADS)

    Zeisel, Steven H.; Story, David L.; Wurtman, Richard J.; Brunengraber, Henri

    1980-08-01

    The uptake of free choline by isolated perfused rat liver was characterized. A saturable uptake mechanism [Ka=0.17± 0.07 mM (SD); Vmax=0.84± 0.16\\ μ mol/min × g dry weight] and a nonsaturable mechanism (through which uptake is proportional to choline concentration in the perfusate) were identified. Most of the choline transported into hepatocytes was converted to betaine, phosphorylcholine, or lecithin. Free choline also accumulated within the intracellular space, suggesting that choline oxidase activity does not always limit choline's uptake by the liver.

  9. Repeated transplantation of hepatocytes prevents fulminant hepatitis in a rat model of Wilson's disease.

    PubMed

    Sauer, Vanessa; Siaj, Ramsi; Stöppeler, Sandra; Bahde, Ralf; Spiegel, Hans-Ullrich; Köhler, Gabriele; Zibert, Andree; Schmidt, Hartmut H J

    2012-02-01

    The outcome of consecutive hepatocyte transplants was explored in a rat model of Wilson's disease before the onset of fulminant hepatitis without preconditioning regimens. Rats received a high-copper diet in order to induce a rapid induction of liver failure. Sham-operated rats (15/15) developed jaundice and fulminant hepatitis, and they died within 4 weeks of first transplantation. Despite the continuation of a high dietary copper challenge, long-term survival was observed for a notable proportion of the transplanted animals (7/18). All survivors displayed normalized levels of hepatitis-associated serum markers and ceruloplasmin oxidase activity by posttransplant days 50 and 98, respectively. The liver copper concentrations, the liver histology, and the expression of marker genes were significantly restored within 4 months of transplantation in comparison with the control group. The high expression of a copper transporter gene (ATPase Cu++ transporting beta polypeptide) in the livers of the survivors indicated a high rate of repopulation by donor hepatocytes. Our data suggest that repeated cell transplantation can overcome the limitations of a single therapy session in rats with severe hepatic disease by functionally restoring the host liver without preconditioning.

  10. Efficient liver repopulation of transplanted hepatocyte prevents cirrhosis in a rat model of hereditary tyrosinemia type I

    PubMed Central

    Zhang, Ludi; Shao, Yanjiao; Li, Lu; Tian, Feng; Cen, Jin; Chen, Xiaotao; Hu, Dan; Zhou, Yan; Xie, Weifen; Zheng, Yunwen; Ji, Yuan; Liu, Mingyao; Li, Dali; Hui, Lijian

    2016-01-01

    Hereditary tyrosinemia type I (HT1) is caused by a deficiency in the enzyme fumarylacetoacetate hydrolase (Fah). Fah-deficient mice and pigs are phenotypically analogous to human HT1, but do not recapitulate all the chronic features of the human disorder, especially liver fibrosis and cirrhosis. Rats as an important model organism for biomedical research have many advantages over other animal models. Genome engineering in rats is limited till the availability of new gene editing technologies. Using the recently developed CRISPR/Cas9 technique, we generated Fah−/− rats. The Fah−/− rats faithfully represented major phenotypic and biochemical manifestations of human HT1, including hypertyrosinemia, liver failure, and renal tubular damage. More importantly, the Fah−/− rats developed remarkable liver fibrosis and cirrhosis, which have not been observed in Fah mutant mice or pigs. Transplantation of wild-type hepatocytes rescued the Fah−/− rats from impending death. Moreover, the highly efficient repopulation of hepatocytes in Fah−/− livers prevented the progression of liver fibrosis to cirrhosis and in turn restored liver architecture. These results indicate that Fah−/− rats may be used as an animal model of HT1 with liver cirrhosis. Furthermore, Fah−/− rats may be used as a tool in studying hepatocyte transplantation and a bioreactor for the expansion of hepatocytes. PMID:27510266

  11. Induction of fatty acid synthesis by pravastatin sodium in rat liver and primary hepatocytes.

    PubMed

    Fujioka, T; Tsujita, Y; Shimotsu, H

    1997-06-11

    We examined the effect of pravastatin sodium (pravastatin), a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on fatty acid synthesis in rat liver. The repeated administration of pravastatin to rats at 250 mg/kg for 7 days led to a 2.8-fold increase in fatty acid synthesis in the liver. The diurnal change of fatty acid synthesis was not affected by the treatment. Hepatic fatty acid synthase activity was increased 3.2-fold, while acetyl-CoA carboxylase activity was not changed by the repeated administration of pravastatin. In rat hepatocytes, the incubation with 2 microg/ml pravastatin for 24 h increased fatty acid synthase activity 1.5-fold, as well as HMG-CoA reductase activity 2.8-fold. These results suggest that HMG-CoA reductase inhibitors might increase fatty acid synthesis in vivo through the induction of hepatic fatty acid synthase.

  12. Reduced expression of mature TGF beta 1 correlates with the suppression of rat hepatocyte apoptosis by the peroxisome proliferator, nafenopin.

    PubMed

    Strange, J; Roberts, R A

    1996-11-11

    Non-genotoxic carcinogens cause cancer without damaging the DNA. Peroxisome proliferators (PPs) are a class of potent rodent non-genotoxic hepatocarcinogens that may act by perturbing hepatocyte growth regulation. Previously, we have shown that although cultured rat hepatocytes degenerate rapidly in culture, their survival can be reversibly maintained by the PP nafenopin. This prolonged survival is associated with a decrease in the number of hepatocytes displaying the chromatin condensation characteristic of apoptosis. The addition of the negative growth regulator TGF beta-1 induced high levels of hepatocyte apoptosis but nafenopin was able to suppress this TGF beta-1 induced apoptosis. These data suggested that increased levels of mature TGF beta-1 may be involved in the signalling of the apoptosis seen in degenerating hepatocyte cultures. To test this hypothesis, we carried out Western blot analyses using a anti-TGF beta 1 antibody. There was an increase (p = 0.014) in expression of mature TGF beta 1 in degenerating rat hepatocyte cultures compared with hepatocyte cultures surviving in the presence of nafenopin. However, there was a concomitant decrease (p = 0.024) in TGF beta 1-latency activated protein (TGF beta 1-LAP), the precursor of the active, mature form. Immunocytochemistry confirmed that TGF beta 1/TGF beta 1-LAP expression was predominantly in the hepatocytes displaying apoptotic morphology although expression was detected also in non-parenchymal liver cells. The immunocytochemistry data indicate that TGF beta 1 is involved during the onset of hepatocyte apoptosis and that the PP nafenopin can impinge on this cell death pathway. TGF beta 1-LAP, probably produced mainly by the non-parenchymal liver cells, may be processed less efficiently to the mature, active form in the presence of nafenopin, although more data are required to confirm this hypothesis.

  13. Cytoprotection by iloprost against paracetamol-induced toxicity in hamster isolated hepatocytes.

    PubMed Central

    Nasseri-Sina, P.; Fawthrop, D. J.; Wilson, J.; Boobis, A. R.; Davies, D. S.

    1992-01-01

    1 The ability of iloprost (ZK36374) to protect hamster isolated hepatocytes from the toxic effects of paracetamol and its reactive metabolite N-acetyl-p-benzoquinoneimine (NABQI) was investigated. The cytoprotection provided by iloprost was compared with that of N-acetyl-L-cysteine. 2 Treatment of hepatocytes with either NABQI (0.4 mM) or paracetamol (2 mM) alone resulted in a considerable loss of cell viability, as assessed by trypan blue exclusion or leakage of lactate dehydrogenase, accompanied by an increase in the percentage of viable cells that were blebbed. N-acetyl-L-cysteine (1.25 mM) pretreatment diminished the loss of cell viability and the percentage of blebbed cells resulting from exposure to NABQI or paracetamol, whereas iloprost (10(-16) M to 10(-10) M) pretreatment reduced only the loss of cell viability, not the percentage of viable cells exhibiting blebbing. Pretreatment with N-acetyl-L-cysteine significantly attenuated the depletion by paracetamol of glutathione and decreased the covalent binding of [14C]-paracetamol to cellular proteins, whereas iloprost was without any such effects. 3 The effects of iloprost and N-acetyl-L-cysteine were also investigated by use of a model of paracetamol toxicity in which it is possible to study the biochemical events leading to cell injury separate from the generation of toxic metabolites. Hamster hepatocytes were incubated with paracetamol (4 mM) for 90 min at 37 degrees C during which metabolism of paracetamol occurs with minimal loss of cell viability.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1373102

  14. Arsenite decreases CYP3A23 induction in cultured rat hepatocytes by transcriptional and translational mechanisms

    SciTech Connect

    Noreault, Trisha L.; Nichols, Ralph C.; Trask, Heidi W.; Wrighton, Steven A.; Sinclair, Peter R.; Evans, Ronald M.; Sinclair, Jacqueline F. . E-mail: JSINC@dartmouth.edu

    2005-12-01

    Arsenic is a naturally occurring, worldwide contaminant implicated in numerous pathological conditions in humans, including cancer and several forms of liver disease. One of the contributing factors to these disorders may be the alteration of cytochrome P450 (CYP) levels by arsenic. In rat and human hepatocyte cultures, arsenic, in the form of arsenite, decreases the induction of several CYPs. The present study investigated whether arsenite utilizes transcriptional or post-transcriptional mechanisms to decrease CYP3A23 in primary cultures of rat hepatocytes. In these cultures, a 6-h treatment with 5 {mu}M arsenite abolished dexamethasone (DEX)-mediated induction of CYP3A23 protein and activity, but did not inhibit general protein synthesis. However, arsenite treatment only reduced DEX-induced levels of CYP3A23 mRNA by 30%. The effects of arsenite on CYP3A23 transcription were examined using a luciferase reporter construct containing 1.4 kb of the CYP3A23 promoter. Arsenite caused a 30% decrease in DEX-induced luciferase expression of this reporter. Since arsenite abolished induction of CYP3A23 protein, but caused only a small decrease in CYP3A23 mRNA, the effects of arsenite on translation of CYP3A23 mRNA were investigated. Polysomal distribution analysis showed that arsenite decreased translation by decreasing the DEX-mediated increase in CYP3A23 mRNA association with polyribosomes. Arsenite did not decrease intracellular glutathione or increase lipid peroxidation, suggesting that the effect of arsenite on CYP3A23 does not involve oxidative stress. Overall, the results suggest that low-level arsenite decreases both transcription and translation of CYP3A23 in primary rat hepatocyte cultures.

  15. Bioactivation and toxicity of acetaminophen in a rat hepatocyte micropatterned coculture system.

    PubMed

    Ukairo, Okechukwu; McVay, Michael; Krzyzewski, Stacy; Aoyama, Simon; Rose, Kelly; Andersen, Melvin E; Khetani, Salman R; Lecluyse, Edward L

    2013-10-01

    We have recently shown that primary rat hepatocytes organized in micropatterned cocultures with murine embryonic fibroblasts (HepatoPac™) maintain high levels of liver functions for at least 4 weeks. In this study, rat HepatoPac was assessed for its utility to study chemical bioactivation and associated hepatocellular toxicity. Treatment of HepatoPac cultures with acetaminophen (APAP) over a range of concentrations (0-15 mM) was initiated at 1, 2, 3, or 4 weeks followed by the assessment of morphological and functional endpoints. Consistent and reproducible concentration-dependent effects on hepatocyte structure, viability, and basic functions were observed over the 4-week period, and were exacerbated by depleting glutathione using buthionine sulfoximine or inducing CYP3A using dexamethasone, presumably due to increased reactive metabolite-induced stress and adduct formation. In conclusion, the results from this study demonstrate that rat HepatoPac represents a structurally and functionally stable hepatic model system to assess the long-term effects of bioactivated compounds.

  16. Expression of rat class I major histocompatibility complex (MHC) alloantigens and hepatocytes and hepatoma cells

    SciTech Connect

    Hunt, J.M.; Desai, P.A.; Chakraborty, S.

    1986-03-05

    Altered expression of Class I MHC alloantigens has been reported for murine tumors, and may be associated with the tumorigenic phenotype of tumor cells. To characterize MHC Class I alloantigen expression on a chemically-induced transplantable rat hepatoma cell line, 17X, derived from a (WF x F344) F/sub 1/ rat, polyvalent anti-F344 and anti-WF rat alloantisera were first used to immunoprecipitate the rat RT1.A Class I MHC alloantigens expressed on primary (WF x F344) F/sub 1/ hepatocyptes in short-term monolayer cultures. Two-dimensional isoelectric focusing and SDS-PAGE of immunoprecipitates from /sup 35/S-methionine-labeled (WF x F344) F/sub 1/ hepatocytes clearly resolved the RT1.A/sup u/ (WF) and RT1.A/sup LvI/ (F344) parental alloantigens. Identical radiolabeling and immunoprecipitation failed to detect either parental alloantigen on the 17X hepatoma cells. However, indirect immunofluorescence and immunoblot analyses demonstrated the presence of parental alloantigens on the 17X cells. Immunization of F344 rats but not of WF rats with 17X cells resulted in antibodies cytotoxic for normal (WF X F344) F/sub 1/ spleen cells in the presence of complement. These findings indicate that a combination of detection techniques will be necessary to characterize altered alloantigen expression on rat hepatoma cells.

  17. Acute effects of cadmium and selenium on glucose output from rat liver hepatocytes using various gluconeogenic precursors.

    PubMed

    Bell, R R; Soliman, M R; Early, J L

    1990-12-17

    Male Sprague-Dawley rats (250-310 g) fasted for 24 h were injected i.p. with either sodium acetate (C2H3NaO2; 1.23 mg/kg, 15 mumol/kg), cadmium acetate (C4H6CdO4; 0.84 mg/kg, 3.6 mumol/kg), sodium selenite (Na2SeO3; 1.6 mg/kg, 9.2 mumol/kg) or cadmium acetate (0.84 mg/kg, 3.6 mumol/kg) and sodium selenite (1.6 mg/kg, 9.2 mumol/kg) simultaneously. Rats were sacrificed 180 min post-treatment and hepatocytes were isolated. An average of 85% cell viability was achieved. Hepatocyte suspension (50 mg cell wt/ml, 1 ml/tube) was incubated for 180 min at 37 degrees/C with 10 mM of one of the following substrates: beta-D(-)fructose, glycerol, DL-alanine, L(+)lactic acid or pyruvic acid. Glucose concentration of the supernatant was measured by a colorimetric method. Cadmium decreased glucose output significantly (P less than 0.05), when lactic acid or alanine was used as substrate, but did significantly (P less than 0.05) increase the output when pyruvic acid, glycerol or fructose was used. Selenium alone significantly increased (P less than 0.05) hepatic glucose output only when fructose was used as substrate. Selenium and cadmium concurrently administered significantly increased (P less than 0.05) hepatic glucose output when pyruvic acid, glycerol or fructose was used as substrate as compared to sodium acetate (control), cadmium or selenium alone. These findings suggest that cadmium and selenium affect the hepatic gluconeogenic pathway and that their effects depend on the gluconeogenic precursor used.

  18. Administration of Escherichia coli endotoxin to rat increases liver mass and hepatocyte volume in vivo.

    PubMed Central

    Qian, D; Brosnan, J T

    1996-01-01

    We have established, in vivo, an increase in liver mass and hepatocyte volume after a single intraperitoneal administration, to fasted rats, of Escherichia coli lipopolysaccharide (0127:B8) at 3 mg/kg. The phenomenon was time- and dose-dependent and could be prevented by treatment with polyclonal antiserum against tumour necrosis factor-alpha (TNF-alpha) before the endotoxin injection. Endotoxin caused an increase of 26% in the hepatic mass compared with fasted controls at 24 h. An increase of 27% in the hepatic water content underlay the altered hepatic mass which could not be accounted for by a change in the volume of hepatic blood and/or interstitial fluid (measured in vivo), suggesting an expansion in the hepatocellular volume. This is supported by an increase of 25% in the K+ content of the endotoxic livers. Morphometric study confirmed a 15% increase in hepatocyte volume after endotoxin administration. The data are discussed in the light of possible metabolic effects of increased hepatocyte volume. PMID:8573081

  19. Morphology and albumin secretion of adult rat hepatocytes cultured on a hydrophobic porous expanded polytetrafluoroethylene membrane.

    PubMed

    Kurosawa, Hiroshi; Yuminamochi, Eri; Yasuda, Ruri; Amano, Yoshifumi

    2003-01-01

    Primary culture of rat hepatocytes was performed on a hydrophobic porous expanded polytetrafluoroethylene (ePTFE) membrane incorporated into the base of a culture dish. Two types of ePTFE membranes, a uniaxially expanded type (ePTFE-1) and a biaxially expanded type (ePTFE-2), could be used as the culture surfaces for hepatocytes. The formation of multicellular aggregates was observed in the culture dish when each membrane type was used. A pore size of 1 mum or higher was adequate for cell adhesion and albumin secretion for both membrane types. The activity of albumin secretion in the dish with the ePTFE membrane was markedly higher than that in the polystyrene dish. Spheroidal multicellular aggregates (spheroids) were observed when hepatocytes were cultured on the ePTFE-1 membrane. The ePTFE-1 membrane maintained the albumin secretion activity for a longer period than the non-expanded PTFE film. It was assumed that the cooperative action of membrane structure and oxygen permeability promoted the formation of cell aggregates and increased the albumin secretion activity.

  20. Trichostatin A, a critical factor in maintaining the functional differentiation of primary cultured rat hepatocytes

    SciTech Connect

    Henkens, Tom . E-mail: Tom.Henkens@vub.ac.be; Papeleu, Peggy; Elaut, Greetje; Vinken, Mathieu; Rogiers, Vera; Vanhaecke, Tamara

    2007-01-01

    Histone deacetylase inhibitors (HDI) have been shown to increase differentiation-related gene expression in several tumor-derived cell lines by hyperacetylating core histones. Effects of HDI on primary cultured cells, however, have hardly been investigated. In the present study, the ability of trichostatin A (TSA), a prototype hydroxamate HDI, to counteract the loss of liver-specific functions in primary rat hepatocyte cultures has been investigated. Upon exposure to TSA, it was found that the cell viability of the cultured hepatocytes and their albumin secretion as a function of culture time were increased. TSA-treated hepatocytes also better maintained cytochrome P450 (CYP)-mediated phase I biotransformation capacity, whereas the activity of phase II glutathione S-transferases (GST) was not affected. Western blot and qRT-PCR analysis of CYP1A1, CYP2B1 and CYP3A11 protein and mRNA levels, respectively, further revealed that TSA acts at the transcriptional level. In addition, protein expression levels of the liver-enriched transcription factors (LETFs) hepatic nuclear factor 4 alpha (HNF4{alpha}) and CCAAT/enhancer binding protein alpha (C/EBP{alpha}) were accordingly increased by TSA throughout culture time. In conclusion, these findings indicate that TSA plays a major role in the preservation of the differentiated hepatic phenotype in culture. It is suggested that the effects of TSA on CYP gene expression are mediated via controlling the expression of LETFs.

  1. The cytochrome P450 inhibitor SKF-525A disrupts autophagy in primary rat hepatocytes.

    PubMed

    Luo, Yong; Yang, Xi; Shi, Qiang

    2016-08-05

    The cytochrome P450 (CYP) inhibitor SKF-525A is commonly used to study drug metabolism and toxicity, particularly hepatotoxicity. By using Western blot and immunofluorescence staining, we unexpectedly found that SKF-525A at 2-20 μM caused remarkable accumulation of microtubule-associated protein light chain 3 II (LC3-II) in primary rat hepatocytes at 1, 4 and 24 h, indicating that autophagy was disrupted. SKF-525A showed no effects on chloroquine induced LC3-II accumulation, suggesting that autophagic flux was blocked, which is further supported by the increased level of the p62 protein after SKF-525A treatment. SKF-525A did not affect proteasome activities or gene expression of LC3-II or p62. Immunofluorescence of green fluorescent protein fused lysosomal-associated membrane protein 1 (LAMP1, a specific protein marker for lysosomes) and LC3-II showed that co-localization of these two proteins was partially abolished by SKF-525A, indicating that autophagosome-lysosome fusion was blocked. The other five CYP inhibitors, metyrapone, 1-aminobenzotriazole, alpha-naphthoflavone, ticlopidine, and ketoconazole, showed no effects in parallel experiments. These findings provide novel insights into the mechanisms by which various CYP inhibitors differentially affect a same drug's toxicity in hepatocytes. The data also indicate that SKF-525A is not an ideal chemical inhibitor for probing the relation between CYP mediated metabolism and toxicity in primary hepatocytes.

  2. Carbon Tetrachloride at Hepatotoxic Levels Blocks Reversibly Gap Junctions between Rat Hepatocytes

    NASA Astrophysics Data System (ADS)

    Saez, J. C.; Bennett, M. V. L.; Spray, D. C.

    1987-05-01

    Electrical coupling and dye coupling between pairs of rat hepatocytes were reversibly reduced by brief exposure to halogenated methanes (CBrCl3, CCl4, and CHCl3). The potency of different halomethanes in uncoupling hepatocytes was comparable to their hepatotoxicity in vivo, and the rank order was the same as that of their tendency to form free radicals. The effect of carbon tetrachloride (CCl4) on hepatocytes was substantially reduced by prior treatment with SKF 525A, an inhibitor of cytochrome P-450, and by exposure to the reducing reagent β -mercaptoethanol. Halomethane uncoupling occurred with or without extracellular calcium and did not change intracellular concentrations of calcium and hydrogen ions or the phosphorylation state of the main gap-junctional protein. Thus the uncoupling appears to depend on cytochrome P-450 oxidative metabolism in which free radicals are generated and may result from oxidation of the gap-junctional protein or of a regulatory molecule that leads to closure of gap-junctional channels. Decreases in junctional conductance may be a rapid cellular response to injury that protects healthy cells by uncoupling them from unhealthy ones.

  3. Increases of intracellular magnesium promote glycodeoxycholate-induced apoptosis in rat hepatocytes.

    PubMed Central

    Patel, T; Bronk, S F; Gores, G J

    1994-01-01

    Retention of bile salts by the hepatocyte contributes to liver injury during cholestasis. Although cell injury can occur by one of two mechanisms, necrosis versus apoptosis, information is lacking regarding apoptosis as a mechanism of cell death by bile salts. Our aim was to determine if the bile salt glycodeoxycholate (GDC) induces apoptosis in rat hepatocytes. Morphologic assessment included electron microscopy and quantitation of nuclear fragmentation by fluorescent microscopy. Biochemical studies included measurements of DNA fragmentation, in vitro endonuclease activity, cytosolic free Ca2+ (Cai2+), and cytosolic free Mg2+ (Mgi2+). Morphologic studies demonstrated typical features of apoptosis in GDC (50 microM) treated cells. The "ladder pattern" of DNA fragmentation was also present in DNA obtained from GDC-treated cells. In vitro endonuclease activity was 2.5-fold greater with Mg2+ than Ca2+. Although basal Cai2+ values did not change after addition of GDC, Mgi2+ increased twofold. Incubation of cells in an Mg(2+)-free medium prevented the rise in Mgi2+ and reduced nuclear and DNA fragmentation. In conclusion, GDC induces apoptosis in hepatocytes by a mechanism promoted by increases of Mgi2+ with stimulation of Mg(2+)-dependent endonucleases. These data suggest for the first time that changes of Mgi2+ may participate in the program of cellular events culminating in apoptosis. Images PMID:7989573

  4. Glycyrrhizic acid modulates t-BHP induced apoptosis in primary rat hepatocytes.

    PubMed

    Tripathi, M; Singh, B K; Kakkar, P

    2009-02-01

    Glycyrrhizic acid (GA) is the main bioactive ingredient of licorice (Glycyrrhiza glabra). The object of this study was to evaluate the protective effects of GA on tert-butyl hydroperoxide (t-BHP) induced oxidative injury leading to apoptosis in cultured primary rat hepatocytes. Throughout the study silymarin was used as positive control. Molecular mechanisms involved in apoptotic pathways induced in hepatocytes by t-BHP at 250 microM were explored in detail. DNA fragmentation, activation of caspases and cytochrome c release were demonstrated. In addition, changes in the mitochondrial membrane potential and ROS generation were detected confirming involvement of mitochondrial pathway. Pre-treatment with GA (4 microg) protected the hepatocytes against t-BHP induced oxidative injury and the results were comparable to the pre-treatment with positive control, i.e. silymarin. The protective potential against cell death was achieved mainly by preventing intracellular GSH depletion, decrease in ROS formation as well as inhibition of mitochondrial membrane depolarization. GA was found to modulate critical end points of oxidative stress induced apoptosis and could be beneficial against liver diseases where oxidative stress is known to play a crucial role.

  5. Effect of L-carnitine on glycogen synthesis and ATP production in cultured hepatocytes of the newborn rat.

    PubMed

    Nishida, N; Sugimoto, T; Takeuchi, T; Kobayashi, Y

    1989-11-01

    Changes in intracellular carnitine, ATP and glycogen concentration were studied when hepatocytes of newborn or adult rats were incubated with oleate, lactate and/or carnitine. Hepatocytes of adult rats appeared to be able to maintain cellular carnitine concentration without exogenous carnitine supplementation. Hepatocytes of newborn rats appeared to be unable to maintain cellular carnitine concentration without carnitine supplementation. Moreover, ATP concentration and glycogen concentration were significantly increased by the carnitine supplement with oleate and/or lactate compared to the unsupplemented group. Increases in both intracellular ATP and carnitine concentration depended on the concentration of carnitine added to the medium. These results suggest that carnitine may be an important factor in glycogen synthesis and ATP production of newborn infants.

  6. L-Methionine Toxicity in Freshly-Isolated Mouse Hepatocytes is Gender-Dependent and Mediated in Part by Transamination

    PubMed Central

    Dever, Joseph T.; Elfarra, Adnan A.

    2008-01-01

    L-methionine (Met) has been implicated in parenteral nutrition-associated cholestasis in infants and, at high levels, causes liver toxicity by mechanisms that are not clear. In this study, Met toxicity was characterized in freshly-isolated male and female mouse hepatocytes incubated with 5-30 mM Met for 0 to 5 h. In male hepatocytes, 20 mM Met was cytotoxic at 4 h as indicated by trypan blue exclusion and LDH leakage assays. Cytotoxicity was preceded by GSH depletion at 3 h without GSSG formation. Exposure to 30 mM Met resulted in increased cytotoxicity and GSH depletion. Interestingly, female hepatocytes were resistant to Met-induced cytotoxicity at these concentrations and showed increased cellular GSH levels compared to hepatocytes exposed to medium alone. The effects of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, and 3-deazaadenosine (3-DA), an inhibitor of the Met transmethylation pathway enzyme S-adenosylhomocysteine hydrolase on Met toxicity in male hepatocytes were then examined. Addition of 0.2 mM AOAA partially blocked Met-induced GSH depletion and cytotoxicity whereas 0.1 mM 3-DA potentiated Met-induced toxicity. Exposure of male hepatocytes to 0.3 mM 3-methylthiopropionic acid (3-MTP), a known Met transamination metabolite, resulted in cytotoxicity and cellular GSH depletion similar to that observed with 30 mM Met whereas incubations with D-methionine resulted in no toxicity. Female hepatocytes were less sensitive to 3-MTP toxicity than males which may partially explain their resistance to Met toxicity. Collectively, these results suggest that Met transamination and not transmethylation plays a major role in Met toxicity in male mouse hepatocytes. PMID:18552130

  7. L-methionine toxicity in freshly isolated mouse hepatocytes is gender-dependent and mediated in part by transamination.

    PubMed

    Dever, Joseph T; Elfarra, Adnan A

    2008-09-01

    L-methionine (Met) has been implicated in parenteral nutrition-associated cholestasis in infants and, at high levels, it causes liver toxicity by mechanisms that are not clear. In this study, Met toxicity was characterized in freshly isolated male and female mouse hepatocytes incubated with 5 to 30 mM Met for 0 to 5 h. In male hepatocytes, 20 mM Met was cytotoxic at 4 h as indicated by trypan blue exclusion and lactate dehydrogenase leakage assays. Cytotoxicity was preceded by reduced glutathione (GSH) depletion at 3 h without glutathione disulfide formation. Exposure to 30 mM Met resulted in increased cytotoxicity and GSH depletion. It is interesting to note that female hepatocytes were resistant to Met-induced cytotoxicity at these concentrations and showed increased cellular GSH levels compared with hepatocytes exposed to medium alone. The effects of amino-oxyacetic acid (AOAA), an inhibitor of Met transamination, and 3-deazaadenosine (3-DA), an inhibitor of the Met transmethylation pathway enzyme S-adenosylhomocysteine hydrolase, on Met toxicity in male hepatocytes were then examined. Addition of 0.2 mM AOAA partially blocked Met-induced GSH depletion and cytotoxicity, whereas 0.1 mM 3-DA potentiated Met-induced toxicity. Exposure of male hepatocytes to 0.3 mM 3-methylthiopropionic acid (3-MTP), a known Met transamination metabolite, resulted in cytotoxicity and cellular GSH depletion similar to that observed with 30 mM Met, whereas incubations with D-methionine resulted in no toxicity. Female hepatocytes were less sensitive to 3-MTP toxicity than males, which may partially explain their resistance to Met toxicity. Taken together, these results suggest that Met transamination and not transmethylation plays a major role in Met toxicity in male mouse hepatocytes.

  8. Adenosine 3',5'-cyclic monophosphate involvement in hepatic triacylglyceride lipase release from prazosin-stimulated primary cultured rat hepatocytes.

    PubMed

    Nakamura, Tetsuya; Morita, Tetsuo

    2014-01-01

    We recently found that hepatic triglyceride lipase (HTGL) was released from primary cultured rat hepatocytes after treatment with prazosin, an antagonist of alpha-1 adrenoceptors. However, the details of prazosin-induced HTGL release remain uncertain. Here we investigated whether changes in cAMP levels in hepatocytes were related to HTGL release from prazosin-stimulated hepatocytes. When hepatocytes were treated with prazosin, cAMP levels during stimulated release of HTGL increased in a time- and dose-dependent manner. Stimulated release of HTGL was suppressed by the adenylate cyclase inhibitors MDL-12,330A and 2',5'-dideoxyadenosine. Further, cAMP-dependent protein kinase A (PKA) activity in prazosin-stimulated hepatocytes also increased in a time- and dose-dependent manner. Moreover, prazosin-stimulated HTGL release was suppressed by the PKA inhibitors H-89 and KT5720. These results suggest that prazosin-stimulated HTGL release from hepatocytes was due to cAMP production and partly due to subsequent PKA activation in hepatocytes.

  9. Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes

    SciTech Connect

    Marion, Tracy L.; Perry, Cassandra H.; St Claire, Robert L.; Brouwer, Kim L.R.

    2012-05-15

    Sandwich-cultured hepatocytes (SCH) are used commonly to investigate hepatic transport protein-mediated uptake and biliary excretion of substrates. However, little is known about the disposition of endogenous bile acids (BAs) in SCH. In this study, four endogenous conjugated BAs common to rats and humans [taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA)], as well as two BA species specific to rodents (α- and β-tauromuricholic acid; α/β TMCA), were profiled in primary rat and human SCH. Using B-CLEAR{sup ®} technology, BAs were measured in cells + bile canaliculi, cells, and medium of SCH by LC-MS/MS. Results indicated that, just as in vivo, taurine-conjugated BA species were predominant in rat SCH, while glycine-conjugated BAs were predominant in human SCH. Total intracellular BAs remained relatively constant over days in culture in rat SCH. Total BAs in control (CTL) cells + bile, cells, and medium were approximately 3.4, 2.9, and 8.3-fold greater in human than in rat. The estimated intracellular concentrations of the measured total BAs were 64.3 ± 5.9 μM in CTL rat and 183 ± 56 μM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16 ± 0.21 μM in CTL rat SCH and 9.61 ± 6.36 μM in CTL human SCH. Treatment of cells for 24 h with 10 μM troglitazone (TRO), an inhibitor of the bile salt export pump (BSEP) and the Na{sup +}-taurocholate cotransporting polypeptide (NTCP), had no significant effect on endogenous BAs measured at the end of the 24-h culture period, potentially due to compensatory mechanisms that maintain BA homeostasis. These data demonstrate that BAs in SCH are similar to in vivo, and that SCH may be a useful in vitro model to study alterations in BA disposition if species differences are taken into account. -- Highlights: ► Bile acids (BAs) were measured in rat and human sandwich-cultured hepatocytes (SCH). ► Cell and medium BA

  10. Effect of Culture Time on the Basal Expression Levels of Drug Transporters in Sandwich-Cultured Primary Rat HepatocytesS⃞

    PubMed Central

    Houghton, Jessica S.; Uyeda, Craig; Grillo, Mark P.; Jin, Lixia

    2011-01-01

    Sandwich-cultured rat hepatocytes are used in drug discovery for pharmacological and toxicological assessment of drug candidates, yet their utility as a functional model for drug transporters has not been fully characterized. To evaluate the system as an in vitro model for drug transport, expression changes of hepatic transporters relative to whole liver and freshly isolated hepatocytes (day 0) were examined by real-time quantitative reverse transcription-polymerase chain reaction for 4 consecutive days of culture. No significant differences in transporter expression levels were observed between freshly isolated hepatocytes and whole liver. Two distinct mRNA profiles were detected over time showing 1) a more than 5-fold decline in levels of uptake transporters such as Na+-taurocholate cotransporting polypeptide (Ntcp), organic anion transporter (Oat) 2, organic anion-transporting polypeptide (Oatp) 1a1, Oatp1a4, and Oatp1b2 and 2) a greater than 5-fold increase of efflux transporters P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-related proteins (Mrp) 1, 2, 3, and 4. In addition, protein levels and functional activities for selected transporters were also determined. Protein levels for Mrp2, Bcrp, P-gp, Ntcp, and Oatp1a4 corresponded to changes in mRNA. Functional activities of Oatps and Oct1 exhibited a 3- and 4-fold decrease on day 2 and day 4, respectively, relative to that on day 0, whereas a more than 10-fold reduction in Oat2 activity was observed. These results indicate that the cell culture conditions used herein did not provide an optimal environment for expression of all hepatic transporters. Significant time-dependent alterations in basal gene expression patterns of transporters were detected compared with those in liver or freshly isolated hepatocytes. Further work and new strategies are required to improve the validity of this model as an in vitro tool for in vivo drug transport or biliary clearance prediction

  11. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes.

    PubMed

    Bhattacharjee, Rajesh; Xiang, Wenpei; Wang, Yinna; Zhang, Xiaoying; Billiar, Timothy R

    2012-06-22

    Tumor necrosis factor α (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF+ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found that cAMP exerts its affect at the proximal level of TNF signaling by inhibiting the formation of the DISC complex upon the binding of TNF to TNFR1. In conclusion, our study shows that cAMP prevents TNF+ActD-induced apoptosis in rat hepatocytes by inhibiting DISC complex formation.

  12. Stimulatory and inhibitory effects of forskolin on adenylate cyclase in rat normal hepatocytes and hepatoma cells.

    PubMed

    Miyamoto, K; Sanae, F; Koshiura, R; Matsunaga, T; Takagi, K; Satake, T; Hasegawa, T

    1989-02-01

    Forskolin synergistically potentiated adenosine 3',5'-cyclic monophosphate formation by prostaglandin E1 (PGE1) in rat normal hepatocytes freshly prepared by collagenase digestion and rat ascites hepatoma AH66 cells, but dose-dependently inhibited the accumulation by PGE1 in AH66F cells. Forskolin activated adenylate cyclase in a dose-dependent manner in homogenates of all cell lines. In normal hepatocytes and AH66 cells, simultaneous addition of forskolin and other adenylate cyclase activators [isoproterenol (IPN), PGE1, guanosine 5'-triphosphate sodium salt (GTP), 5'-guanylylimidodiphosphate sodium salt (Gpp (NH)p), NaF, cholera toxin, islet activating protein and MnCl2] gave greater than additive responses. On the other hand, in AH66F cells, the effect of forskolin on adenylate cyclase was hardly influenced by GTP, but forskolin diminished the activities induced by high concentrations of GTP to that by the diterpene alone. Forskolin also significantly inhibited the PGE1-stimulated and the guanine nucleotide binding regulatory protein-stimulated activities. Because AH66F cells were insensitive to IPN, the combination with forskolin and IPN gave similar activity to that obtained with the diterpene alone. The effect of forskolin on the activation by manganese ion was neither synergistic nor inhibitory but was additive in AH66F cells. These results suggest that forskolin promotes the interaction between the stimulatory guanine nucleotide binding regulatory protein and the catalytic unit in normal hepatocytes and AH66 cells, but in AH66F cells forskolin interferes with the coupling of the two components of adenylate cyclase.

  13. DEPLETION OF CELLULAR PROTEIN THIOLS AS AN INDICATOR OF ARYLATION IN ISOLATED TROUT HEPATOCYTES EXPOSED TO 1,4-BENZOQUINONE

    EPA Science Inventory

    A method for the measurement of protein thiols (PrSH), un-reacted as well as oxidized, i.e. dithiothreitol recoverable, was adapted for the determination of PrSH depletion in isolated rainbow trout hepatocytes exposed to an arylating agent, 1,4-benzoquinone (BQ). Toxicant analysi...

  14. Combining a Laboratory Practical Class with a Computer Simulation: Studies on the Synthesis of Urea in Isolated Hepatocytes.

    ERIC Educational Resources Information Center

    Bender, David A.

    1986-01-01

    Describes how a computer simulation is used with a laboratory experiment on the synthesis of urea in isolated hepatocytes. The simulation calculates the amount of urea formed and the amount of ammonium remaining as the concentrations of ornithine, citrulline, argininosuccinate, arginine, and aspartate are altered. (JN)

  15. Selective protection of normal hepatocytes by indocyanine green in photodynamic therapy for the hepatoma of rat

    NASA Astrophysics Data System (ADS)

    Gu, Ying; Li, Junheng; Guo, Zhong-He

    1993-03-01

    Using hepatocarcinoma transplanted rats, the present study made consecutive observation for the color change and indocyanine green (ICG) absorption peak of the normal liver and tumor tissues after intravenous injection of ICG. The normal liver tissue of the rat was found to turn violet-green soon after ICG injection and the optic density (OD) of ICG-characteristic spectral peak of the tissue homogenate reached its maximum value at 35 minutes post-injection, while neither color change nor OD value increase was noticed in the tissue of transplanted hepatocarcinoma, suggesting that there is a specific absorption of ICG by the normal liver tissue. Chemiluminescentoassay revealed inhibited luminal chemiluminescence by ICG, indicating the depression of singlet oxygen and reactive oxygen species (ROS) oxidation during HPD photosensitization by ICG. In PDT of the hepatocarcinoma, the irradiated area was examined under microscope and auto-microimage analysis system after ICG administration. For tumor-free tissue, the photosensitization induced necrotic area was found smaller in those with than those without ICG administration, whereas the tumor killing effect was almost the same of the two. It is suggested that ICG may offer selective protection for healthy hepatocytes without diminishing the destruction of tumor cells. The protection of healthy hepatocytes by ICG is thought to be in accordance with the amount of ICG in the cell and the distribution of light energy.

  16. Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes

    SciTech Connect

    Vance, J.E.; Vance, D.E.

    1986-05-01

    The role of phospholipid biosynthesis in lipoprotein secretion from cultured rat hepatocytes has been investigated. In liver, phosphatidylcholine (PC) is made both by the CDP-choline pathway and by the methylation of phosphatidyl-ethanolamine (PE), which in turn is derived from both serine (via phosphatidylserine) and ethanolamine (via CDP-ethanol-amine). Monolayer cultures of rat hepatocytes were incubated in the presence of (methyl-/sup 3/H)choline, (2-/sup 3/H)ethanolamine or (3-/sup 3/H)serine. The specific radioactivity of the phospholipids derived from each of these precursors was measured in the cells and in the secreted lipoproteins of the culture medium. The specific radioactivities of PC and PE derived from (1-/sup 3/H)ethanolamine were markedly lower (approximately 1/2 and less than 1/10, respectively) in the secreted phospholipids than in the cellular phospholipids. Thus, ethanolamine was not an effective precursor of the phospholipids in lipoproteins. On the contrary, the specific radioactivity of PC made from (methyl-/sup 3/H)choline was approximately equal in cells and lipoproteins. In addition, over the first 4 h of incubation with (3-/sup 3/H)serine, the specific radioactivities of PC and PE were significantly higher in the lipoproteins than in the cells. These data indicate that specific pools of phospholipids are selected on the basis of their routes of biosynthesis, for secretion into lipoproteins.

  17. Supercooling as a viable non-freezing cell preservation method of rat hepatocytes.

    PubMed

    Usta, O Berk; Kim, Yeonhee; Ozer, Sinan; Bruinsma, Bote G; Lee, Jungwoo; Demir, Esin; Berendsen, Tim A; Puts, Catheleyne F; Izamis, Maria-Louisa; Uygun, Korkut; Uygun, Basak E; Yarmush, Martin L

    2013-01-01

    Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4(o)C) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4(o)C) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 (o)C). We find that there exists an optimum temperature (-4(o)C) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.

  18. Transplantation speed offers early hepatocyte engraftment in acute liver injured rats: A translational study with clinical implications.

    PubMed

    Ho, Cheng-Maw; Chen, Ya-Hui; Chien, Chin-Sung; Ho, Yi-Tian; Ho, Shu-Li; Hu, Rey-Heng; Chen, Hui-Ling; Lee, Po-Huang

    2015-05-01

    The impact of the rate of intraportal hepatocyte transplantation on early engraftment and repopulation is unclear. The aim of this study was to address this and to improve the engraftment and repopulation efficiencies of hepatocyte transplantation for the treatment of a rat model of acute liver failure in a clinically useful way without preconditioning. Acute hepatic injury was induced into Sprague-Dawley rats with D-galactosamine. Hepatocytes were infused intraportally over a period of 30, 70, or 100 seconds to study early engraftment (2 days) and repopulation (7 days). Three groups had significant differences in hepatocyte engraftment (P = 0.018) and repopulation efficiencies (P = 0.037), and an infusion over a period of 70 seconds produced superior outcomes. After the 70-second infusion, the transplanted cells immediately transmigrated the sinusoidal endothelial layer and rarely accumulated in the portal venules, with liver function improving significantly. The mean first peak pressures, without significant differences, were 14.8 ± 6.5, 17.7 ± 3.7, and 13.6 ± 3.0 mm Hg in the 30-, 70-, and 100-second groups, respectively. Differential hepatocyte transfusion rates contributed to accelerated early engraftment and repopulation in rats with acute liver injury. These proof-of-concept findings are of clinical significance because they are easy to translate into practice.

  19. P21waf-1/cip-1/sdi-1 is expressed at G1 phase in primary culture of hepatocytes from old rats, presumably preventing the cells from entering the S phase of the cell cycle.

    PubMed

    Sawada, N; Kojima, T; Obata, H; Saitoh, M; Isomura, H; Kokai, Y; Satoh, M; Mori, M

    1996-11-21

    To elucidate whether p21waf-1/cip-1/sdi-1 expression is associated with loss of growth potential of hepatocytes of old rats, we determined p21waf-1/cip-1/sdi-1 expression of hepatocytes from old (30 months) rats during the cell cycle in primary culture. A high level of expression of p21waf-1/cip-1/sdi-1 was detected at the G1 phase in old-rat hepatocytes, but after the S phase in young-rat hepatocytes. Consistently, the incidence of the cells positive for p21waf-1/cip-1/sdi-1 in nuclei before entering the S phase was significantly higher in old-rat hepatocytes than in young-rat hepatocytes. These results account for the loss of growth potential of old-rat hepatocytes in vitro and the marked retardation of regeneration of liver in old rats in vivo.

  20. Glycyrrhizic acid attenuates CCl4-induced hepatocyte apoptosis in rats via a p53-mediated pathway

    PubMed Central

    Guo, Xiao-Ling; Liang, Bo; Wang, Xue-Wei; Fan, Fu-Gang; Jin, Jing; Lan, Rui; Yang, Jing-Hui; Wang, Xiao-Chun; Jin, Lei; Cao, Qin

    2013-01-01

    AIM: To investigate the effect of glycyrrhizic acid (GA) on carbon tetrachloride (CCl4)-induced hepatocyte apoptosis in rats via a p53-dependent mitochondrial pathway. METHODS: Forty-five male Sprague-Dawley rats were randomly and equally divided into three groups, the control group, the CCl4 group, and the GA treatment group. To induce liver fibrosis in this model, rats were given a subcutaneous injection of a 40% solution of CCl4 in olive oil at a dose of 0.3 mL/100 g body weight biweekly for 8 wk, while controls received the same isovolumetric dose of olive oil by hypodermic injection, with an initial double-dose injection. In the GA group, rats were also treated with a 40% solution of CCl4 plus 0.2% GA solution in double distilled water by the intraperitoneal injection of 3 mL per rat three times a week from the first week following previously published methods, with modifications. Controls were given the same isovolumetric dose of double distilled water. Liver function parameters, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. Pathologic changes in the liver were detected by hematoxylin and eosin staining. Collagen fibers were evaluated by Sirius red staining. Hepatocyte apoptosis was investigated using the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) assay and the cleaved caspase-3 immunohistochemistry assay. The expression levels of p53 and apoptosis-related proteins were evaluated by immunohistochemistry or Western blotting analysis. RESULTS: After 8 wk of treatment, GA significantly reduced serum activity of ALT (from 526.7 ± 57.2 to 342 ± 44.8, P < 0.05) and AST (from 640 ± 33.7 to 462.8 ± 30.6, P < 0.05), attenuated the changes in liver histopathology and reduced the staging score (from 3.53 ± 0.74 to 3.00 ± 0.76, P < 0.05) in CCl4-treated rats. GA markedly reduced the positive area of Sirius red and the ratio of the hepatic fibrotic region (from 7

  1. Dexamethasone blocks arachidonate biosynthesis in isolated hepatocytes and cultured hepatoma cells

    SciTech Connect

    Marra, C.A.; de Alaniz, M.J.; Brenner, R.R.

    1986-03-01

    The effect of dexamethasone on the incorporation and conversion of (1-14C)eicosa-8,11,14-trienoic acid to arachidonic acid in isolated hepatocytes and in hepatoma tissue culture (HTC) cells was studied. In both kinds of cells, no changes in the exogenous acid incorporation were found when the hormone was added to the incubation media at 0.1 or 0.2 mM concentration, while the biosynthesis of arachidonic acid was significantly depressed. The effect on the biosynthesis was faster in isolated normal liver cells (60 min) than in tumoral cells (120 min) and reached an inhibition of ca. 50% after 3 hr of treatment. The addition of cycloheximide (10(-6) M) also caused a marked decrease in the biosynthesis of this polyunsaturated fatty acid, but when dexamethasone was added to the media simultaneously with cycloheximide, a synergistic action was not observed. The results obtained show that protein synthesis would be involved in the modulation of the biosynthesis of arachidonic acid by glucocorticoids. The changes in the delta 5 desaturation of labeled 20:3 omega 6 to arachidonic acid correlated with changes in the fatty acid composition in isolated cells.

  2. Expression profiles uncover the relationship between erythropoietin and cell proliferation in rat hepatocytes after a partial hepatectomy.

    PubMed

    Zhang, Jihong; Yang, Yajuan; He, Tingting; Liu, Yunqing; Zhou, Yun; Chen, Yongkang; Xu, Cunshuan

    2014-09-01

    Erythropoietin (EPO) has a beneficial effect on hepatic cell proliferation during liver regeneration. However, the underlying mechanism has not yet been elucidated. To uncover the proliferation response of EPO in rat liver regeneration after partial hepatectomy (PH) at the cellular level, hepatocytes (HCs) were isolated using Percoll density gradient centrifugation. The genes of the EPO-mediated signaling pathway and the target genes of the transcription factor (TF) in the pathway were identified in a pathway and TF database search. Their expression profiles were then detected using Rat Genome 230 2.0 Microarray. The results indicated that the EPO-mediated signaling pathway is involved in 19 paths and that 124 genes participate, of which 32 showed significant changes and could be identified as liver regeneration-related genes. In addition, 443 targets regulated by the TFs of the pathway and 60 genes associated with cell proliferation were contained in the array. Subsequently, the synergetic effect of these genes in liver regeneration was analyzed using the E(t) mathematical model based on their expression profiles. The results demonstrated that the E(t) values of paths 3, 8, 12 and 14-17 were significantly strengthened in the progressing phase of liver regeneration through the RAS/MEK/ERK or PI3K/AκT pathways. The synergetic effect of the target genes, in parallel with target-related cell proliferation, was also enhanced 12-72 h after PH, suggesting a potential positive effect of EPO on HC proliferation during rat liver regeneration. These data imply that the EPO receptor may allow EPO to promote HC proliferation through paths 3, 8, 12 and 14-17, mediating the RAS/MEK/ERK and PI3K/AκT pathways in rat liver regeneration after PH.

  3. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    SciTech Connect

    Bhattacharjee, Rajesh; Xiang, Wenpei; Wang, Yinna; Zhang, Xiaoying

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. Black-Right-Pointing-Pointer cAMP blocks NF-{kappa}B activation induced by TNF and actinomycin D. Black-Right-Pointing-Pointer cAMP blocks DISC formation following TNF and actinomycin D exposure. Black-Right-Pointing-Pointer cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor {alpha} (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found

  4. Feasibility of direct oxygenation of primary-cultured rat hepatocytes using polyethylene glycol-decorated liposome-encapsulated hemoglobin (LEH).

    PubMed

    Naruto, Hirosuke; Huang, Hongyun; Nishikawa, Masaki; Kojima, Nobuhiko; Mizuno, Atsushi; Ohta, Katsuji; Sakai, Yasuyuki

    2007-10-01

    We tested the short-term efficacy of liposome-encapsulated hemoglobin (LEH) in cultured rat hepatocytes. Supplementation with LEH (20% of the hemoglobin concentration of blood) did not lower albumin production in static culture, and completely reversed the cell death and deterioration in albumin production caused by an oxygen shortage in 2D flat-plate perfusion bioreactors.

  5. Biliary copper excretion by hepatocyte lysosomes in the rat. Major excretory pathway in experimental copper overload

    SciTech Connect

    Gross, J.B. Jr.; Myers, B.M.; Kost, L.J.; Kuntz, S.M.; LaRusso, N.F.

    1989-01-01

    We investigated the hypothesis that lysosomes are the main source of biliary copper in conditions of hepatic copper overload. We used a rat model of oral copper loading and studied the relationship between the biliary output of copper and lysosomal hydrolases. Male Sprague-Dawley rats were given tap water with or without 0.125% copper acetate for up to 36 wk. Copper loading produced a 23-fold increase in the hepatic copper concentration and a 30-65% increase in hepatic lysosomal enzyme activity. Acid phosphatase histochemistry showed that copper-loaded livers contained an increased number of hepatocyte lysosomes; increased copper concentration of these organelles was confirmed directly by both x ray microanalysis and tissue fractionation. The copper-loaded rats showed a 16-fold increase in biliary copper output and a 50-300% increase in biliary lysosomal enzyme output. In the basal state, excretory profiles over time were similar for biliary outputs of lysosomal enzymes and copper in the copper-loaded animals but not in controls. After pharmacologic stimulation of lysosomal exocytosis, biliary outputs of copper and lysosomal hydrolases in the copper-loaded animals remained coupled: injection of colchicine or vinblastine produced an acute rise in the biliary output of both lysosomal enzymes and copper to 150-250% of baseline rates. After these same drugs, control animals showed only the expected increase in lysosomal enzyme output without a corresponding increase in copper output. We conclude that the hepatocyte responds to an increased copper load by sequestering excess copper in an increased number of lysosomes that then empty their contents directly into bile. The results provide direct evidence that exocytosis of lysosomal contents into biliary canaliculi is the major mechanism for biliary copper excretion in hepatic copper overload.

  6. Low concentration of mercury induces autophagic cell death in rat hepatocytes.

    PubMed

    Chatterjee, Sarmishtha; Ray, Atish; Mukherjee, Sandip; Agarwal, Soumik; Kundu, Rakesh; Bhattacharya, Shelley

    2014-08-01

    In the present study, we attempted to elucidate the induction of autophagy in rat hepatocytes by a low concentration of mercury. Hepatocytes treated with different doses of mercuric chloride (HgCl2; 1, 2.5, 5 and 10 µM) and at different time intervals (0 min, 30 min, 1 h, 2 h and 4 h) show autophagic cell death only at 5 µM HgCl2 within 30 min of incubation. At 1 and 2.5 µM HgCl2, no cell death is recorded, while apoptosis is found at 10 µM HgCl2, as evidenced by the activation of caspase 3. Autophagic cell death is confirmed by the presence of monodansylcadaverine (MDC) positive hepatocytes which is found to be highest at 1 h. Atg5-Atg12 covalent-conjugation triggers the autophagic pathway within 30 min of 5 µM HgCl2 treatment and continues till 4 h of incubation. In addition, damage-regulated autophagy modulator (DRAM) expression gradually increases from 30 min to 4 h of treatment with mercury and a corresponding linear decrease in p53 has been observed. It is concluded that a low concentration (5 µM HgCl2) of mercury induces autophagy or nonapoptotic programmed cell death following an Atg5-Atg12 covalent-conjugation pathway, which is modulated by DRAM in a p53-dependent manner.

  7. [Influence of ionizing radiation on enzymatic activity and state of nucleus-nucleolar apparatus in rat hepatocytes].

    PubMed

    Nersesova, L S; Gazariants, M G; Mkrtchian, Z S; Meliksetian, G O; Pogosian, L G; Pogosian, S A; Pogosian, L L; Karalova, E M; Avetisian, A S; Abroian, l O; Karalian, Z A; Akopian, Zh I

    2013-01-01

    The effects of a single exposure of rats to the whole-body roentgen irradiation at the doses of 3.5 Gy and 4.5 Gy on the activity of creatine kinase, purine nucleoside phosphorylase, alanine aminotransferase, aspartate aminotransferase, as well as on the state of the nuclear-nucleolar apparatus in rat hepatocytes on the 6th and 13th days after radiation exposure have been studied. Irradiation at the above doses induced changes in the levels of enzymatic activity of different values and different directions within the same time periods, as well as oscillating changes in this type of enzymatic activity over time. This demonstrates various radiosensitivity and adaptation abilities of these enzymatic activities. The changes in the enzymatic activity significantly correspond to the changes in the morphometric indices of nuclear-nucleolar apparatus of hepatocytes, as well as the distribution of hepatocytes within the ploidy classes: in particular, stabilization of the enzymatic activity on the 13th day after irradiation correlates with the increased transcriptional activity, which is detectable through the increased number of nucleoli per nucleus and the expanded space of a hepatocyte nucleus. The compensation mechanisms are likely to be targeted at the changes in the functional activity of surviving hepatocytes, rather than at the replacement of the damaged cells by the new ones.

  8. L-methionine-dl-sulfoxide metabolism and toxicity in freshly isolated mouse hepatocytes: gender differences and inhibition with aminooxyacetic acid.

    PubMed

    Dever, Joseph T; Elfarra, Adnan A

    2008-11-01

    L-methionine-dl-sulfoxide (MetO) is an L-methionine (Met) metabolite, but its role in Met metabolism and toxicity is not clear. In this study, MetO uptake, metabolism to Met, cytotoxicity, and glutathione (GSH) and glutathione disulfide (GSSG) status were characterized in freshly isolated mouse hepatocytes incubated at 37 degrees C with 0 to 30 mM MetO for 0 to 5 h. In male hepatocytes, dose-dependent cytotoxicity concomitant with GSH depletion without GSSG formation occurred after exposure to 20 or 30 mM MetO but not after exposure to 10 mM MetO. Interestingly, female hepatocytes exposed to 30 mM MetO showed no cytotoxicity and exhibited increased intracellular GSH levels compared with control hepatocytes. Male hepatocytes had approximately 2-fold higher levels of intracellular Met-d-O or Met-l-O after MetO (30 mM) exposure for 0 to 1.5 h compared with female hepatocytes. In hepatocytes of both genders, Met-l-O was detected at nearly 5-fold higher levels than Met-d-O, and no significant increase in cellular Met levels was detected. Addition of aminooxyacetic acid (AOAA), an inhibitor of transamination reactions, to MetO-exposed male hepatocytes resulted in higher cellular Met-d-O and Met-l-O levels and decreased the cytotoxicity of MetO. Interestingly, exposure of control male hepatocytes to AOAA selectively increased cellular Met-d-O levels to levels similar to those observed after exposure to MetO (30 mM). Analysis of MetO transamination activity by glutamine transaminase K in mouse liver cytosol revealed similar rates of MetO transamination in cytosol of both genders. Taken together, these results provide evidence for stereoselective oxidation of Met to Met-d-O under physiological conditions and suggest a major role for MetO transamination in MetO metabolism and toxicity.

  9. Insulin phosphorylates calmodulin in preparations of solubilized rat hepatocyte insulin receptors

    SciTech Connect

    Sacks, D.B.; McDonald, J.M.

    1987-05-01

    It has previously been shown that insulin stimulates the phosphorylation of calmodulin in adipocyte insulin receptor preparations. Here they demonstrate that insulin also stimulates the phosphorylation of calmodulin in wheat germ lectin-enriched insulin receptor preparations obtained from rat hepatocytes. Standard phosphorylation assays were performed at 30C in the presence of 50mM Tris-HCl (pH 7.5), 0.1% (v/v) Triton X-100, 1mM EGTA, 50 M (el-TSP)ATP, 5mM MgCl2, 0.25 M polylysine, 1.2 M calmodulin and various CaS and insulin concentrations. The phosphorylation of calmodulin was determined by SDS-PAGE and autoradiography. Phosphorylation of calmodulin had an absolute requirement for insulin receptors, insulin and certain basic proteins. Phosphorylation was maximal above 13 nM insulin and at submicromolar CaS concentrations, whereas supramicromolar CaS concentrations were inhibitory. As was observed in the adipocyte insulin receptor system, calmodulin phosphorylation was dependent upon the presence of co-factors, such as polylysine, histone H/sub f/2b and protamine sulfate. The role played by these co-factors has not yet been established. These data suggest that both CaS and calmodulin participate in post receptor insulin events in hepatocytes.

  10. Nitric-oxide dioxygenase function of human cytoglobin with cellular reductants and in rat hepatocytes.

    PubMed

    Gardner, Anne M; Cook, Matthew R; Gardner, Paul R

    2010-07-30

    Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b(5) were found to support a high NOD activity. Cygb-NOD activity shows respective K(m) values for ascorbate, cytochrome b(5), NO, and O(2) of 0.25 mm, 0.3 microm, 40 nm, and approximately 20 microm and achieves a k(cat) of 0.5 s(-1). Ascorbate and cytochrome b(5) reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m(-1) s(-1) and 3 x 10(6) m(-1) s(-1), respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar k(cat) of 1.2 s(-1), a K(m)(NO) of 40 nm, and a k(cat)/K(m)(NO) (k'(NOD)) value of 3 x 10(7) m(-1) s(-1), demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O(2)] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b(5) as reductants.

  11. Comparative metabolism of honokiol in mouse, rat, dog, monkey, and human hepatocytes.

    PubMed

    Jeong, Hyeon-Uk; Kim, Ju-Hyun; Kong, Tae Yeon; Choi, Won Gu; Lee, Hye Suk

    2016-04-01

    Honokiol has antitumor, antioxidative, anti-inflammatory, and antithrombotic effects. Here we aimed to identify the metabolic profile of honokiol in mouse, rat, dog, monkey, and human hepatocytes and to characterize the enzymes responsible for the glucuronidation and sulfation of honokiol. Honokiol had a high hepatic extraction ratio in all five species, indicating that it was extensively metabolized. A total of 32 metabolites, including 17 common and 15 different metabolites, produced via glucuronidation, sulfation, and oxidation of honokiol allyl groups were tentatively identified using liquid chromatography-high resolution quadrupole Orbitrap mass spectrometry. Glucuronidation of honokiol to M8 (honokiol-4-glucuronide) and M9 (honokiol-2'-glucuronide) was the predominant metabolic pathway in hepatocytes of all five species; however, interspecies differences between 4- and 2'-glucuronidation of honokiol were observed. UGT1A1, 1A8, 1A9, 2B15, and 2B17 played major roles in M8 formation, whereas UGT1A7 and 1A9 played major roles in M9 formation. Human cDNA-expressed SULT1C4 played a major role in M10 formation (honokiol-2'-sulfate), whereas SULT1A1*1, 1A1*2, and 1A2 played major roles in M11 formation (honokiol-4-sulfate). In conclusion, honokiol metabolism showed interspecies differences.

  12. Isolation and characterization of endosomes from rat liver

    SciTech Connect

    Kennedy, G.C.

    1987-01-01

    Three fractions of rat liver endosomes, called 50 Kg Light, 50 Kg Heavy, and 150 Kg have been isolated on 16% Percoll gradients. The 50 Kg Heavy fraction accumulates ligand as a function of time after injection, using either /sup 125/I-asialoorosomucoid (/sup 125/I-ASOR) or /sup 125/I-immunoglobulin A (/sup 125/I-IgA) as ligands. A pulse-chase protocol was also used to study the kinetics of ligand entry into the endosomal compartments. A double-label, 3,3'-diaminobenzidine (DAB)-induced density shift protocol was used to study the internalization of two ligands with different destinations in the hepatocyte. Rats were injected intraportally with /sup 125/I-ASOR-HRP and /sup 131/I-IgA and the liver was fractionated at various times post-injection. The three ligand-containing endosomal fractions were isolated and each subjected to the DAB shift procedure. This treatment causes organelles containing /sup 125/I-ASOR-HRP and another ligand occupying the same compartment to shift to a higher density. Thus, information on whether the /sup 131/I-IgA is colocalized or segregated from the /sup 125/I-ASOR-HRP can be obtained. The authors have used an instantaneous pulse, temperature shift protocol to study the heterogeneity of these three endosomal fractions isolated from rat liver.

  13. Inactivation of PEMT2 in hepatocytes initiated by DENA in fasted/refed rats

    SciTech Connect

    Marengo, Barbara; Bottini, Consuelo; La Porta, C.A.M.; Domenicotti, Cinzia; Tessitore, Luciana . E-mail: tessitore@pharm.unipmn.it

    2006-07-21

    Phosphatidylethanolamine N-methyltransferase (PEMT) is the enzyme that converts phosphatidylethanolamine (PE) into phosphatidylcholine. We have previously shown that PEMT suppressed hepatoma growth by triggering apoptosis. We investigate whether PEMT controlled cell death and cell proliferation triggered by fasting/refeeding and whether it is a marker of early preneoplastic lesions. The induction of programmed cell death and suppression of cell proliferation by fasting were associated with enhanced PEMT expression and activity, and with a decrease in CTP:phosphocholine cytidylyltransferase expression. Refeeding returned the liver growth and expression of CTP:phosphocholine cytidylyltransferase to control levels, while the expression of PEMT decreased to below control values. After DENA administration, PEMT protein, evaluated by Western blotting, slightly increased, but it remained below control levels. The treatment with 20 mg/kg DENA to refed rats induced the appearance of initiated hepatocytes that were negative for PEMT expression. Present findings indicate that PEMT is a novel tumour marker for early liver preneoplastic lesions.

  14. Liver tissue engineering based on aggregate assembly: efficient formation of endothelialized rat hepatocyte aggregates and their immobilization with biodegradable fibres.

    PubMed

    Pang, Y; Montagne, K; Shinohara, M; Komori, K; Sakai, Y

    2012-12-01

    To realize long-term in vitro culture of hepatocytes at a high density while maintaining a high hepatic function for aggregate-based liver tissue engineering, we report here a novel culture method whereby endothelialized rat hepatocyte aggregates were formed using a PDMS microwell device and cultured in a perfusion bioreactor by introducing spacers between aggregates to improve oxygen and nutrient supply. Primary rat hepatocyte aggregates around 100 µm in diameter coated with human umbilical vein endothelial cells were spontaneously and quickly formed after 12 h of incubation, thanks to the continuous supply of oxygen by diffusion through the PDMS honeycomb microwell device. Then, the recovered endothelialized rat hepatocyte aggregates were mixed with biodegradable poly-l-lactic acid fibres in suspension and packed into a PDMS-based bioreactor. Perfusion culture of 7 days was successfully achieved with more than 73.8% cells retained in the bioreactor. As expected, the fibres acted as spacers between aggregates, which was evidenced from the enhanced albumin production and more spherical morphology compared with fibre-free packing. In summary, this study shows the advantages of using PDMS-based microwells to form heterotypic aggregates and also demonstrates the feasibility of spacing tissue elements for improving oxygen and nutrient supply to tissue engineering based on modular assembly.

  15. Potential hepatoprotective effects of new Cuban natural products in rat hepatocytes culture.

    PubMed

    Rodeiro, I; Donato, M T; Martínez, I; Hernández, I; Garrido, G; González-Lavaut, J A; Menéndez, R; Laguna, A; Castell, J V; Gómez-Lechón, M J

    2008-08-01

    The protective effects of five Cuban natural products (Mangifera indica L. (MSBE), Erythroxylum minutifolium, Erythroxylum confusum, Thalassia testudinum and Dictyota pinnatifida extracts and mangiferin) on the oxidative damage induced by model toxicants in rat hepatocyte cultures were studied. Cells were pre-incubated with the natural products (5-200 microg/mL) for 24 h. Then hepatotoxins (tert-butyl hydroperoxide, ethanol, carbon tetrachloride and lipopolysaccharide) were individually added and post-incubated for another 24 h. After treatments, cell viability was determined using the MTT assay. Mangiferin and MSBE exhibited the highest cytoprotective potential (EC50 between 50 and 125 microg/mL), followed by T. testudinum and Erythroxylum extracts, whereas no significant protective effects was produced by Dictyota extract treatment. Antioxidant properties of the natural products against lipid peroxidation and GSH depletion induced by tert-butyl hydroperoxide were then investigated. The results show that at 36 h pre-treatment of cells with mangiferin or MSBE, concentrations of T. testudinum and Erythroxylum extracts ranging from 25 to 100 microg/mL significantly inhibited lipid peroxidation induced by tert-butyl hydroperoxide (100 and 250 microM) and increased the GSH levels reduced by the toxicant. D. pinnatifida inhibited lipid peroxidation, but did not preserve GSH levels. In conclusion, MSBE, E. minutifolium, E. confusum and T. testudinum extracts and mangiferin showed hepatoprotective activity against induced damage in all the experimental series, where mangiferin and the extracts of MSBE and T. testudinum were the best candidates to inhibit "in vitro" damage to rat hepatocytes. This hepatoprotective effect found could be associated with the antioxidant properties observed for the products.

  16. Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells

    PubMed Central

    Pfeiffer, Elisa; Kegel, Victoria; Zeilinger, Katrin; Hengstler, Jan G; Nüssler, Andreas K; Seehofer, Daniel

    2015-01-01

    Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 106 KC, 2.7 × 105 LEC and 4.7 × 105 HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4–5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor. PMID:25394621

  17. Administration of antisense DNA for hepatocyte growth factor causes an depressive and anxiogenic response in rats.

    PubMed

    Wakatsuki, Masatoshi; Akiyoshi, Jotaro; Ichioka, Shugo; Tanaka, Yoshihiro; Tsuru, Jusen; Matsushita, Hirotaka; Hanada, Hiroaki; Isogawa, Koichi

    2007-12-01

    Hepatocyte growth factor (HGF) is induced in neurons during ischemia and is neuroprotective against post-ischemic delayed neuronal death in the hippocampus. HGF might play an important role in the maturation and functioning of these neurons in the hippocampus. Our aim was to determine what effect HGF antisense has on depression and anxiety in rats. HGF antisense was infused at a constant rate into cerebral lateral ventricles and its effect on anxiety in rats was monitored. In forced swimming test, rats that received antisense DNA increased the length of time that they were immobile in the water. In the elevated plus maze test, the black and white box test and conditioned fear test, HGF antisense administration caused all indicators of anxiety to increase. Number of HGF-positive cells in C1 of hippocampus was significantly decreased in the HGF antisense-infused group compared to the vehicle- and scrambled oligonucleotide-treated group. No significant effect on general locomotor activity was seen. These results indicate that inhibition of HGF induces an increase in depression and anxiety-related behaviors suggesting a depressive and anxiogenic-like effect.

  18. Gene expression profiles of hepatocytes treated with La (NO3) 3 of rare earth in rats

    PubMed Central

    Zhao, Hui; Hao, Wei-Dong; Xu, Hou-En; Shang, Lan-Qin; Lu, You-Yong

    2004-01-01

    AIM: To compare the gene expression between La (NO3) 3-exposed and control rats in vivo. METHODS: Rats were fed La (NO3) 3 once daily at a dose of 20 mg/kg for one month by gavage. Gene expression of hepatocytes was detected using mRNA differential display (DD) technique and cDNA microarray and compared between treated and control groups. RESULTS: Six differentially expressed sequence tags were cloned by DD, of which five were up regulated and one was down regulated in treated rats. Two sequences were determined. One band was novel. The other shared 100% sequence homology with AU080263 Sugano mouse brain mncb Mus musculus cDNA clone MNCb-5435 5’. With DNA microarray, 136 differentially expressed genes were identified including 131 over-expressed genes and 5 under-expressed genes. Most of these differentially expressed genes were cell signal and transmission genes, genes associated with metabolism, protein translation and synthesis. CONCLUSION: La (NO3) 3 could change the expression levels of some kinds of genes. Further analysis of the differentially expressed genes would be helpful for understanding the wide biological effect spectrum of rare earth elements. PMID:15162537

  19. In vitro metabolism of [(14)C]-benalaxyl in hepatocytes of rats, dogs and humans.

    PubMed

    Nallani, Gopinath C; ElNaggar, Shaaban F; Shen, Li; Chandrasekaran, Appavu

    2017-03-01

    The in vitro comparative animal metabolism study is now a data requirement under EU Directive 1107/2009 for registration of plant protection products. This type of study helps determine the extent of metabolism of a chemical in each surrogate species and whether any unique human metabolite(s) are formed. In the present study, metabolism of racemic [(14)C]-benalaxyl, a fungicide was investigated in cryopreserved rat, dog and human hepatocytes. The metabolites generated were identified/characterized by LC/MS/MS with radiometric detection and comparison with reference standards. [(14)C]-glucuronide conjugates of benalaxyl metabolites in rat, dog and human hepatocytes were confirmed via additional experiments in which known reference standards were incubated with dog liver microsomes in the presence of UDPGA. After 4 h of incubation, benalaxyl was extensively metabolized in all the species with the following trend: dog (100%) > human (86%) > rat (75%). In all species, the major metabolic pathways consisted of hydroxylation of the methyl group in the xylene moiety to 2-hydroxymethyl-benalaxyl, further oxidation to its carboxylic acid analogue (benalaxyl-2-benzoic acid), and hydrolysis of the methyl ester to yield benalaxyl acid or 2-hydroxymethyl benalaxyl acid. In addition, glucuronidation of phase I metabolites occurred in all species, to a higher extent in dog hepatocytes in which 2-hydroxymethyl-benalaxyl-glucuronide conjugate constituted the most significant metabolite. No major unique metabolite was observed in human hepatocytes. Also, benalaxyl did not undergo stereo-selective metabolism in rat or human hepatocytes.

  20. Effects of metal ions on lipid peroxidation in cultured rat hepatocytes loaded with alpha-linolenic acid.

    PubMed

    Furono, K; Suetsuga, T; Sugihara, N

    1996-06-07

    We investigated the ability of various redox-active metal ions to induce lipid peroxidation in normal and alpha-linolenic acid-loaded (LNA-loaded) cultured rat hepatocytes. Lipid peroxidation was estimated by the accumulation of malondialdehyde (MDA) in the culture medium. At low concentrations induction was highest with ferrous ions (Fe), whereas at high concentrations, vanadium (V) and copper ions (Cu) had the greatest effect on both groups of hepatocytes. With any one of the three metal ions, the extent of lipid peroxidation in LNA-loaded hepatocytes was several times greater compared to normal cells. In addition, upon the addition of Fe or V, LNA-loaded hepatocytes were injured whereas normal cells were not. The addition of Cu caused substantial cell injury in normal hepatocytes, and even greater injury in LNA-loaded cells. The prevention of lipid peroxidation in LNA-loaded hepatocytes by addition of an antioxidant like N,N'-diphenyl-p-phenylene-diamine (DPPD) almost completely prevented Fe- and V-induced cell injury, and reduced Cu-induced cell injury. alpha-Tocopherol behaved in a way similar to but less effective than DPPD. .OH radical scavengers such as mannitol and dimethyl sulfoxide (DMSO) had no effect on lipid peroxidation induced by any metal ions in LNA-loaded hepatocytes. Addition of cadmium ions (Cd), which required the lowest concentration to cause cell injury, induced a slight increase in lipid peroxidation in normal hepatocytes, but did not induce lipid peroxidation to the same extent as seen in LNA-loaded cells treated with any of the three metal ions already mentioned. The inhibition of lipid peroxidation by DPPD scarcely protected LNA-loaded hepatocytes from Cd-induced cell injury. None of the other metal ions including aluminum (Al), chromium (Cr), manganese (Mn), nickel (Ni), lead (Pb), and tin (Sn) ions, effectively induced lipid peroxidation in either group of hepatocytes, except cobalt ions (Co), which had a peroxidative effect in LNA

  1. Effect of improved hydrogen supply on energy state, ureogenesis and gluconeogenesis in isolated hepatocytes.

    PubMed

    Letko, G; Halangk, W

    1986-01-01

    The cellular energy state as well as ureogenesis and gluconeogenesis as ATP-consuming processes were studied as a function of hydrogen supply of mitochondria in isolated hepatocytes. Even though the ATP turnover was quite different during ureogenesis and gluconeogenesis and in the absence of these syntheses both the safranine signals (as a probe for the mitochondrial membrane potential) and the total ATP contents of the cells did not vary to a great extent. Partial uncoupling by FCCP destroyed the mitochondrial membrane potential and decreased the cellular ATP totally. Under these conditions the presence of ornithine plus lactate maintained a measurable membrane potential and ATP level. In order to demonstrate the relevance of hydrogen supply of mitochondria to the syntheses of glucose and urea, the load on energy metabolism was additionally increased by partial uncoupling, and the cells were incubated in a parallel mode in Krebs-Henseleit buffer and an 80 mM succinate containing medium, the latter forcing succinate into the cells. After partial uncoupling, ureogenesis and gluconeogenesis as well as the ATP level were seen to decrease markedly in Krebs-Henseleit buffer, whereas in the succinate medium the improved hydrogen supply of mitochondria counteracted the drain by FCCP, and substantially higher rates of the syntheses and elevated ATP levels were maintained.

  2. Amelioration of Hyperbilirubinemia in Gunn Rats after Transplantation of Human Induced Pluripotent Stem Cell-Derived Hepatocytes

    PubMed Central

    Chen, Yong; Li, Yanfeng; Wang, Xia; Zhang, Wei; Sauer, Vanessa; Chang, Chan-Jung; Han, Bing; Tchaikovskaya, Tatyana; Avsar, Yesim; Tafaleng, Edgar; Madhusudana Girija, Sanal; Tar, Krisztina; Polgar, Zsuzsanna; Strom, Stephen; Bouhassira, Eric E.; Guha, Chandan; Fox, Ira J.; Roy-Chowdhury, Jayanta; Roy-Chowdhury, Namita

    2015-01-01

    Summary Hepatocyte transplantation has the potential to cure inherited liver diseases, but its application is impeded by a scarcity of donor livers. Therefore, we explored whether transplantation of hepatocyte-like cells (iHeps) differentiated from human induced pluripotent stem cells (iPSCs) could ameliorate inherited liver diseases. iPSCs reprogrammed from human skin fibroblasts were differentiated to iHeps, which were transplanted into livers of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-deficient Gunn rats, a model of Crigler-Najjar syndrome 1 (CN1), where elevated unconjugated bilirubin causes brain injury and death. To promote iHep proliferation, 30% of the recipient liver was X-irradiated before transplantation, and hepatocyte growth factor was expressed. After transplantation, UGT1A1+ iHep clusters constituted 2.5%–7.5% of the preconditioned liver lobe. A decline of serum bilirubin by 30%–60% and biliary excretion of bilirubin glucuronides indicated that transplanted iHeps expressed UGT1A1 activity, a postnatal function of hepatocytes. Therefore, iHeps warrant further exploration as a renewable source of hepatocytes for treating inherited liver diseases. PMID:26074313

  3. Effects of nutritional and hormonal factors on the metabolism of retinol-binding protein by primary cultures of rat hepatocytes

    SciTech Connect

    Dixon, J.L.; Goodman, D.S.

    1987-01-01

    Studies were conducted to explore hormonal and nutritional factors that might be involved in the regulation of retinol-binding protein (RBP) synthesis and secretion by the liver. The studies employed primary cultures of hepatocytes from normal rats. When cells were cultured in Dulbecco's modified Eagle's medium alone, a high rate of RBP secretion was observed initially, which declined and became quite low by 24 hr. Supplementing the medium with amino acids maintained RBP and albumin secretion at moderate (but less than initial) rates for at least 3 days. Further addition of dexamethasone maintained the production and secretion rates of RBP, transthyretin, and albumin close to the initial rates for up to 3-5 days in culture as measured by radioimmunoassay. Hormonally treated hepatocytes produced and secreted RBP, transthyretin, and albumin at both absolute and relative rates similar to physiological values, as estimated from rates reported by others from studies in vivo and with perfused livers. Glucagon addition partially maintained the secretion rates of these 3 proteins, but less effectively than did dexamethasone. A number of other hormones, added singly or in combination, did not affect RBP production or secretion. Addition of retinol to the cultured normal hepatocytes was without effect upon RBP secretion. These studies show that supplementing the culture medium of hepatocytes with amino acids and dexamethasone maintains RBP production and secretion for several days. In normal hepatocytes, with ample supply of retinol available within the cell, addition of exogenous retinol does not appear to influence RBP metabolism or secretion by the cells.

  4. Use of in situ hybridization to identify collagen and albumin mRNAs in isolated mouse hepatocytes.

    PubMed Central

    Saber, M A; Zern, M A; Shafritz, D A

    1983-01-01

    We present a simple and improved method for in situ localization of albumin and collagen mRNAs in isolated mouse hepatocytes. The cells were isolated by collagenase perfusion, mincing, and differential centrifugation. Nick-translated 3H-labeled mouse albumin cDNA (pmalb-2) and chicken pro-alpha 2(I) collagen cDNA (pCg45) probes were then hybridized with the cells in silane-treated microcentrifuge tubes. The cells were transferred and fixed to a microscope slide and hybridization was evaluated semiquantitatively by counting exposure of grains in autoradiographic emulsion placed over the cells. With this method of in situ hybridization, all hepatocytes appear to have significant, but highly variable, amounts of albumin mRNA. In addition, type I procollagen mRNA appears to be present at low abundance in hepatocytes. These results indicate that in situ hybridization can effectively demonstrate the presence of specific low- or high-abundance mRNAs in isolated well-differentiated eukaryotic cells. Images PMID:6575392

  5. Site-specificity of ethanol-induced dephosphorylation of rat hepatocyte keratins 8 and 18: A 31P NMR study.

    PubMed

    Eckert, B S; Yeagle, P L

    1996-01-01

    Chronic feeding of ethanol to rats results in disorganization of the keratin intermediate filament network within hepatocytes. Previous studies from this laboratory have shown that intermediate filament organization in cultured cells is related to the phosphorylation state of the proteins. Therefore, we have examined the phosphorylation state of hepatocyte keratins from control and ethanol-fed rats. Feeding ethanol to rats results in dephosphorylation of one site on keratin 8 and one site on keratin 18 at all time points beginning with 6 weeks of ethanol treatment. Dephosphorylation was detected by phosphate analysis and by two-dimensional electrophoresis in which a change in isoelectric point of keratins from ethanol-fed rats was observed. These observations indicate that dephosphorylation of keratins in ethanol-fed animals may be an early step in alcoholic hepatitis which has occurred by 6 weeks of ethanol treatment. To further characterize keratin dephosphorylation in ethanol-fed rats, we used 31P NMR spectroscopy to classify the dephosphorylation site(s). Hepatocyte keratins were purified and solubilized in 9.5 M urea, 10 mM Tris-Cl, pH 8.1. 31P NMR spectra were obtained at 109 MHz, in 10 mm tubes at 30 degrees C. Samples of hepatocyte keratins were phosphorylated with A-kinase, protein kinase C, casein kinase II or Ca/CAM kinase and these samples were analyzed by 31P NMR spectroscopy. The resulting spectra were used as standards to compare the 31P chemical shifts of the resonances produced by these kinases with the phosphorus resonances of control and experimental samples. The 31P NMR spectrum of control hepatocyte keratins shows three resonances at 0.7, 4 and 5 ppm. In vitro phosphorylation by A-kinase produces a resonance at 4 ppm which is distinctly different from the resonance produced by each of the other kinases. In hepatocyte keratins from ethanol-fed animals, the resonance at 4 ppm was missing from the spectrum. These observations indicate that the

  6. Hepatocyte growth factor and transforming growth factor beta regulate 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in rat hepatocyte primary cultures.

    PubMed Central

    Joaquin, M; Rosa, J L; Salvadó, C; López, S; Nakamura, T; Bartrons, R; Gil, J; Tauler, A

    1996-01-01

    Hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-beta) are believed to be of major importance for hepatic regeneration after liver damage. We have studied the effect of these growth factors on fructose 2,6-bisphosphate (Fru-2,6-P2) levels and the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-BPase) in rat hepatocyte primary cultures. Our results demonstrate that HGF activates the expression of the 6PF2K/Fru-2,6-BPase gene by increasing the levels of its mRNA. As a consequence of this activation, the amount of 6PF2K/Fru-2,6-BPase protein and 6-phosphofructo-2-kinase activity increased, which was reflected by a rise in Fru-2,6-P2 levels. In contrast, TGF-beta decreased the levels of 6PF2K/Fru-2,6-BPase mRNA, which led to a decrease in the amount of 6PF2K/Fru-2,6-BPase protein and Fru-2,6-P2. The different actions of HGF and TGF-beta on 6PF2K/Fru-2,6-BPase gene expression are concomitant with their effect on cell proliferation. Here we show that, in the absence of hormones, primary cultures of hepatocytes express the F-type isoenzyme. In addition, HGF increases the expression of this isoenzyme, and dexamethasone activates the L-type isoform. HGF and TGF-beta were able to inhibit this activation. PMID:8660288

  7. Hepatoprotective effects of Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] on alcohol-damaged primary rat hepatocyte culture in vitro.

    PubMed

    Jiang, Wenhua; Bian, Yuzhu; Wang, Zhenghui; Chang, Thomas Ming Swi

    2017-02-01

    We have prepared a novel nanobiotherapeutic, Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase], which not only transports both oxygen and carbon dioxide but also a therapeutic antioxidant. Our previous study in a severe sustained 90 min hemorrhagic shock rat model shows that it has a hepatoprotective effect. We investigate its hepatoprotective effect further in this present report using an alcohol-damaged primary hepatocyte culture model. Results show that it significantly reduced ethanol-induced AST release, lipid peroxidation, and ROS production in rat primary hepatocytes culture. It also significantly enhanced the viability of ethanol-treated hepatocytes. Thus, the result shows that Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] also has some hepatoprotective effects against alcohol-induced injury in in vitro rat primary hepatocytes cell culture. This collaborate our previous observation of its hepatoprotective effect in a severe sustained 90-min hemorrhagic shock rat model.

  8. Effects of Epigallocatechin Gallate on Tert-Butyl Hydroperoxide-Induced Mitochondrial Dysfunction in Rat Liver Mitochondria and Hepatocytes

    PubMed Central

    Endlicher, Rene; Sobotka, Ondrej; Drahota, Zdenek

    2016-01-01

    Epigallocatechin gallate (EGCG) is a green tea antioxidant with adverse effects on rat liver mitochondria and hepatocytes at high doses. Here, we assessed whether low doses of EGCG would protect these systems from damage induced by tert-butyl hydroperoxide (tBHP). Rat liver mitochondria or permeabilized rat hepatocytes were pretreated with EGCG and then exposed to tBHP. Oxygen consumption, mitochondrial membrane potential (MMP), and mitochondrial retention capacity for calcium were measured. First, 50 μM EGCG or 0.25 mM tBHP alone increased State 4 Complex I-driven respiration, thus demonstrating uncoupling effects; tBHP also inhibited State 3 ADP-stimulated respiration. Then, the coexposure to 0.25 mM tBHP and 50 μM EGCG induced a trend of further decline in the respiratory control ratio beyond that observed upon tBHP exposure alone. EGCG had no effect on MMP and no effect, in concentrations up to 50 μM, on mitochondrial calcium retention capacity. tBHP led to a decline in both MMP and mitochondrial retention capacity for calcium; these effects were not changed by pretreatment with EGCG. In addition, EGCG dose-dependently enhanced hydrogen peroxide formation in a cell- and mitochondria-free medium. Conclusion. Moderate nontoxic doses of EGCG were not able to protect rat liver mitochondria and hepatocytes from tBHP-induced mitochondrial dysfunction. PMID:28074116

  9. SELENIUM COMPOUNDS MODULATE THE ACTIVITY OF RECOMBINANT RAT ASIII-METHYLTRANSFERASE AND THE METHYLATION OF ARSENITE BY RAT AND HUMAN HEPATOCYTES

    EPA Science Inventory

    Abstract

    Formation of methylated metabolites is a critical step in the metabolism of inorganic arsenic or selenium. We have previously shown that under conditions of a concurrent exposure selenite inhibits methylation of arsenite by cultured rat hepatocytes. Here, we com...

  10. Embryonic mouse STO cell-derived xenografts express hepatocytic functions in the livers of nonimmunosuppressed adult rats.

    PubMed

    Zhang, Mingjun; Joseph, Brigid; Gupta, Sanjeev; Guest, I; Xu, Meng; Sell, Stewart; Son, Kyung-Hwa; Koch, Katherine S; Leffert, Hyam L

    2005-02-01

    Cells derived from embryonic mouse STO cell lines differentiate into hepatocytes when transplanted into the livers of nonimmunosuppressed dipeptidylpeptidase IV (DPPIV)-negative F344 rats. Within 1 day after intrasplenic injection, donor cells moved rapidly into the liver and were found in intravascular and perivascular sites; by 1 month, they were intrasinusoidal and also integrated into hepatic plates with approximately 2% efficiency and formed conjoint bile canaliculi. Neither donor cell proliferation nor host inflammatory responses were observed during this time. Detection of intrahepatic mouse COX1 mitochondrial DNA and mouse albumin mRNA in recipient rats indicated survival and differentiation of donor cells for at least 3 months. Mouse COX1 targets were also detected intrahepatically 4-9 weeks after STO cell injection into nonimmunosuppressed wild-type rats. In contrast to STO-transplanted rats, mouse DNA or RNA was not detectable in untreated or mock-transplanted rats or in rats injected with donor cell DNA. In cultured STO donor cells, DPPIV and glucose-6-phosphatase activities were observed in small clusters; in contrast, mouse major histocompatibility complex class I H-2Kq, H-2Dq, and H-2Lq and class II I-Aq markers were undetectable in vitro before or after interferon gamma treatment. Together with H-2K allele typing, which confirmed the Swiss mouse origin of the donor cells, these observations indicate that mouse-derived STO cell lines can differentiate along hepatocytic lineage and engraft into rat liver across major histocompatibility barriers.

  11. Effects of chlorpromazine on Na+-K+-ATPase pumping and solute transport in rat hepatocytes

    SciTech Connect

    Van Dyke, R.W.; Scharschmidt, B.F.

    1987-11-01

    Inhibition of Na+-K+-ATPase and sodium-dependent bile acid transport has been suggested as a mechanism for the cholestasis produced by certain drugs such as chlorpromazine. We examined the effects of chlorpromazine (and in selected studies, two of its metabolites) on Na+-K+-ATPase cation pumping (ouabain-suppressible /sup 86/Rb uptake), exchangeable intracellular sodium content, membrane potential (assessed by /sup 36/Cl- distribution), and sodium-dependent transport of taurocholate and alanine in primary cultures of rat hepatocytes. Chlorpromazine (10-300 microM), 7,8-dihydroxychlorpromazine (10-300 microM), and ouabain (0.1-2 mM), but not chlorpromazine sulfoxide, produced a concentration-dependent decrease in Na+-K+-ATPase cation pumping and an increase in intracellular sodium content. Chlorpromazine (100 microM) and ouabain (0.75 mM) also modestly decreased hepatocyte membrane potential. In further studies, chlorpromazine (75 and 100 microM) and ouabain (0.1, 0.5, and 0.75 mM) decreased initial sodium-dependent uptake rates of taurocholate and alanine by 18-63%. Although the steady-state intracellular content of alanine was decreased 25-53% by both agents, chlorpromazine increased the steady-state content of taurocholate by 171% and decreased taurocholate efflux, apparently related to partitioning of taurocholate into a large, slowly turning over intracellular pool. These studies provide direct evidence that chlorpromazine inhibits Na+-K+-ATPase cation pumping in intact cells and that partial inhibition of Na+-K+-ATPase cation pumping is associated with a reduction of both the electrochemical sodium gradient and sodium-dependent solute transport. These effects of chlorpromazine may contribute to chlorpromazine-induced cholestasis in animals and humans.

  12. Toxicokinetics of acrylamide in primary rat hepatocytes: coupling to glutathione is faster than conversion to glycidamide.

    PubMed

    Watzek, Nico; Scherbl, Denise; Schug, Markus; Hengstler, Jan G; Baum, Matthias; Habermeyer, Michael; Richling, Elke; Eisenbrand, Gerhard

    2013-08-01

    Acrylamide (AA), classified as class 2A carcinogen (probably carcinogenic to humans) by the International Agency for Research on Cancer (IARC), is formed during heating of food from reducing carbohydrates and asparagine by Maillard reaction chemistry. After dietary uptake, AA is in part metabolically converted into the proximate genotoxic phase I metabolite glycidamide (GA). GA reacts with nucleophilic base positions in DNA, primarily forming N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) adducts. In a competing phase II biotransformation pathway AA, as well as its phase I metabolite GA, is coupled to glutathione (GSH). The GSH coupling products are further biotransformed and excreted via urine as mercapturic acids (MA), N-acetyl-S-(2-carbamoylethyl)cysteine (AAMA), and N-acetyl-S-(2-hydroxy-2-carbamoylethyl)cysteine (GAMA). In the present study, hepatic biotransformation pathways and DNA adduct formation were studied in primary rat hepatocytes, incubated with AA (0.2-2,000 μM) for up to 24 h. Contents of AA-GSH, GA, AAMA, and GAMA were measured in the cell culture medium after solid phase extraction (SPE). N7-GA-Gua adducts in DNA of hepatocytes were determined by HPLC-ESI-MS/MS after lysis of the cells and neutral thermal hydrolysis. Formation of AA-GSH was linear with AA concentration and incubation time and became detectable already at 0.2 μM (4 h). In contrast to AA, GA was not detected before 16 h incubation at 10-fold higher AA concentration (2 μM). In summary, the rate of AA-GSH formation was found to be about 1.5-3 times higher than that of GA formation. N7-GA-Gua adducts were found only at the highest AA concentration tested (2,000 μM).

  13. Effects of chlorpromazine on Na+-K+-ATPase pumping and solute transport in rat hepatocytes.

    PubMed

    Van Dyke, R W; Scharschmidt, B F

    1987-11-01

    Inhibition of Na+-K+-ATPase and sodium-dependent bile acid transport has been suggested as a mechanism for the cholestasis produced by certain drugs such as chlorpromazine. We examined the effects of chlorpromazine (and in selected studies, two of its metabolites) on Na+-K+-ATPase cation pumping (ouabain-suppressible 86Rb uptake), exchangeable intracellular sodium content, membrane potential (assessed by 36Cl- distribution), and sodium-dependent transport of taurocholate and alanine in primary cultures of rat hepatocytes. Chlorpromazine (10-300 microM), 7,8-dihydroxychlorpromazine (10-300 microM), and ouabain (0.1-2 mM), but not chlorpromazine sulfoxide, produced a concentration-dependent decrease in Na+-K+-ATPase cation pumping and an increase in intracellular sodium content. Chlorpromazine (100 microM) and ouabain (0.75 mM) also modestly decreased hepatocyte membrane potential. In further studies, chlorpromazine (75 and 100 microM) and ouabain (0.1, 0.5, and 0.75 mM) decreased initial sodium-dependent uptake rates of taurocholate and alanine by 18-63%. Although the steady-state intracellular content of alanine was decreased 25-53% by both agents, chlorpromazine increased the steady-state content of taurocholate by 171% and decreased taurocholate efflux, apparently related to partitioning of taurocholate into a large, slowly turning over intracellular pool. These studies provide direct evidence that chlorpromazine inhibits Na+-K+-ATPase cation pumping in intact cells and that partial inhibition of Na+-K+-ATPase cation pumping is associated with a reduction of both the electrochemical sodium gradient and sodium-dependent solute transport. These effects of chlorpromazine may contribute to chlorpromazine-induced cholestasis in animals and humans.

  14. A new flavanone isolated from rhizoma smilacis glabrae and the structural requirements of its derivatives for preventing immunological hepatocyte damage.

    PubMed

    Chen, T; Li, J; Cao, J; Xu, Q; Komatsu, K; Namba, T

    1999-02-01

    From the rhizome of Smilax glabra Roxb., a new flavanone was isolated and named as smitilbin (1), together with 6 known compounds, engeletin (2), astilbin (3), dihydroquercetin (4), eurryphin (5), resveratrol (6), and 5-O-caffeoylshikimic acid (7). These compounds were applied to the assay of liver nonparenchymal cells (NPC) against hepatocytes (HC) isolated from mice with an immunological liver injury. Against the NPC-caused elevation of ALT (alanine transminase) in culture supernatant from HC, the pretreatment of NPC with flavanoids (1-3) dose-dependently blocked the ALT release while 4, the aglycone of 3, did not. The chromone 5 showed a much stronger inhibition. Compound 6 also showed the activity. However, 1-7 did not show any suppression of NPC or CCl4-induced ALT release when they were used to pretreat HC. These results suggest that compounds 1-3, 5, and 6 could protect the hepatocyte damage from NPC through selectively producing the dysfunction of NPC with an essential requirement of rhamnose, and the chromone part in their structures may be critical for exhibiting the activity rather than through protecting the hepatocyte membranes.

  15. New physiologically-relevant liver tissue model based on hierarchically cocultured primary rat hepatocytes with liver endothelial cells.

    PubMed

    Xiao, Wenjin; Perry, Guillaume; Komori, Kikuo; Sakai, Yasuyuki

    2015-11-01

    To develop an in vitro liver tissue equivalent, hepatocytes should be cocultured with liver non-parenchymal cells to mimic the in vivo physiological microenvironments. In this work, we describe a physiologically-relevant liver tissue model by hierarchically organizing layers of primary rat hepatocytes and human liver sinusoidal endothelial cells (TMNK-1) on an oxygen-permeable polydimethylsiloxane (PDMS) membrane, which facilitates direct oxygenation by diffusion through the membrane. This in vivo-mimicking hierarchical coculture was obtained by simply proceeding the overlay of TMNK-1 cells on the hepatocyte layer re-formed on the collagen immobilized PDMS membranes. The comparison of hepatic functionalities was achieved between coculture and sandwich culture with Matrigel, in the presence and absence of direct oxygenation. A complete double-layered structure of functional liver cells with vertical contact between hepatocytes and TMNK-1 was successfully constructed in the coculture with direct oxygen supply and was well-maintained for 14 days. The hepatocytes in this hierarchical culture exhibited improved survival, functional bile canaliculi formation, cellular level polarization and maintenance of metabolic activities including Cyp1A1/2 activity and albumin production. By contrast, the two cell populations formed discontinuous monolayers on the same surfaces in the non-oxygen-permeable cultures. These results demonstrate that (i) the direct oxygenation through the PDMS membranes enables very simple formation of a hierarchical structure consisting of a hepatocyte layer and a layer of TMNK-1 and (ii) we may include other non-parenchymal cells in this format easily, which can be widely applicable to other epithelial organs.

  16. Uptake and glutathione conjugation of ethacrynic acid and efflux of the glutathione adduct by periportal and perivenous rat hepatocytes.

    PubMed

    Tirona, R G; Tan, E; Meier, G; Pang, K S

    1999-12-01

    We assessed the impact of zonal factors on the hepatic reduced glutathione (GSH) conjugation of ethacrynic acid (EA). Uptake of EA by enriched periportal (PP) and perivenous (PV) rat hepatocytes was characterized by both saturable (V(max)(uptake) = 3.4 +/- 1.7 and 3. 2 +/- 0.8 nmol/min/mg protein and K(m)(uptake) = 51 +/- 13 and 44 +/- 15 microM) and nonsaturable (12 +/- 5 and 12 +/- 3 microl/min/mg protein) components. Values for the overall GSH conjugation rates of EA (200 microM) were similar among the zonal hepatocytes and resembled those for the influx transport rates. In the absence of the hepatocyte membrane, GSH conjugation in PV and PP hepatocyte cytosol was similar, but a higher perivenous GSH conjugation activity toward EA (PV/PP of 2.4) that mirrored the higher PV/PP ratios of immunodetectable GSTs Ya (1.7) and Yb2 (2.5) was found in cell lysates obtained by the dual-digitonin-pulse perfusion technique. The GSH conjugation rates in the subcellular fragments were, however, much greater than those observed for intact hepatocytes. Efflux rates of the glutathione conjugate EA-SG from zonal hepatocytes were similar, as were levels of the immunodetectable multidrug-resistance protein 2/canalicular multispecific organic anion transporter (Mrp2/cMoat) in the 100,000g pellets. The composite results suggest that the GSTs responsible for EA metabolism are more abundant in the PV region, albeit that the gradient of enzymatic activities is shallow. Despite the existence of zonal metabolic activity, the overall GSH conjugation rate of EA is homogeneous among cells because the reaction is rate limited by uptake, which occurs evenly. Results on EA-SG efflux suggest the acinar homogeneity in Mrp2/cMoat function for canalicular transport.

  17. Induction of cMrp/cMoat gene expression by cisplatin, 2-acetylaminofluorene, or cycloheximide in rat hepatocytes.

    PubMed

    Kauffmann, H M; Keppler, D; Kartenbeck, J; Schrenk, D

    1997-10-01

    The human multidrug-resistance-associated protein (MRP), a member of the adenosine triphosphate (ATP)-binding cassette transporter superfamily, is frequently overexpressed in tumor cells resistant to antineoplastic drugs. In the rat, two Mrp isoforms have been identified, Mrp and cMrp. cMrp, also called Mrp2 or cMoat (canalicular multispecific organic anion transporter), is expressed in the canalicular membrane of rat hepatocytes and mediates the excretion of glucuronate, sulfate, and glutathione conjugates into bile. We investigated the expression of cMrp and Mrp in rat hepatocytes in primary culture. Treatment with the chemical carcinogen 2-acetylaminofluorene (2-AAF), the antineoplastic drug cisplatin, and the protein-synthesis inhibitor cycloheximide led to a dose-dependent and time-dependent increase in cmrp gene expression. A 347-base pair cmrp complementary DNA (cDNA) probe served to demonstrate the induction of cmrp messenger RNA (mRNA) with 40 micromol/L 2-AAF, 5 micromol/L cisplatin, or 5 micromol/L cycloheximide. An analogous response was obtained for the increase in cMrp protein. Mrp mRNA was below the detection limit in Northern blots of RNA from liver and hepatocyte cultures, in contrast to rat testis mRNA which served as a positive control. Immunofluorescence microscopy of cultured hepatocytes was used to visualize cMrp in the plasma membrane. Treatment with 2-AAF led to a marked increase in the immunofluorescence signal confirming the cMrp-inducing potency of 2-AAF. In conclusion, the inducing effect of the compounds studied may reflect a general inducibility of hepatic cMrp by a variety of cytotoxic, carcinogenic, and chemotherapeutic agents which is likely to be of relevance for the acquisition of multidrug resistance during chemotherapy and in the process of chemical carcinogenesis in the liver.

  18. Evidence for a substrate cycle between AMP and adenosine in isolated hepatocytes.

    PubMed Central

    Bontemps, F; Van den Berghe, G; Hers, H G

    1983-01-01

    The effect of adenosine on the metabolism of prelabeled adenine nucleotides was investigated in isolated hepatocytes. Adenosine caused an approximately equal to 2-fold increase in the ATP content of the cells. This effect was in part counteracted by an increased rate of adenine nucleotide catabolism that could be explained by a stimulation of both AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) and the cytoplasmic 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) because of the increased concentration of ATP. The unexpected finding that labeled adenosine was formed immediately after the addition of the unlabeled nucleoside could be explained by the trapping effect of adenosine. An accumulation of labeled adenosine was observed also in the presence of 5-iodotubercidin, a potent inhibitor of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20). Under these conditions, there was a decrease in the concentration of ATP in the cell and a 2- to 3-fold increase in the rate of formation of allantoin. This formation of adenosine was only slightly decreased by inhibition of the membranous 5'-nucleotidase; it led to the accumulation of S-adenosylhomocysteine in the presence of coformycin and an excess of L-homocysteine. It was concluded that, under basal conditions, the cytoplasmic 5'-nucleotidase present in the liver cell continuously produces adenosine, which is immediately reconverted into AMP by adenosine kinase, without giving rise to allantoin. This futile cycle between AMP and adenosine amounts to at least 20 nmol/min per g of liver and, thus, exceeds the basic rate of allantoin formation. PMID:6304684

  19. Differential effects of long-term exposure to Aroclor 1254 on lipid secretion by primary cultures of adult rat hepatocytes

    SciTech Connect

    Mendoza-Figueroa, T.; Hernandez, A.; Lopez, L.

    1992-06-01

    PCBs produce hepatic triglyceride (TG) accumulation (fatty liver) in experimental animals and humans exposed accidentally and occupationally. It has been suggested that this effect could be due to a block in TG secretion. On the other hand, increased levels of plasmatic TG and cholesterol have been described in rats after dietary exposure to Aroclor 1254 (Aro) and other PCBs; hypertriglyceridemia and hypertension have been also described in humans exposed for long periods to low concentrations of PCBs. Since the study of hepatic lipid metabolism and its alteration by toxic chemicals is complicated in the whole animal, short term cultures of adult rat hepatocytes have been used. We have described a system for the long term culture of adult rat hepatocytes which for several weeks maintain differentiated functions, like fatty acid and TG synthesis and their export to the culture medium. In this paper we used this culture system to study the effect of long-term exposure to micromolar concentrations of Aro on the secretion of lipids by cultured hepatocytes. 27 refs., 4 figs., 1 tab.

  20. Osmolyte and Na+ transport balances of rat hepatocytes as a function of hypertonic stress.

    PubMed

    Wehner, F; Tinel, H

    2000-11-01

    The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an influx of Na+ that is then exchanged for K+ via stimulation of Na+/K+-adenosine triphosphatase (ATPase). In this study, we analysed the activation pattern of the Na+ transporters underlying RVI as a function of the degree of hypertonic stress. In confluent primary cultures, four hypertonic conditions were tested (changes from 300 to 327, 360, 400 or 450 mosmol/l) and the activities of Na+ conductance, Na+/H+ antiport, Na+-K+-2Cl- symport and Na+/K+-ATPase were quantified using intracellular microelectrodes, microfluorometry and time-dependent, furosemide- or ouabain-sensitive 86Rb+ uptake, respectively. Neither Na+ conductance nor Na+-K+-2Cl- symport responded to 327 mosmol/A. At 360, 400 and 450 mosmol/l, uptake via these transporters would lead to increases of cell Na+ by 33.0, 49.0 and 49.0 and by 4.5, 10.4 and 9.2 mmol/l per 10 min, respectively. In contrast, Na+/H+ antiport exhibited 65% of its maximal activation already at 327 mosmol/l. At the four osmolarities tested, this transporter would augment cell Na+ by 6.9, 8.9, 9.8 and 10.6 mmol/l per 10 min. The sums of Na+ import were consistent with the amounts of Na+ exported via Na+/K+-ATPase plus the actual increases of cell Na+ (21.2, 58.5, 63.6 and 68.3 mmol/l per 10 min and 2.2, 4.0, 6.3 and 8.2 mmol/l, respectively). In addition, these elevations of cell Na+ plus the increases of cell K+ (via Na+/K+-ATPase) that amounted to 5.0, 6.5, 17.5 and 18.4 mmol/l were consistent with the increases of intracellular osmotic (cationic) activity of 2.5, 11.5, 21.0 and 28.5 mmol/l, respectively, computed from RVI data. It is concluded that the principle of rat hepatocyte RVI, i.e. an initial uptake of Na+ that is then exchanged for K+ via Na+/K+-ATPase, is realized over the entire range of 9-50% hypertonicity tested. The set-point for the activation of RVI clearly lies below 327 mosmol/l. Na+/H+ antiport is the most sensitive Na+ importer

  1. Synergistic effects of food colors on the toxicity of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) in primary cultured rat hepatocytes.

    PubMed

    Ashida, H; Hashimoto, T; Tsuji, S; Kanazawa, K; Danno, G

    2000-06-01

    The synergistic effect of food additives or food colors on the toxicity of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was investigated using primary cultured rat hepatocytes. When hepatocytes from rats fed a standard diet were treated with a mixture of four major food additives (sorbitol, sodium L(+)-glutamate, benzoic acid, and propylene glycol) or a mixture of six typical artificial food colors (erythrosine, allura red, new coccine, brilliant blue, tertrazine, and fast green), the in vitro treated food-color mixture itself showed cytotoxicity: the reduction of cell viability and decreases in the activities of gluconeogenesis and ureogenesis. The food-color mixture enhanced cytotoxicity of Trp-P-1 obviously. We then investigated the effects of in vivo-dosed food additives or food colors on Trp-P-1-caused toxicity. Hepatocytes were isolated and cultured from rats fed a diet containing a mixture of food additives or a mixture of food colors with half the amount of their respective acceptable daily intake for 4 wk. Trp-P-1 was administered to the hepatocytes at various concentrations for 12 h. Synergistic effects of in vivo-dosed food additives and food colors were not observed on Trp-P-1-caused cytotoxicity as estimated by a loss of cell viability and the reductions of DNA and protein syntheses. On the contrary, we have observed that in vivo administered food colors synergistically facilitated to reduce the activities of gluconeogenesis and ureogenesis in Trp-P-1-treated hepatocytes. These results suggest that the daily intake of artificial food colors may impair hepatic functions such as gluconeogenesis and ureogenesis, when dietary carcinogens are exposed to the liver cells.

  2. Isolation and co-culture of rat parenchymal and non-parenchymal liver cells to evaluate cellular interactions and response

    PubMed Central

    Bale, Shyam Sundhar; Geerts, Sharon; Jindal, Rohit; Yarmush, Martin L.

    2016-01-01

    The liver is a central organ in the human body, and first line of defense between host and external environment. Liver response to any external perturbation is a collective reaction of resident liver cells. Most of the current in vitro liver models focus on hepatocytes, the primary metabolic component, omitting interactions and cues from surrounding environment and non-parenchymal cells (NPCs). Recent studies suggest that contributions of NPCs are vital, particularly in disease conditions, and outcomes of drugs and their metabolites. Along with hepatocytes, NPCs–Kupffer (KC), sinusoidal endothelial (LSEC) and stellate cells (SC) are major cellular components of the liver. Incorporation of primary cells in in vitro liver platforms is essential to emulate the functions of the liver, and its overall response. Herein, we isolate individual NPC cell fractions from rat livers and co-culture them in a transwell format incorporating primary rat hepatocytes with LSECs, SCs, and KCs. Our results indicate that the presence and contributions of multiple cells within the co-culture capture the interactions between hepatocytes and NPC, and modulates the responses to inflammatory stimulus such as LPS. The isolation and co-culture methods could provide a stable platform for creating in vitro liver models that provide defined functionality beyond hepatocytes alone. PMID:27142224

  3. Comparative pharmacodynamics of CYP2B induction by DDT, DDE, and DDD in male rat liver and cultured rat hepatocytes.

    PubMed

    Nims, R W; Lubet, R A; Fox, S D; Jones, C R; Thomas, P E; Reddy, A B; Kocarek, T A

    1998-03-27

    In this study the pharmacodynamics were characterized of rat hepatic cytochrome P-450 2B (CYP2B) induction by the pesticide DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane] and its metabolites DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], which is bioretained, and DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane], which is metabolized further and therefore less prone to bioaccumulate. DDT, DDE, and DDD were each found to be pure phenobarbital-type cytochrome P-450 inducers in the male F344/NCr rat, causing induction of hepatic CYP2B and CYP3A, but not CYP1A. The ED50 values for CYP2B induction (benzyloxyresorufin O-dealkylation) by DDT, DDE, and DDD were, respectively, 103, 88, and > or = 620 ppm in diet (14 d of exposure). The efficacies (Emax values) for induction of benzyloxyresorufin O-dealkylation by DDT, DDE, and DDD were 24-, 22-, and > or = 1-fold, respectively, compared to control values. The potencies of the three congeners for CYP2B induction appeared also to be similar, with EC50 values (based on total serum DDT equivalents) of 1.5, 1.8, and > or = 0.51 microM, respectively. The EC50 values based on DDT equivalents in hepatic tissue were 15, 16, and > or = 5.9 micromol/kg liver tissue, respectively. In primary cultures of adult rat hepatocytes, DDT, DDE, and DDD each displayed ability to induce total cellular RNA coding for CYP2B (ED50 values of 0.98, 0.83, and > or = 2.7 microM, respectively). These results suggest that DDT, DDE, and DDD each possess a high degree of intrinsic CYP2B-inducing ability for rat liver, despite marked differences in bioretention among the congeners.

  4. The effect of oleic and palmitic acid on induction of steatosis and cytotoxicity on rat hepatocytes in primary culture.

    PubMed

    Moravcová, A; Červinková, Z; Kučera, O; Mezera, V; Rychtrmoc, D; Lotková, H

    2015-01-01

    In vitro models serve as a tool for studies of steatosis. Palmitic and oleic acids can induce steatosis in cultured hepatocytes. The aim of our study was to verify steatogenic and cytotoxic effects of palmitic acid (PA), oleic acid (OA) and their combinations as well as their impact on functional capacity of rat primary hepatocytes. Hepatocytes were exposed to OA or PA (0.125-2 mmol/l) or their combination at ratios of 3:1, 2:1 or 1:1 at the final concentrations of 0.5-1 mmol/l. Both OA and PA caused a dose-dependent increase in triacylglycerol content in hepatocytes. PA was more steatogenic at 0.25 and 0.5 mmol/l while OA at 0.75 and 1 mmol/l. PA exhibited a dose-dependent cytotoxic effect associated with ROS production, present markers of apoptosis and necrosis and a decrease in albumin production. OA induced a damage of the cytoplasmic membrane from 1 mM concentration. Mixture of OA and PA induced lower cytotoxicity with less weakened functional capacity than did PA alone. Extent of steatosis was comparable to that after exposure to OA alone. In conclusion, OA or combination of OA with PA is more suitable for simulation of simple steatosis than PA alone.

  5. Protective Role of Morin, a Flavonoid, against High Glucose Induced Oxidative Stress Mediated Apoptosis in Primary Rat Hepatocytes

    PubMed Central

    Kapoor, Radhika; Kakkar, Poonam

    2012-01-01

    Apoptosis is an early event of liver damage in diabetes and oxidative stress has been linked to accelerate the apoptosis in hepatocytes. Therefore, the compounds that can scavenge ROS may confer regulatory effects on high-glucose induced apoptosis. In the present study, primary rat hepatocytes were exposed to high concentration (40 mM) of glucose. At this concentration decreased cell viability and enhanced ROS generation was observed. Depleted antioxidant status of hepatocytes under high glucose stress was also observed as evident from transcriptional level and activities of antioxidant enzymes. Further, mitochondrial depolarisation was accompanied by the loss of mitochondrial integrity and altered expression of Bax and Bcl-2. Increased translocation of apoptotic proteins like AIF (Apoptosis inducing factor) & Endo-G (endonuclease-G) from its resident place mitochondria to nucleus was also observed. Cyt-c residing in the inter-membrane space of mitochondria also translocated to cytoplasm. These apoptotic proteins initiated caspase activation, DNA fragmentation, chromatin condensation, increased apoptotic DNA content in glucose treated hepatocytes, suggesting mitochondria mediated apoptotic mode of cell death. Morin, a dietary flavonoid from Psidium guajava was effective in increasing the cell viability and decreasing the ROS level. It maintained mitochondrial integrity, inhibited release of apoptotic proteins from mitochondria, prevented DNA fragmentation, chromatin condensation and hypodiploid DNA upon exposure to high glucose. This study confirms the capacity of dietary flavonoid Morin in regulating apoptosis induced by high glucose via mitochondrial mediated pathway through intervention of oxidative stress. PMID:22899998

  6. Fluorometric assessment of acetaminophen-induced toxicity in rat hepatocyte spheroids seeded on micro-space cell culture plates.

    PubMed

    Sanoh, Seigo; Santoh, Masataka; Takagi, Masashi; Kanayama, Tatsuya; Sugihara, Kazumi; Kotake, Yaichiro; Ejiri, Yoko; Horie, Toru; Kitamura, Shigeyuki; Ohta, Shigeru

    2014-09-01

    Hepatotoxicity induced by the metabolic activation of drugs is a major concern in drug discovery and development. Three-dimensional (3-D) cultures of hepatocyte spheroids may be superior to monolayer cultures for evaluating drug metabolism and toxicity because hepatocytes in spheroids maintain the expression of various metabolizing enzymes and transporters, such as cytochrome P450 (CYP). In this study, we examined the hepatotoxicity due to metabolic activation of acetaminophen (APAP) using fluorescent indicators of cell viability and intracellular levels of glutathione (GSH) in rat hepatocyte spheroids grown on micro-space cell culture plates. The mRNA expression levels of some drug-metabolizing enzymes were maintained during culture. Additionally, this culture system was compatible with microfluorometric imaging under confocal laser scanning microscopy. APAP induced a decrease in intracellular ATP at 10mM, which was blocked by the CYP inhibitor 1-aminobenzotriazole (ABT). APAP (10mM, 24h) decreased the levels of both intracellular ATP and GSH, and GSH-conjugated APAP (APAP-GSH) were formed. All three effects were blocked by ABT, confirming a contribution of APAP metabolic activation by CYP to spheroid toxicity. Fluorometric imaging of hepatocyte spheroids on micro-space cell culture plates may allow the screening of drug-induced hepatotoxicity during pharmaceutical development.

  7. Hepatocyte Lysosomal Membrane Stabilization by Olive Leaves against Chemically Induced Hepatocellular Neoplasia in Rats

    PubMed Central

    Abdel-Hamid, N. M.; El-Moselhy, M. A.; El-Baz, A.

    2011-01-01

    Extensive efforts are exerted looking for safe and effective chemotherapy for hepatocellular carcinoma (HCC). Specific and sensitive early biomarkers for HCC still in query. Present work to study proteolytic activity and lysosomal membrane integrity by hepatocarcinogen, trichloroacetic acid (TCA), in Wistar rats against aqueous olive leaf extract (AOLE).TCA showed neoplastic changes as oval- or irregular-shaped hepatocytes and transformed, vesiculated, and binucleated liver cells. The nuclei were pleomorphic and hyperchromatic. These changes were considerably reduced by AOLE. The results added, probably for the first time, that TCA-induced HCC through disruption of hepatocellular proteolytic enzymes as upregulation of papain, free cathepsin-D and nonsignificant destabilization of lysosomal membrane integrity, a prerequisite for cancer invasion and metastasis. AOLE introduced a promising therapeutic value in liver cancer, mostly through elevating lysosomal membrane integrity. The study substantiated four main points: (1) the usefulness of proteolysis and lysosomalmembrane integrity in early prediction of HCC. (2) TCA carcinogenesis is possibly mediated by lysosomal membrane destabilization, through cathepsin-D disruption, which could be reversed by AOLE administration. (3) A new strategy for management of HCC, using dietary olive leaf system may be a helpful phytotherapeutic trend. (4) A prospective study on serum proteolytic enzyme activity may introduce novel diagnostic tools. PMID:21994869

  8. Statins alter the hepatobiliary transport of unconjugated and conjugated bilirubin in sandwich-cultured rat hepatocytes.

    PubMed

    Szabó, Mónika; Veres, Zsuzsa; Bátai-Konczos, Attila; Kékesi, Orsolya; Kis, Emese; Szabó, Kitti; Jemnitz, Katalin

    2014-09-01

    Several studies have reported that statins occasionally cause impairment of liver functions characterized by elevated serum bilirubin levels, which might be due to altered function of the multidrug resistance-associated proteins (Mrp2/3). We aimed to study the modulation of the hepatobiliary transport of bilirubin by four statin derivatives, atorvastatin, fluvastatin, pravastatin, and rosuvastatin in sandwich-cultured rat hepatocytes. All statins except pravastatin significantly inhibited the uptake of bilirubin. The biliary efflux of bilirubin conjugates was increased by pravastatin and rosuvastatin concentration dependently. Rosuvastatin stimulated not only the Mrp2 mediated biliary, but the Mrp3 mediated sinusoidal elimination, resulting in decreased intracellular bilirubin accumulation. The significantly induced Mrp2/3 protein levels (ranging from 1.5 to 1.8-fold) accounted for the elevated efflux. Cell polarization, the formation of biliary network was also significantly increased by fluvastatin, pravastatin and rosuvastatin (151%, 216% and 275% of the control, respectively). The simultaneous inhibition of the uptake and the stimulation of the sinusoidal and canalicular elimination may explain, at least in part, the clinical observation of elevated serum bilirubin levels. In conclusion, our results suggest that in spite of the elevated serum bilirubin levels, the altered Mrp2 and Mrp3 functions by statins is probably not associated with hepatotoxic effects.

  9. Relationship between autophagy and the intracellular degradation of asialoglycoproteins in cultured rat hepatocytes

    SciTech Connect

    Kindberg, G.M.; Refsnes, M.; Christoffersen, T.; Norum, K.R.; Berg, T.

    1987-05-25

    The relationship between autophagy and the intracellular distribution of endocytosed asialoorosomucoid was studied in cultured rat hepatocytes. Overt autophagy was induced by shifting the cells to a minimal salt medium. Incubation in minimal salt medium led to the formation of buoyant lysosomes at the expense of denser lysosomes manifested as a dual distribution of these organelles in Nycodenz gradients. Asialoorosomucoid was labeled with /sup 125/I-tyramine cellobiose. The labeled degradation products formed from this ligand are trapped at the site of degradation and may therefore serve as markers for the subgroup of lysosomes involved in the degradation. In control cells the degradation of the ligand was initiated in a light prelysosomal compartment and continued in denser lysosomes. In cells with high autophagic activity, the degradation of labeled asialoorosomucoid took place exclusively in a buoyant group of lysosomes. These results suggest that degradation of endocytosed ligand takes place in the same secondary lysosomes as substrate sequestered by autophagic mechanisms. These light lysosomes represent a subgroup of active lysosomes which are gradually recruited from dense bodies. Data are also presented that indicate that insulin may prevent the change in buoyant density brought about by incubation in deficient medium.

  10. Antioxidant effect of a fermented powder of Lady Joy bean in primary rat hepatocytes.

    PubMed

    La Marca, Margherita; Pucci, Laura; Bollini, Roberto; Russo, Rossella; Sparvoli, Francesca; Gabriele, Morena; Longo, Vincenzo

    2015-03-01

    The role and beneficial effects of plant and food extracts against various diseases induced by oxidative stress have received much attention in recent years. Legumes are rich in bioactive compounds, and some studies suggest a correlation between their consumption and a reduced incidence of diseases. Primary cultures of rat hepatocytes were used to investigate whether and how an extract obtained from a fermented powder of bean named Lady Joy (Phaseolus vulgaris L.) is able to regulate antioxidant and detoxifying enzymes through the NRF2 pathway, inhibit NF-kB activation, and reduce H2O2-induced endoplasmic reticulum (ER) stress. All of the antioxidant and detoxifying enzymes studied were significantly up-regulated by Lady Joy treatment. Western blot showed that Nrf2 was activated by Lady Joy treatment. Also, cells treated with this fermented bean were partially protected against NF-kB activation resulting from H2O2 stress. As a link between oxidative stress and ER dysfunction is hypothesized, we verified whether Lady Joy was able to protect cells from H2O2-induced ER stress, by studying the response of the proteins CHOP, BiP and caspase 12. The results of this study show that Lady Joy can induce the Nrf2 pathway, inhibit NF-kB, and protect ER from stress induced by H2O2.

  11. Human circulating monocytes internalize 125I-insulin in a similar fashion to rat hepatocytes: relevance to receptor regulation in target and nontarget tissues.

    PubMed

    Grunberger, G; Robert, A; Carpentier, J L; Dayer, J M; Roth, A; Stevenson, H C; Orci, L; Gorden, P

    1985-08-01

    Circulating monocytes bind 125I-insulin in a specific fashion and have been used to analyze the ambient receptor status in humans. When freshly isolated circulating monocytes are incubated with 125I-insulin and examined by electron microscopic autoradiography, approximately 18% of the labeled material is internalized after 15 minutes at 37 degrees C. By 2 hours at 37 degrees C, approximately one half of the 125I-insulin is internalized. Internalization occurs also at 15 degrees C but at a slower rate. Furthermore, the monocytes bind and internalize 125I-insulin in a manner that mirrors that of major target tissues, such as rat hepatocytes. These data suggest that the insulin receptor of the circulating monocyte might be regulated by adsorptive endocytosis in a manner analogous to that of target tissue, such as the liver.

  12. Expression and release of the latent transforming growth factor beta binding protein by hepatocytes from rat liver.

    PubMed

    Roth, S; Schurek, J; Gressner, A M

    1997-06-01

    In very recent studies it was established that transforming growth factor beta (TGF-beta), likely to be the most relevant fibrogenic cytokine and regulator of cell proliferation, differentiation, and matrix metabolism, is expressed by hepatocytes (parenchymal cell [PC]) and secreted from cultured PC in a latent form incapable of receptor binding. The structural composition of the latent TGF-beta complex secreted by cultured PC is unknown. In some TGF-beta expressing cell types this cytokine is released as a large molecular weight complex containing in addition to the TGF-beta latency associated peptide (LAP) a disulfide bonded latent TGF-beta binding protein (LTBP), of which the existence and function in liver is hitherto unknown. This study is directed to the identification of LTBP expression in rat PC. Cells were isolated from rat liver with the collagenase method and analyzed for LTBP before and during culture under standard conditions using alkaline phosphatase anti-alkaline phosphatase (APAAP) immunostainings, metabolic labeling, messenger RNA (mRNA) detection (reverse-transcription polymerase chain reaction [RT-PCR]) and sequencing, and immunoblotting of gel chromatographically separated cell extracts and conditioned media, respectively. APAAP immunostainings applying a specific polyclonal LTBP-antiserum (ab 39) indicated expression of LTBP in PC of liver in situ and freshly isolated PC but a strong expression in cultured PC. Transcripts of LTBP-1 were detected by RT-PCR and confirmed by sequence analyses. Metabolic labeling of PC with [35S]-Met/Cys followed by immunoprecipitation of cell lysates with LTBP antiserum confirmed the synthesis of the high molecular mass complex of 250 kd containing LTBP with a molecular mass of 160 kd. Latent TGF-beta complexes, associated with LTBP related proteins, could be separated from both extracts and conditioned media of PC by gel filtration chromatography. They confirmed the release of the large latent TGF-beta complex

  13. Effect of Brewing Duration on the Antioxidant and Hepatoprotective Abilities of Tea Phenolic and Alkaloid Compounds in a t-BHP Oxidative Stress-Induced Rat Hepatocyte Model.

    PubMed

    Braud, Laura; Peyre, Ludovic; de Sousa, Georges; Armand, Martine; Rahmani, Roger; Maixent, Jean-Michel

    2015-08-17

    Tea is an interesting source of antioxidants capable of counteracting the oxidative stress implicated in liver diseases. We investigated the impact of antioxidant molecules provided by a mixture of teas' leaves (green, oolong, pu-erh) after different infusion durations in the prevention of oxidative stress in isolated rat hepatocytes, by comparison with pure epigallocatechin-3-gallate (EGCG), the main representative of tea catechins. Dried aqueous tea extracts (ATE) obtained after 5, 15 and 30 min infusion time were characterized for total polyphenols (gallic acid equivalent), catechins, gallic acid and caffeine (HPLC-DAD/ESI-MS) contents, and for scavenging ability against 2,2-diphenyl-1-picrylhydrazyl free radical. Hepatoprotection was evaluated through hepatocyte viability tests using tert-butyl hydroperoxide as a stress inducer, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, neutral red uptake, real-time cellular impedance) and mitochondrial function tests. We showed that a 5-min incubation time is sufficient for an optimal bioaccessibility of tea compounds with the highest antioxidative ability, which decreases for longer durations. A 4-h pretreatment of cells with ATE significantly prevented cell death by regulating reactive oxygen species production and maintaining mitochondrial integrity. Pure EGCG, at doses similar in ATE (5-12 µM), was inefficient, suggesting a plausible synergy of several water-soluble tea compounds to explain the ATE beneficial effects.

  14. Inhibition by an extract of Ocimum sanctum of DNA-binding activity of 7,12-dimethylbenz[a]anthracene in rat hepatocytes in vitro.

    PubMed

    Prashar, R; Kumar, A; Hewer, A; Cole, K J; Davis, W; Phillips, D H

    1998-06-19

    Ocimum sanctum is a traditional medicinal plant. Previous studies have shown that extracts of O. sanctum inhibit the induction of skin papillomas in mice by 7,12-dimethylbenz[a]anthracene (DMBA). In the present study, primary cultures of rat hepatocytes were treated with 0-500 microg of O. sanctum extract for 24 h and then with DMBA (10 or 50 microg) for 18 h. Cells were then harvested and their DNA was isolated and analyzed by 32P-postlabelling. A significant reduction in the levels of DMBA-DNA adducts was observed in all cultures pretreated with O. sanctum extract. This effect was more pronounced at the lower dose of DMBA (10 microg). Hepatocytes which were treated with the highest dose of extract (500 microg) showed a maximum reduction of 93% in the mean values of DMBA-DNA adducts. The viability of the cells was not adversely affected by pretreatment with extract. Our findings suggest that O. sanctum leaf extract blocks or suppresses the events associated with chemical carcinogenesis by inhibiting metabolic activation of the carcinogen.

  15. Enhanced Cell Survival and Yield of Rat Small Hepatocytes by Honeycomb-Patterned Films

    NASA Astrophysics Data System (ADS)

    Tsukiyama, Shusaku; Matsushita, Michiaki; Tanaka, Masaru; Tamura, Hitoshi; Todo, Satoru; Yamamoto, Sadaaki; Shimomura, Masatsugu

    2008-02-01

    Surface designing of substrate to regulate cell adhesion and function in nano and micro scale is a critical issue in biomaterial science. In this study, we describe the fabrication of highly regular patterned porous films (honeycomb-patterned film) formed by a simply casting technique, and the culture of mature hepatocytes and small hepatocytes on the films. The pore size of the honeycomb-patterned films used was 6, 12, and 16 µm. We evaluated the effect of the honeycomb-patterned films on the morphology, cell yield, survival and the differentiated hepatic function (albumin production) of the both hepatocytes. Both hepatocytes attached on the flat films appeared to spread well, showing a typical monolayer morphology. They peeled off from the films at 7 days in culture on the flat films. On the other hand, spreading of the each hepatocytes was restricted on the honeycomb-patterned films at 3 and 7 days in culture. The cell yield and survival of the each hepatocytes increased with increasing culture time. Small hepatocyte on the pore sizes of 16 µm showed the highest cell yield (approximately 3 times). Albumin production of mature hepatocyte on the pore sizes of 16 µm (224.1.3 ±157 ng ml-1 well-1 at 1 day in culture, 369.5 ±222 ng ml-1 well-1 at 3 days in culture) was higher than that of the hepatocytes on the flat films (119.3 ±9.3 ng ml-1 well-1 at 1 day in culture, 262.8 ±47.3 ng ml-1 well-1 at 3 days in culture), although that of small hepatocytes on the honeycomb-patterned films (pore size: 16 µm) was similar on the flat film. These results indicated that both the surface topography and the pore size of the honeycomb-patterned film affected the hepatic metabolic function.

  16. Isolation and cultivation of adult primary bovine hepatocytes from abattoir derived liver

    PubMed Central

    Ehrhardt, Sonja; Schmicke, Marion

    2016-01-01

    The aim was to establish a cell culture of adult primary bovine hepatocytes obtained from liver following slaughter and to cultivate the cells in a sandwich culture. Cells and medium samples were taken after separation of cells (day 0), during monolayer (days 1, 2 and 3) and during sandwich culture (days 1, 2, 3, 4, 7, 10 and 14). The mRNA expression of BAX, BCL2L, FAS, IGF-1 and GHR1A was measured as well as urea and LDH. Hepatocytes were obtained by using a two-step collagenase perfusion and were purified thereafter by density gradient centrifugation. The viability was 68.2 ± 9.5 %. In sandwich culture, cells have a typical polygonal hepatocyte-like shape, build cell-cell contacts, and show irregularity of cell borders suggesting bile canaliculi generation. The BAX mRNA expression increased on day 1 as well but decreased steadily until day 3 and remained constant for 14 days. Urea- and LDH-concentrations increased from day 4 to day 7. In conclusion, we found that it is possible to gather viable primary hepatocytes from adult bovine liver after slaughter, and that cells gathered this way show typical morphologies, urea-production and low LDH-leakage especially at day 4 in a sandwich system. PMID:28275320

  17. Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells.

    PubMed

    Kegel, Victoria; Deharde, Daniela; Pfeiffer, Elisa; Zeilinger, Katrin; Seehofer, Daniel; Damm, Georg

    2016-03-30

    Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the "gold standard" for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine(1). Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique. Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation. Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

  18. In vivo cell lineage analysis during chemical hepatocarcinogenesis in rats using retroviral-mediated gene transfer: evidence for dedifferentiation of mature hepatocytes.

    PubMed

    Gournay, Jérôme; Auvigne, Isabelle; Pichard, Virginie; Ligeza, Catherine; Bralet, Marie-Pierre; Ferry, Nicolas

    2002-06-01

    Feeding adult rats with a diet containing 2-acetylaminofluorene (2-AAF) results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. Although oval cells may be facultative liver stem cells, the actual relationship between oval cells and liver cancer has not been clearly established in vivo. Our goal was to label hepatic cells in vivo using retroviral vectors and follow their fate during the early steps of chemically induced hepatocarcinogenesis. Oval cell proliferation was induced by continuous feeding with a carcinogenic diet containing 2-AAF. We used two different strategies to genetically label hepatic cells: (a) labeling of proliferating cells in rats fed 2-AAF by injecting recombinant retroviral vectors containing the beta-galactosidase gene either in a peripheral vein or in the common bile duct at the peak of oval cell proliferation and (b) prelabeling of hepatocytes by intravenously injecting recombinant vectors 1 day after partial hepatectomy and 1 week before subsequent administration of 2-AAF. Using the first strategy, transgene expression occurred in both oval cells and hepatocytes. Using the second strategy, we could selectively label, and hence study the fate of, differentiated hepatocytes. In the latter case, we observed clusters of beta-galactosidase-positive hepatocytes, some of them also expressing preneoplastic markers such as gamma-glutamyl transpeptidase as well as the placental form of glutathione-S-transferase. These results demonstrate that preneoplastic foci can originate from mature hepatocytes and are consistent with the hypothesis that dedifferentiation of mature hepatocytes may occur during the course of carcinogenic regimen.

  19. Sex and strain differences in the hepatocyte primary culture/DNA repair test

    SciTech Connect

    McQueen, C.A.; Way, B.M. )

    1991-01-01

    The hepatocyte primary culture (HPC)/DNA repair test was developed using hepatocytes isolated from male F-344 rats. A number of genetic polymorphisms have been shown to occur in inbred strains of rats, which may lead to variation in biotransformation of xenobiotics resulting in differences in susceptibility to genotoxins. The effect of the strain utilized as a source of hepatocytes was investigated with female Lewis, F-344, and DA rats. Variation was observed when hepatocytes from the three strains were exposed to aflatoxin B{sub 1} (AFB{sub 1}). No clearcut strain differences were seen when cells were exposed to diethylnitrosamine (DEN) or 2-acetylaminofluorene. These results demonstrate that both the strain and the sex of the animal used as a source of hepatocytes can affect the HPC/DNA repair test.

  20. An in vivo role of Mrp2 in the rat hepatocytes by immunocytochemistry for amoxicillin using the transporter-deficient EHBR.

    PubMed

    Fujiwara, Kunio; Shin, Masashi; Yoshizaki, Yohei; Miyazaki, Tsubasa; Saita, Tetsuya

    2012-06-01

    An in vivo role of the multidrug resistant-associated protein (Mrp2) in rat hepatocytes was examined by immunocytochemistry (ICC) for amoxicillin (AMPC) by the use of the transporter-deficient Eisai hyperbilirubinemic rats (EHBR). The ICC revealed that in the liver of EHBR at 3-h post-administration, amoxicillin accumulated in the cytoplasmic pools and nuclei of the hepatocytes in a characteristic granular morphology on the bile capillaries. However, no amoxicillin was observed on the surface of the lumina ranging from the bile capillaries to the interlobular bile ducts. The drug persisted at least for 6-h after administration. In contrast, in the control rat liver at 3-h post-administration, AMPC-adsorption occurred on such luminal surface, while AMPC accumulated to a less level in both the cytoplasm and nuclei of the hepatocytes. The drug completely disappeared in the hepatocytes at 6-h post-administration. These results strongly suggest that AMPC taken up into the cytoplasm of the hepatocytes excretes via Mrp2 into the bile flow. Furthermore, electron microscopy demonstrated that the lower electron density areas in large sizes, corresponding to the cytoplasmic pools in ICC for AMPC, occurred in the cytoplasm peripheral to the nuclei of the hepatocytes in EHBR at 3-h post-administration, and then disappeared 24 h after administration.

  1. Hepatocyte growth factor attenuates pancreatic damage in caerulein-induced pancreatitis in rats.

    PubMed

    Warzecha, Z; Dembiński, A; Konturek, P C; Ceranowicz, P; Konturek, S J; Tomaszewska, R; Schuppan, D; Stachura, J; Nakamura, T

    2001-10-26

    Hepatocyte growth factor (HGF) overexpression was reported in experimental and clinical acute pancreatitis. These observations prompted us to determine the effect of HGF administration on the development of caerulein-induced pancreatitis in rats. Acute pancreatitis was induced by s.c. infusion of caerulein (10 microg/kg/h) for 5 h. HGF was administrated twice (30 min before caerulein or saline infusion and 3 h later) at the doses: 0.4, 2, 10 or 50 microg/kg s.c. Immediately after cessation of caerulein or saline infusion, the pancreatic blood flow, plasma amylase and lipase activity, plasma cytokines concentration, cell proliferation, and morphological signs of pancreatitis were examined. Caerulein administration induced acute edematous pancreatitis manifested by 41% decrease in DNA synthesis, 53% inhibition of pancreatic blood flow, a significant increase in plasma amylase and lipase activity, plasma interleukin-1beta and interleukin-6 concentration, as well as, the development of the histological signs of pancreatic damage (edema, leukocyte infiltration, and vacuolization). Administration of HGF without induction of pancreatitis increased plasma interleukin-10. Treatment with HGF, during induction of pancreatitis, increased plasma interleukin-10 and attenuated the pancreatic damage, what was manifested by histological improvement of pancreatic integrity, the partial reversion of the drop in DNA synthesis and pancreatic blood flow, and the reduction in pancreatitis evoked increase in plasma amylase, lipase, and interleukin-1beta and interleukin-6 levels. HGF administrated at the dose 2 microg/kg exhibited a similar beneficial effect as administration of HGF at the doses 10 or 50 microg/kg. Treatment with HGF at the dose 0.4 microg/kg was less effective. We conclude that: (1) administration of HGF attenuates pancreatic damage in caerulein-induced pancreatitis; (2) this effect seems to be related to the increase in production of interleukin-10, the reduction in

  2. Toxicity of green tea extracts and their constituents in rat hepatocytes in primary culture.

    PubMed

    Schmidt, M; Schmitz, H-J; Baumgart, A; Guédon, D; Netsch, M I; Kreuter, M-H; Schmidlin, C B; Schrenk, D

    2005-02-01

    Recent reports on sporadic cases of liver disorders (acute hepatitis, icterus, hepatocellular necrosis) after ingestion of dietary supplements based on hydro-alcoholic extracts from green tea leaves led to restrictions of the marketing of such products in certain countries of the EU. Since green tea is considered to exert a number of beneficial health effects, and, therefore, green tea products are widely used as dietary supplements, we were interested in the possible mechanism of hepatotoxicity of green tea extracts and in the components involved in such effects. Seven hours after seeding on collagen, rat hepatocytes in primary culture were treated with various hydro-alcoholic green tea extracts (two different native 80% ethanolic dry extracts and an 80% ethanolic dry extract cleared from lipophilic compounds). Cells were washed, and reduction of resazurin, used as a viability parameter monitoring intact mitochondrial function, was determined. It was found that all seven green tea extracts examined enhanced resazurin reduction significantly at a concentration range of 100-500 microg/ml medium, while a significant decrease was observed at 1-3mg/ml medium. Decreased levels were concomitant with abundant necrosis as observed by microscopic inspection of the cultures and with increased leakage of lactate dehydrogenase activity from the cells. In a separate series of experiments, the green tea constituents (-)-epicatechin, (-)-epigallocatechin-3-gallate, caffeine and theanine were tested at concentrations reflecting their levels in a typical green tea extract. Synthetic (+)-epigallocatechin (200 microM) was used for comparison. Cytotoxicity was found with (-)-epigallocatechin-3-gallate only. The concomitant addition of 0.25 mM ascorbate/0.05 mM alpha-tocopherol had no influence on cytotoxicity. In conclusion, our results suggest that high concentrations of green tea extract can exert acute toxicity in rat liver cells. (-)-Epigallocatechin-3-gallate seems to be a key

  3. Amylin impairment of insulin effects on glycogen synthesis and phosphoenolpyruvate carboxykinase gene expression in rat primary cultured hepatocytes.

    PubMed Central

    Baqué, S; Guinovart, J J; Gómez-Foix, A M

    1994-01-01

    The ability of amylin to impair hepatic insulin action is controversial. We have found that the effect of amylin in primary cultured hepatocytes is strongly dependent on the culture conditions. Only in hepatocytes preincubated in the presence of fetal serum did amylin, at concentrations ranging from 1 to 100 nM, reduce insulin-stimulated glycogen synthesis rate and glycogen accumulation without showing direct effects. Neither basal glycogen synthase nor glycogen phosphorylase activity was modified by amylin treatment. Nevertheless, amylin (100 nM) blocked the activation of glycogen synthase by insulin. Amylin also proved capable of opposing the reduction in the expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene induced by insulin, whereas the basal mRNA level of PEPCK was unaffected by amylin treatment. Thus, these results show that, in cultured rat hepatocytes, amylin is indeed able to interfere with insulin regulation of glycogenesis and PEPCK gene expression, favouring the hypothesis that amylin may modulate liver sensitivity to insulin. Images Figure 3 PMID:7998979

  4. Effects of Aflatoxin B1 and Fumonisin B1 on the Viability and Induction of Apoptosis in Rat Primary Hepatocytes

    PubMed Central

    Ribeiro, Deise H. B.; Ferreira, Fabiane L.; da Silva, Valéria N.; Aquino, Simone; Corrêa, Benedito

    2010-01-01

    The present study evaluated the effect of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) either alone, or in association, on rat primary hepatocyte cultures. Cell viability was assessed by flow cytometry after propidium iodine intercalation. DNA fragmentation and apoptosis were assessed by agarose gel electrophoresis and acridine orange and ethidium bromide staining. At the concentrations of AFB1 and FB1 used, the toxins did not decrease cell viability, but did induce apoptosis in a concentration and time-dependent manner. PMID:20480051

  5. Tryptophan pyrolysis products, Trp-P-1 and Trp-P-2 induce apoptosis in primary cultured rat hepatocytes.

    PubMed

    Ashida, H; Shiotani, B; Adachi, H; Hashimoto, T; Kanazawa, K; Danno, G

    1998-11-01

    The cytotoxicity of heterocyclic amines, dietary carcinogens derived from cooked foods, to primary cultured rat hepatocytes was studied. A tryptophan pyrolysis product, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was the most cytotoxic of 11 compounds tested. Trp-P-1 was found to induce apoptosis as measured by morphological changes in nuclear chromatin and internucleosomal DNA fragmentation. 3-Amino-1-methyl-5H-pyrido[4,3-b] indole (Trp-P-2) showed a moderate apoptotic effect, and other compounds had a similar but weaker effect.

  6. Electron microscopic identification of putative liver stem cells and intermediate hepatocytes following periportal necrosis induced in rats by allyl alcohol.

    PubMed

    Sell, S

    1997-01-01

    The ultrastructural characteristics of the putative liver stem cells that repopulate the necrotic periportal zones after allyl alcohol induced liver injury are described. Periportal liver cell necrosis was induced in adult female Sprague-Dawley rats by i.p. injection with 0.62 mmol/kg of allyl alcohol. Electron microscopic examination of the livers was carried out at 33, 57, 81 and 129 h after injection. After periportal necrosis small nondescript intraportal cells (putative liver stem cells) as well as three type of "progenitor" cells are seen: type I, immature "precursor" cells; type II, bile duct-like; and type III, hepatocyte-like, with numerous cells of intermediate type between type I and type III. The periportal necrotic zone (zone I) is reconstituted largely by an increase in hepatocyte-like cells containing mitochondria, lysosomes, lipid-filled vacuoles, rare peroxisomes, prominent endoplasmic reticulum and lateral microvilli (type III cells) with a relatively small number of type I (immature) cells participating. The type III cells display different degrees of differentiation; the less mature are termed "restitutive" and the more mature "transitional" hepatocytes to emphasize the probable relationship between these cell types. Immature ductular cells (type II cells) are seen located basally within hyperplastic ducts in the periportal zone. It is postulated that hepatocyte restitution after periportal necrosis is accomplished by proliferation and differentiation of stem cells with both biliary and hepatic potential that specifically differentiate into hepatic cells through "restitutive" and "transitional" intermediates. These postulated liver stem cells may be intraportal cells seen 33-57 h after injury that precede the type I and type III hepatic precursors seen later.

  7. The involvement of the docking protein Gab1 in mitogenic signalling induced by EGF and HGF in rat hepatocytes.

    PubMed

    Aasrum, Monica; Odegård, John; Sandnes, Dagny; Christoffersen, Thoralf

    2013-12-01

    Grb2-associated binder (Gab) family proteins are docking molecules that can interact with receptor tyrosine kinases (RTKs) and cytokine receptors and bind several downstream signalling proteins. Studies in several cell types have shown that Gab1 may have a role in signalling mediated by the two RTKs epidermal growth factor (EGF) receptor (EGFR) and Met, the receptor for hepatocyte growth factor (HGF), but the involvement of Gab1 in EGFR and Met signalling has not been directly compared in the same cell. We have studied mechanisms of activation and role in mitogenic signalling of Gab1 in response to EGF and HGF in cultured rat hepatocytes. Gab1, but not Gab2, was expressed in the hepatocytes and was phosphorylated upon stimulation with EGF or HGF. Depletion of Gab1, using siRNA, decreased the ERK and Akt activation, cyclin D1 expression, and DNA synthesis in response to both EGF and HGF. Studies of mechanisms of recruitment to the receptors showed that HGF induced co-precipitation of Gab1 and Met while EGF induced binding of Gab1 to Grb2 but not to EGFR. Gab1 activation in response to both EGF and HGF was dependent on PI3K. While EGF activated Gab1 and Shc equally, within the same concentration range, HGF very potently and almost exclusively activated Gab1, having only a minimal effect on Shc. Collectively, our results strongly suggest that although Gab1 interacts differently with EGFR and Met, it is involved in mitogenic signalling mediated by both these growth factor receptors in hepatocytes.

  8. Hepatocyte membrane water permeability measured by silicone layer filtering centrifugation.

    PubMed

    Gradilone, Sergio A; Ochoa, J Elena; García, Fabiana; Larocca, M Cecilia; Pellegrino, José M; Marinelli, Raúl A

    2002-03-01

    We previously found that hepatocytes are able to control their osmotic membrane water permeability (P(f)) by regulating the number of surface aquaporin water channels. Hepatocyte P(f) has been assessed by phase-contrast microscopy and cell image analysis, an established but relatively laborious procedure. We report here an alternative method to assess hepatocyte P(f) based on a single silicone layer filtering centrifugation system. Isolated rat hepatocytes were incubated in hypotonic or isotonic buffers containing (3)H(2)O as a tracer and, then, were filtered by rapid centrifugation through a silicone layer down to a lysis layer. Osmotically driven radioactivity (i.e., (3)H(2)O) within hepatocytes was calculated as the difference between the dpm in lysis media measured under hypotonic and isotonic conditions. The P(f) calculated from the initial slope of the radioactivity-versus-time curve was 18 microm/s at 4 degrees C. Hepatocytes treated with dibutyryl cyclic AMP, to increase P(f) through the plasma membrane insertion of aquaporins, showed an increased P(f) value of 37 microm/s. The aquaporin blocker dimethyl sulfoxide selectively prevented the agonist-induced hepatocyte P(f). These data are in good agreement with the corresponding values determined by quantitative phase-contrast microscopy; thus, the method developed allows the rapid and reliable measurement of hepatocyte P(f).

  9. Protective Effect of Aframomum melegueta phenolics Against CCl4-Induced Rat Hepatocytes Damage; Role of Apoptosis and Pro-inflammatory Cytokines inhibition

    PubMed Central

    El-Halawany, Ali M.; Dine, Riham Salah El; El Sayed, Nesrine S.; Hattori, Masao

    2014-01-01

    Aframomum melegueta is a commonly used African spice. Through a hepatoprotective bioassay-guided isolation, the chloroform fraction of A.melegueta seeds yielded one new diarylheptanoid named 3-(S)-acetyl-1-(4′-hydroxy-3′, 5′-di methoxyphenyl)-7-(3″,4″, 5″-trihydroxyphenyl)heptane (1), and two new hydroxyphenylalkanones, [8]-dehydrogingerdione (2) and [6]-dehydroparadol (3), in addition to six known compounds (4–9). The hepatoprotective effect of A. melegueta methanol extract, sub-fractions and isolated compounds was investigated using carbon tetrachloride (CCl4)-induced liver injury in a rat hepatocytes model. The methanol, chloroform extracts and compounds 1, 5, 8 and 9 of A. melegueta significantly inhibited the elevated serum alanine aminotransferase (ALT), thiobarbituric acid reactive substances (TBARS), tumor necrosis factor (TNFα), interleukin-1beta (Il-1β), caspase3 and 9 and enhanced the reduced liver glutathione (GSH) level caused by CCl4 intoxication. These results indicate that A.melegueta extracts, and isolated compounds play a protective role in CCl4 induced acute liver injury which might be due to elevated antioxidative defense potentials, suppressed inflammatory responses and apoptosis of liver tissue. PMID:25077538

  10. Regulation of cytochrome P450 expression by inhibitors of hydroxymethylglutaryl-coenzyme A reductase in primary cultured rat hepatocytes and in rat liver.

    PubMed

    Kocarek, T A; Reddy, A B

    1996-11-01

    It was previously demonstrated that treatment of primary cultured rat hepatocytes with lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, induced the mRNAs for several cytochromes P450 (P450s), including CYP2B1/2, CYP3A1/2, and CYP4A. In this study, we have compared the effects of lovastatin with those of three additional HMG-CoA reductase inhibitors (simvastatin, pravastatin, and the structurally dissimilar drug fluvastatin) on P450 expression in primary cultured rat hepatocytes, and we have also characterized the effects of in vivo treatment with fluvastatin on P450 expression in rat liver. Treatment of cultured hepatocytes with lovastatin, simvastatin, or fluvastatin increased CYP2B1/2, CYP3A1/2, and CYP4A mRNA and immunoreactive protein levels over the dose range (3 x 10(-6) to 3 x 10(-5) M) required to increase the amount of HMG-CoA reductase mRNA. The increases in CYP2B1/2 levels produced by 3 x 10(-5) M fluvastatin treatment were larger than those produced by lovastatin or simvastatin treatment or by treatment with 10(-4) M phenobarbital. In contrast, treatment of cultured hepatocytes with 3 x 10(-5) M lovastatin, simvastatin, or fluvastatin increased CYP3A1/2 and CYP4A mRNA and immunoreactive protein to lower levels than those produced by treatment with 10(-5) M dexamethasone or 10(-4) M ciprofibrate. Treatment of cultured hepatocytes with pravastatin had little or no effect on the amount of any of the P450s examined, although this drug induced HMG-CoA reductase mRNA as effectively as did fluvastatin. Incubation of hepatocytes with 10(-4) M fluvastatin increased CYP1A1 mRNA to 67% of the level induced by treatment with 10(-5) M beta-naphthoflavone. Doses of 50 or 100 mg/ kg/day fluvastatin administered for 3 days to rats increased the hepatic levels of CYP2B1/2 and CYP4A mRNA and immunoreactive protein, although to much lower levels than those produced by treatment with phenobarbital or ciprofibrate, respectively. Treatment of

  11. In vitro biocompatibility of polypyrrole/PLGA conductive nanofiber scaffold with cultured rat hepatocytes

    NASA Astrophysics Data System (ADS)

    Chu, Xue-Hui; Xu, Qian; Feng, Zhang-Qi; Xiao, Jiang-Qiang; Li, Qiang; Sun, Xi-Tai; Cao, Yang; Ding, Yi-Tao

    2014-09-01

    To intruduce conductive biomaterial into liver tissue engineering, a conductive nanofiber scaffold, polypyrrole/poly(lactic-co-glycolic)acid(PLGA), was designed and prepared via electro-spinning and oxidative polymerization. Effects of the scaffold on hepatocyte adhesion, viability and function were then investigated. SEM revealed pseudopodium formation and abundant extracellular matrix on the surface of PLGA membrane and polypyrrole/PLGA membrane. The adhesion rate, cellular activity, urea synthesis and albumin secretion of the hepatocytes cultured on polypyrrole/PLGA group were similar to those on the PLGA group, but were significantly higher than those on the control group. There were no significant differences in concentrations of LDH and TNF-α among three groups. These results suggested the potential application of this conductive nanofiber scaffold as a suitable substratum for hepatocyte culturing in liver tissue engineering.

  12. Stimulation of fatty acid oxidation by a 3-thia fatty acid reduces triacylglycerol secretion in cultured rat hepatocytes.

    PubMed

    Skrede, S; Bremer, J; Berge, R K; Rustan, A C

    1994-08-01

    The present work shows that when mitochondrial beta-oxidation is stimulated by the hypolipemic, non-beta-oxidizable fatty acid analogue tetradecylthioacetic acid, there is a decrease in the secretion of triacylglycerol in cultured rat hepatocytes. In order to study the effects of tetradecylthioacetic acid in cells with different fatty acid oxidation rates, cells were grown without or with L-carnitine supplement or with addition of the beta-oxidation inhibitor L-aminocarnitine. In cells grown without and with L-carnitine in the medium, the oxidation of [1-14C]oleic acid was stimulated by tetradecylthioacetic acid, whereas it was not significantly changed by palmitic acid. In cells grown with L-aminocarnitine, oxidation of [1-14C]oleic acid was almost abolished both in the absence and in presence of tetradecylthioacetic acid. The effect of tetradecylthioacetic acid and palmitic acid on incorporation of [1-14C]oleic acid into triacylglycerol was similar under all conditions. In the presence of L-carnitine, secretion of oleic acid-labeled triacylglycerol was reduced significantly more by tetradecylthioacetic acid than by palmitic acid. The effects of tetradecylthioacetic acid and palmitic acid on secretion of oleic acid-labeled triacylglycerol were reversed in cells grown with L-aminocarnitine, where palmitic acid was the stronger inhibitor. These results were substantiated by determination of mass of triacylglycerol secreted. It is concluded that tetradecylthioacetic acid reduces secretion of triacylglycerol from rat hepatocytes mainly by acutely stimulating fatty acid oxidation.

  13. Determination of the Mutagenicity Potential of Supermint Herbal Medicine by Single Cell Gel Electrophoresis in Rat Hepatocytes

    PubMed Central

    Kalantari, Heibatullah; Rezaei, Mohsen; Mahdavinia, Masoud; Kalantar, Mojtaba; Amanpour, Zivar; varnaseri, golnaz

    2012-01-01

    Purpose: The increasing use of herbal drugs and their easy availability have necessitated the use of mutagenicity test to analyze their toxicity and safety. The aim of this study was to evaluate the genotoxicity of Supermint herbal medicine in DNA breakage of rat hepatocytes in comparison with sodium dichromate by single cell gel electrophoresis technique or comet assay. Methods: Hepatocytes were prepared from male wistar rats and were counted and kept in a bioreactor for 30 minutes. Then cells were exposed to the Supermint herbal medicine at doses of 125, 250 and 500 µl/ml. Buffer 4 (incubation buffer) and sodium dichromate were used as negative and positive control for one hour respectively. Then cell suspension with low melting point agarose were put on precoated slides and covered with agarose gel. Then lysing, electrophoresis, neutralization and staining were carried out. Finally the slides were analyzed with fluorescence microscope. The parameter under this analysis was the type of migration which was determined according to Kobayashi pattern. Results: With increased dose of Supermint herbal medicine the DNA damage was slightly increased (P<0001). Conlusion: In overall compared to the positive control significant differences is observed which convinced that the crude extract of Supermint in vitro did not have mutagenic effect. Conlusion: In overall compared to the positive control significant differences is observed which convinced that the crude extract of Supermint in vitro did not have mutagenic effect. PMID:24312800

  14. Initial heme uptake from albumin by short-term cultured rat hepatocytes is mediated by a transport mechanism differing from that of other organic anions.

    PubMed

    Noyer, C M; Immenschuh, S; Liem, H H; Muller-Eberhard, U; Wolkoff, A W

    1998-07-01

    Although it is known that circulating heme accumulates in liver cells, the process by which heme enters hepatocytes is only partly understood. Hemopexin and a putative hemopexin receptor on hepatocyte membranes may mediate the uptake process. However, whether there are sufficient hemopexin receptors on rat hepatocytes to account for the bulk of heme entering cells is unknown. It is likely that heme may be transferred directly from albumin with the help of a plasma membrane heme transporter. To clarify the transport mechanism of heme into liver cells, we studied the uptake by short-term cultured rat hepatocytes of 55Fe-heme incubated with rat serum albumin. In these cells, the initial uptake of 55Fe-heme at 37 degrees C was five- to eightfold higher than that at 4 degrees C, linear for at least 5 minutes, and saturable. The Km of heme uptake was 0.95 +/- 0.27 micromol/L, and the Vmax was 0.12 +/- 0.01 pmol/min/mg protein (n = 3). Neither isosmotic substitution of sucrose for NaCl in the medium nor adenosine triphosphate (ATP) depletion, perturbations that are known to reduce uptake of bilirubin, sulfobromophthalein (BSP), and taurocholate, had any influence on 55Fe-heme uptake. In addition, heme uptake was not reduced in the presence of a greater than 500-fold molar excess of BSP. These results indicate that hepatocytes take up heme by a process that is distinct from that of these other organic anions.

  15. Development of an in vitro high content imaging assay for quantitative assessment of CAR-dependent mouse, rat, and human primary hepatocyte proliferation.

    PubMed

    Soldatow, Valerie; Peffer, Richard C; Trask, O Joseph; Cowie, David E; Andersen, Melvin E; LeCluyse, Edward; Deisenroth, Chad

    2016-10-01

    Rodent liver tumors promoted by constitutive androstane receptor (CAR) activation are known to be mediated by key events that include CAR-dependent gene expression and hepatocellular proliferation. Here, an in vitro high content imaging based assay was developed for quantitative assessment of nascent DNA synthesis in primary hepatocyte cultures from mouse, rat, and human species. Detection of DNA synthesis was performed using direct DNA labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU). The assay was multiplexed to enable direct quantitation of DNA synthesis, cytotoxicity, and cell count endpoints. An optimized defined medium cocktail was developed to sensitize hepatocytes to cell cycle progression. The baseline EdU response to defined medium was greatest for mouse, followed by rat, and then human. Hepatocytes from all three species demonstrated CAR activation in response to the CAR agonists TCPOBOP, CITCO, and phenobarbital based on increased gene expression for Cyp2b isoforms. When evaluated for a proliferation phenotype, TCPOBOP and CITCO exhibited significant dose-dependent increases in frequency of EdU labeling in mouse and rat hepatocytes that was not observed in hepatocytes from three human donors. The observed species differences are consistent with CAR activators inducing a proliferative response in rodents, a key event in the liver tumor mode of action that is not observed in humans.

  16. Regulation of Bcl-2 and Bcl-xL anti-apoptotic protein expression by nuclear receptor PXR in primary cultures of human and rat hepatocytes.

    PubMed

    Zucchini, Nathalie; de Sousa, Georges; Bailly-Maitre, Béatrice; Gugenheim, Jean; Bars, Rémi; Lemaire, Géraldine; Rahmani, Roger

    2005-08-15

    The pregnane X receptor (PXR) plays a major role in the protection of the body by regulating the genes involved in the metabolism and elimination of potentially toxic xeno- and endobiotics. We previously described that PXR activator dexamethasone protects hepatocytes from spontaneous apoptosis. We hypothesise a PXR-dependent co-regulation process between detoxication and programmed cell death. Using primary cultured human and rat hepatocytes, we investigated to determine if PXR is implicated in the regulation of Bcl-2 and Bcl-xL, two crucial apoptosis inhibitors. In the present study we demonstrated that the treatment of primary cultured hepatocytes with PXR agonists increased hepatocyte viability and protects them from staurosporine-induced apoptosis. The anti-apoptotic capacity of PXR activation was correlated with Bcl-2 and Bcl-xL induction at both the transcriptional and protein levels in man and rats, respectively. The inhibition of PXR expression by antisense oligonucleotide abolished PXR activators Bcl-xL induction. Accordingly, PXR overexpression in HepG2 cells led to bcl-2 induction upon clotrimazole treatment and protects cells against Fas-induced apoptosis. Our results demonstrate that PXR expression is required for Bcl-2 and Bcl-xL up-regulation upon PXR activators treatment in human and rat hepatocytes. They also suggest that PXR may protect the liver against chemicals by simultaneously regulating detoxication and the apoptotic pathway.

  17. Receptor binding and cell-mediated metabolism of (/sup 125/I)monoiodoglucagon by isolated canine hepatocytes

    SciTech Connect

    Hagopian, W.A.; Tager, H.S.

    1984-07-25

    A reverse-phase HPLC method has been developed to purify /sup 125/I-labeled products resulting from the chloramine-T-based iodination of glucagon. In addition the products ((/sup 125/I)iodoTyr/sup 10/ /sup 13/)glucagon, ((/sup 125/I)iodoTyr/sup 13/)glucagon, and ((/sup 125/I)iodoTyr/sup 10/)glucagon) have been used to study the receptor binding of glucagon and the cell-mediated metabolism of the hormone by isolated canine hepatocytes. It was concluded that (a) not withstanding apparent differences in affinities exhibited by the three peptides, the interactions with the glucagon receptor are functionally equivalent, and (b) the cell-mediated metabolism of receptor-bound glucagon involves the formation of hormone-derived peptides in which the biologically important NH/sub 2/-terminal region of the hormone has been modified by limited proteolytic cleavage.

  18. Liver Progenitors Isolated from Adult Healthy Mouse Liver Efficiently Differentiate to Functional Hepatocytes In Vitro and Repopulate Liver Tissue.

    PubMed

    Tanimizu, Naoki; Ichinohe, Norihisa; Ishii, Masayuki; Kino, Junichi; Mizuguchi, Toru; Hirata, Koichi; Mitaka, Toshihiro

    2016-12-01

    It has been proposed that tissue stem cells supply multiple epithelial cells in mature tissues and organs. However, it is unclear whether tissue stem cells generally contribute to cellular turnover in normal healthy organs. Here, we show that liver progenitors distinct from bipotent liver stem/progenitor cells (LPCs) persistently exist in mouse livers and potentially contribute to tissue maintenance. We found that, in addition to LPCs isolated as EpCAM(+) cells, liver progenitors were enriched in CD45(-) TER119(-) CD31(-) EpCAM(-) ICAM-1(+) fraction isolated from late-fetal and postnatal livers. ICAM-1(+) liver progenitors were abundant by 4 weeks (4W) after birth. Although their number decreased with age, ICAM-1(+) liver progenitors existed in livers beyond that stage. We established liver progenitor clones derived from ICAM-1(+) cells between 1 and 20W and found that those clones efficiently differentiated into mature hepatocytes (MHs), which secreted albumin, eliminated ammonium ion, stored glycogen, and showed cytochrome P450 activity. Even after long-term culture, those clones kept potential to differentiate to MHs. When ICAM-1(+) clones were transplanted into nude mice after retrorsine treatment and 70% partial hepatectomy, donor cells were incorporated into liver plates and expressed hepatocyte nuclear factor 4α, CCAAT/enhancer binding protein α, and carbamoylphosphate synthetase I. Moreover, after short-term treatment with oncostatin M, ICAM-1(+) clones could efficiently repopulate the recipient liver tissues. Our results indicate that liver progenitors that can efficiently differentiate to MHs exist in normal adult livers. Those liver progenitors could be an important source of new MHs for tissue maintenance and repair in vivo, and for regenerative medicine ex vivo. Stem Cells 2016;34:2889-2901.

  19. Cold preservation of isolated hepatocytes in UW solution: experimental studies on the respiratory activity at 0 degrees C.

    PubMed

    Llarrull, Maria Soledad; Pizarro, Maria Dolores; Scandizzi, Angel L; Bottai, Hebe; Guibert, Edgardo E; Rodriguez, Joaquin V

    2007-01-01

    To date, little attention has been paid to the role of the gas milieu in preservation solutions and its effect on cell viability. Dissolved O2 in the preservation media may be an important parameter to consider. In this study we polarographically measured the O2 concentration in air-equilibrated UW solution at 0 degrees C, as well as the respiratory activity of isolated hepatocytes cold-preserved in this solution up to 72 hours. To perform measurements at 0 degrees C, it was first necessary to characterize the sensor behavior at low temperatures. We verified that the sensor response is still linear at this temperature but the rate of response is significantly slower. The O2 solubility in UW-air solution at 0 degrees C was determined using a modified physical method and it was 410 microM O2, which, as expected, is lower than the solubility in water at the same temperature (453 microM O2). Isolated hepatocytes cold-stored in UW-air solution retained a measurable respiratory activity during a period of 72 hours. The O2 consumption rate was 0.48 +/- 0.13 nmol/O2/min/10(6) cells, which represents 1% of the control value at 36 degrees C (61.46 +/- 14.61 nmol/O2/min/10(6) cells). The respiratory activity and cell viability were well maintained during the preservation period. At present, preservation conditions need to be improved for cells to remain functionally active. Dissolved O2 may be required for energy re-synthesis but it also leads to an increment in reactive oxygen species. The O2 concentration in the preservation solution should be carefully controlled, reaching a compromise between cell requirement and toxicity.

  20. Physiological oxygen tensions modulate expression of the mdr1b multidrug-resistance gene in primary rat hepatocyte cultures.

    PubMed Central

    Hirsch-Ernst, K I; Kietzmann, T; Ziemann, C; Jungermann, K; Kahl, G F

    2000-01-01

    P-Glycoprotein transporters encoded by mdr1 (multidrug resistance) genes mediate extrusion of an array of lipophilic xenobiotics from the cell. In rat liver, mdr transcripts have been shown to be expressed mainly in hepatocytes of the periportal region. Since gradients in oxygen tension (pO(2)) may contribute towards zonated gene expression, the influence of arterial and venous pO(2) on mRNA expression of the mdr1b isoform was examined in primary rat hepatocytes cultured for up to 3 days. Maximal mdr1b mRNA levels (100%) were observed under arterial pO(2) after 72 h, whereas less than half-maximal mRNA levels (40%) were attained under venous pO(2). Accordingly, expression of mdr protein and extrusion of the mdr1 substrate rhodamine 123 were maximal under arterial pO(2) and reduced under venous pO(2). Oxygen-dependent modulation of mdr1b mRNA expression was prevented by actinomycin D, indicating transcriptional regulation. Inhibition of haem synthesis by 25 microM CoCl(2) blocked mdr1b mRNA expression under both oxygen tensions, whereas 80 microM desferrioxamine abolished modulation by O(2). Haem (10 microM) increased mdr1b mRNA levels under arterial and venous pO(2). In hepatocytes treated with 50 microM H(2)O(2), mdr1b mRNA expression was elevated by about 1.6-fold at venous pO(2) and 1.5-fold at arterial pO(2). These results support the conclusion that haem proteins are crucial for modulation of mdr1b mRNA expression by O(2) in hepatocyte cultures and that reactive oxygen species may participate in O(2)-dependent signal transduction. Furthermore, the present study suggests that oxygen might be a critical modulator for zonated secretion of mdr1 substrates into the bile. PMID:10947958

  1. Lack of formic acid production in rat hepatocytes and human renal proximal tubule cells exposed to chloral hydrate or trichloroacetic acid.

    PubMed

    Lock, Edward A; Reed, Celia J; McMillan, Joellyn M; Oatis, John E; Schnellmann, Rick G

    2007-02-12

    The industrial solvent trichloroethylene (TCE) and its major metabolites have been shown to cause formic aciduria in male rats. We have examined whether chloral hydrate (CH) and trichloroacetic acid (TCA), known metabolites of TCE, produce an increase in formic acid in vitro in cultures of rat hepatocytes or human renal proximal tubule cells (HRPTC). The metabolism and cytotoxicity of CH was also examined to establish that the cells were metabolically active and not compromised by toxicity. Rat hepatocytes and HRPTC were cultured in serum-free medium and then treated with 0.3-3mM CH for 3 days or 0.03-3mM CH for 10 days, respectively and formic acid production, metabolism to trichloroethanol (TCE-OH) and TCA and cytotoxicity determined. No increase in formic acid production in rat hepatocytes or HRPTC exposed to CH was observed over and above that due to chemical degradation, neither was formic acid production observed in rat hepatocytes exposed to TCA. HRPTC metabolized CH to TCE-OH and TCA with a 12-fold greater capacity to form TCE-OH versus TCA. Rat hepatocytes exhibited a 1.6-fold and three-fold greater capacity than HRPTC to form TCE-OH and TCA, respectively. CH and TCA were not cytotoxic to rat hepatocytes at concentrations up to 3mM/day for 3 days. With HRPTC, one sample showed no cytotoxicity to CH at concentrations up to 3mM/day for 10 days, while in another cytotoxicity was seen at 1mM/day for 3 days. In summary, increased formic acid production was not observed in rat hepatocytes or HRPTC exposed to TCE metabolites, suggesting that the in vivo response cannot be modelled in vitro. CH was toxic to HRPTC at millimolar concentrations/day over 10 days, while glutathione derived metabolites of TCE were toxic at micromolar concentrations/day over 10 days [Lock, E.A., Reed, C.J., 2006. Trichloroethylene: mechanisms of renal toxicity and renal cancer and relevance to risk assessment. Toxicol. Sci. 19, 313-331] supporting the view that glutathione derived

  2. GENE EXPRESSION ALTERATIONS OBSERVED IN PRIMARY CULTURED RAT HEPATOCYTES AFTER TREATMENT WITH CHLORINATED OR CHLORINATED AND OZONATED DRINKING WATER FROM EAST FORK LAKE, OHIO

    EPA Science Inventory

    Drinking water from East Fork Lake was spiked with iodide and bromide, disinfected with chlorine or ozone + chlorine, concentrated ~100-fold using reverse osmosis, and volatile disinfection by-products (DBPs) added back. Primary rat hepatocytes were exposed to full-strength, 1:10...

  3. Cytotoxicity of luteolin in primary rat hepatocytes: the role of CYP3A-mediated ortho-benzoquinone metabolite formation and glutathione depletion.

    PubMed

    Shi, Fuguo; Zhao, Peng; Li, Xiaobing; Pan, Hong; Ma, Shiping; Ding, Li

    2015-11-01

    Luteolin (LUT), an active ingredient in traditional Chinese medicines and an integral part of the human diet, has shown promising pharmacological activities with a great potential for clinical use. The purpose of this study was to evaluate the role of cytochrome P450 (CYP450)-mediated reactive ortho-benzoquinone metabolites formation and glutathione (GSH) depletion in LUT-induced cytotoxicity in primary rat hepatocytes. A reactive ortho-benzoquinone metabolite was identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in rat liver microsomes (RLMs) and rat hepatocytes. Using a specific chemical inhibitor method, the CYP3A subfamily was found to be responsible for the reactive metabolite formation in RLMs. Induction of CYP3A by dexamethasone enhanced LUT-induced cytotoxicity, whereas inhibition of CYP3A by ketoconazole (Keto) decreased the cytotoxicity. The cytotoxicity and cell apoptosis induced by LUT were related to the amount of reactive metabolite formation. Furthermore, Keto inhibited the LUT-induced GSH exhaustion. The cytotoxicity was significantly enhanced by pretreatment with L-buthionine sulfoximine to deplete the intracellular GSH. A time course experiment showed that GSH depletion by LUT was not via oxidation of GSH and occurred prior to the increase in 2', 7'-dichlorofluorescein in hepatocytes. Collectively, these data suggest that CYP3A-mediated reactive metabolite formation plays a critical role in LUT-induced hepatotoxicity, and the direct GSH depletion is an initiating event in LUT-mediated cytotoxicity in primary rat hepatocytes.

  4. INTEGRATED DISINFECTION BY-PRODUCTS (DBP) MIXTURES RESEARCH: GENE EXPRESSION ALTERATIONS IN PRIMARY RAT HEPATOCYTE CULTURES EXPOSED TO DBP MIXTURES FORMED BY CHLORINATION AND OZONATION/POSTCHLORINATION

    EPA Science Inventory

    What is the study?
    This study was designed to provide data on the in vitro toxicity of water concentrates containing complex mixtures of DBPs. Rat hepatocytes in primary culture were exposed for 24 hr to full strength, 1:10 or 1:20 dilutions of chlorination or ozonation/chl...

  5. Transplantation of Porcine Hepatocytes Cultured with Polylactic Acid-O-Carboxymethylated Chitosan Nanoparticles Promotes Liver Regeneration in Acute Liver Failure Rats

    PubMed Central

    Chen, Zhong; Chang, Renan; Guan, Weijun; Cai, Hongyu; Tang, Fei; Zhu, Wencai; Chen, Jiahui

    2011-01-01

    In this study, free porcine hepatocytes suspension (Group A), porcine hepatocytes embedded in collagen gel (Group B), porcine hepatocytes cultured with PLA-O-CMC nanoparticles and embedded in collagen gel (Group C), and PLA-O-CMC nanoparticles alone (Group D) were transplanted into peritoneal cavity of ALF rats, respectively. The result showed that plasma HGF levels were elevated post-transplantation with a peak at 12 hr. The rats in Group C showed highest plasma HGF levels at 2, 6, 12, 24 and 36 hr post-transplantation and lowest HGF level at 48 hr. Plasma VEGF levels were elevated at 48 hr post-transplantation with a peak at 72 hr. The rats in Group C showed highest plasma HGF levels at 48, 72, and 96 hr post-transplantation. The liver functions in Group C were recovered most rapidly. Compared with Group B, Group C had significant high liver Kiel 67 antigen labeling index (Ki-67 LI) at day 1 post-HTx (P < .05). Ki-67 LI in groups B and C was higher than that in groups A and D at days 5 and 7 post-HTx. In conclusion, intraperitoneal transplantation of porcine hepatocytes cultured with PLA-O-CMC nanoparticles and embedded in collagen gel can promote significantly liver regeneration in ALF rats. PMID:21603218

  6. Influence of low-power laser radiation on the activity of some membraneous and mitochondrial enzymes of hepatocytes in rats

    NASA Astrophysics Data System (ADS)

    Cieslar, Grzegorz; Adamek, Mariusz; Sieron, Aleksander; Kaminski, Marcin

    1995-01-01

    It was observed in some experiments that visible laser radiation activates the enzymatic function of mitochondria, while infrared laser radiation affects the enzymatic activity of cellular membranes. The aim of the study was to estimate the activity of some membranous as well as mitochondrial enzymes of hepatocytes in rats irradiated with infrared laser. Experimental material consisted of 38 Wistar rats divided into 2 groups -- a studied group exposed to infrared laser radiation and a control group, in which no irradiation was made. A semiconductive infrared laser (wavelength -- 904 nm, mean power -- 8.9 mW) was used. The clean-shaven skin of the right infracostal region of animals was irradiated 5 minutes daily for 15 consecutive days. After finishing the experiment in the preparations from obtained segments of the left liver lobe, the enzymatic activity of succinate dehydrogenase (SDH, EC 1.3.99.1), lactic dehydrogenase (LDH, EC 1.1.1.27), Mg2+ dependent ATP-ase (ATP-ase Mg2+, EC 3.1.3.2.) and acid phosphatase (AcP, EC 3.6.1.8.) was estimated with the use of histochemical methods. In the case of SDH and LDH the increase of enzymatic activity was observed in all 3 zones of liver cluster, especially in male rats. In the case of ATP-ase Mg2+ and AcP the increase of enzymatic activity in biliary canaliculi of hepatocytes in all zones of the liver cluster was observed. On the basis of the obtained results it was proved that infrared laser radiation activates significantly the enzymatic activity of most of the analyzed enzymes, which means that it affects not only properties of biological membranes but also activates the oxidoreductive processes of organism, as it has been observed for visible laser radiation. On the basis of the spectrum of energetic levels in macromolecules (Jablonski's diagram) the mechanisms of availed results are discussed both for enzymes possessing and not possessing chromatophores.

  7. Comparative gene expression profiles induced by PPAR{gamma} and PPAR{alpha}/{gamma} agonists in rat hepatocytes

    SciTech Connect

    Rogue, Alexandra; Renaud, Marie Pierre; Claude, Nancy; Guillouzo, Andre; Spire, Catherine

    2011-07-01

    Species-differential toxic effects have been described with PPAR{alpha} and PPAR{gamma} agonists between rodent and human liver. PPAR{alpha} agonists (fibrates) are potent hypocholesterolemic agents in humans while they induce peroxisome proliferation and tumors in rodent liver. By contrast, PPAR{gamma} agonists (glitazones) and even dual PPAR{alpha}/{gamma} agonists (glitazars) have caused idiosyncratic hepatic and nonhepatic toxicities in human without evidence of any damage in rodent during preclinical studies. The mechanisms involved in such differences remain largely unknown. Several studies have identified the major target genes of PPAR{alpha} agonists in rodent liver while no comprehensive analysis has been performed on gene expression changes induced by PPAR{gamma} and dual PPAR{alpha}/{gamma} agonists. Here, we investigated transcriptomes of rat hepatocytes after 24 h treatment with two PPAR{gamma} (troglitazone and rosiglitazone) and two PPAR{alpha}/{gamma} (muraglitazar and tesaglitazar) agonists. Although, hierarchical clustering revealed a gene expression profile characteristic of each PPAR agonist class, only a limited number of genes was specifically deregulated by glitazars. Functional analyses showed that many genes known as PPAR{alpha} targets were also modulated by both PPAR{gamma} and PPAR{alpha}/{gamma} agonists and quantitative differences in gene expression profiles were observed between these two classes. Moreover, most major genes modulated in rat hepatocytes were also found to be deregulated in rat liver after tesaglitazar treatment. Taken altogether, these results support the conclusion that differential toxic effects of PPAR{alpha} and PPAR{gamma} agonists in rodent liver do not result from transcriptional deregulation of major PPAR target genes but rather from qualitative and/or quantitative differential responses of a small subset of genes.

  8. Cytokine and chemokine expression associated with steatohepatitis and hepatocyte proliferation in rats fed ethanol via total enteral nutrition.

    PubMed

    Ronis, Martin J J; Butura, Angelica; Korourian, Soheila; Shankar, Kartik; Simpson, Pippa; Badeaux, Jamie; Albano, Emanuele; Ingelman-Sundberg, Magnus; Badger, Thomas M

    2008-03-01

    To determine the temporal relationship between alcohol-induced changes in cytokines and chemokines, development of liver pathology and stimulation of hepatocyte proliferation, male Sprague-Dawley rats were intragastrically fed low carbohydrate-containing ethanol (EtOH) diets via total enteral nutrition (TEN) for up to 49 d. Induction of EtOH metabolism and appearance of steatosis preceded development of oxidative stress, inflammation, and cell death. A transitory peak of tumor necrosis factor (TNFalpha) and interferon gamma (IFN gamma) was observed at 14 d followed by reduced expression of TNFalpha, IFN gamma and another Th1 cytokine IL-12 accompanied by reduced expression of the Th1 regulators T-bet and STAT4. After 35-49 d of EtOH, at a time when hepatocyte proliferation was stimulated, IL-12 returned to control values and a second peak of TNFalpha occurred. The Th2 cytokine IL-4 remained suppressed throughout the study and was accompanied by reductions in the Th2 regulator GATA3. There was no temporal effect of EtOH on expression of IL-6 or TGFbeta. IL-5 and IL-13 mRNA were undetectable. Chemokine CXCL-2 expression increased progressively up to 35 d and preceded the appearance of inflammatory infiltrates. These data suggest that steatosis, increased ethanol metabolism, a transient induction of the innate immune response and suppression of Th2 responses were acute consequences of ethanol treatment and were followed by suppression of Th1 responses. However, the majority of necrosis, apoptosis and a late peak of TNFalpha only occurred after 6-7 weeks of ethanol, coincided with the appearance of inflammatory infiltrates and were associated with stimulation of hepatocyte proliferation.

  9. Effect of chronic ethanol feeding on glutathione and functional integrity of mitochondria in periportal and perivenous rat hepatocytes.

    PubMed Central

    García-Ruiz, C; Morales, A; Ballesta, A; Rodés, J; Kaplowitz, N; Fernández-Checa, J C

    1994-01-01

    Chronic ethanol feeding selectively impairs the translocation of cytosol GSH into the mitochondrial matrix. Since ethanol-induced liver cell injury is preferentially localized in the centrilobular area, we examined the hepatic acinar distribution of mitochondrial GSH transport in ethanol-fed rats. Enriched periportal (PP) and perivenous (PV) hepatocytes from pair- and ethanol-fed rats were prepared as well as mitochondria from these cells. The mitochondrial pool size of GSH was decreased in both PP and PV cells from ethanol-fed rats either as expressed per 10(6) cells or per microliter of mitochondrial matrix volume. The rate of reaccumulation of mitochondrial GSH and the linear relationship of mitochondrial to cytosol GSH from ethanol-fed mitochondria were lower for both PP and PV cells, effects observed more prominently in the PV cells. Mitochondrial functional integrity was lower in both PP and PV ethanol-fed rats, which was associated with decreased cellular ATP levels and mitochondrial membrane potential, effects which were greater in the PV cells. Mitochondrial GSH depletion by ethanol feeding preceded the onset of functional changes in mitochondria, suggesting that mitochondrial GSH is critical in maintaining a functionally competent organelle and that the greater depletion of mitochondrial GSH by ethanol feeding in PV cells could contribute to the pathogenesis of alcoholic liver disease. PMID:8040260

  10. A role for lipid rafts in the protection afforded by docosahexaenoic acid against ethanol toxicity in primary rat hepatocytes.

    PubMed

    Aliche-Djoudi, Fatiha; Podechard, Normand; Collin, Aurore; Chevanne, Martine; Provost, Emilie; Poul, Martine; Le Hégarat, Ludovic; Catheline, Daniel; Legrand, Philippe; Dimanche-Boitrel, Marie-Thérèse; Lagadic-Gossmann, Dominique; Sergent, Odile

    2013-10-01

    Previously, we demonstrated that eicosapentaenoic acid enhanced ethanol-induced oxidative stress and cell death in primary rat hepatocytes via an increase in membrane fluidity and lipid raft clustering. In this context, another n-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), was tested with a special emphasis on physical and chemical alteration of lipid rafts. Pretreatment of hepatocytes with DHA reduced significantly ethanol-induced oxidative stress and cell death. DHA protection could be related to an alteration of lipid rafts. Indeed, rafts exhibited a marked increase in membrane fluidity and packing defects leading to the exclusion of a raft protein marker, flotillin. Furthermore, DHA strongly inhibited disulfide bridge formation, even in control cells, thus suggesting a disruption of protein-protein interactions inside lipid rafts. This particular spatial organization of lipid rafts due to DHA subsequently prevented the ethanol-induced lipid raft clustering. Such a prevention was then responsible for the inhibition of phospholipase C-γ translocation into rafts, and consequently of both lysosome accumulation and elevation in cellular low-molecular-weight iron content, a prooxidant factor. In total, the present study suggests that DHA supplementation could represent a new preventive approach for patients with alcoholic liver disease based upon modulation of the membrane structures.

  11. Suppression of CYP2B Induction by Alendronate-Mediated Farnesyl Diphosphate Synthase Inhibition in Primary Cultured Rat Hepatocytes

    PubMed Central

    Jackson, Nancy M.; Kocarek, Thomas A.

    2008-01-01

    We previously reported that squalestatin 1-mediated induction of CYP2B expression is attributable to squalene synthase inhibition and accumulation of an endogenous isoprenoid(s) that is capable of activating the constitutive androstane receptor. To determine whether squalestatin 1-mediated CYP2B induction is strictly dependent upon the biosynthesis of farnesyl pyrophosphate (FPP), the substrate for squalene synthase, the effects of alendronate, a nitrogen-containing bisphosphonate inhibitor of farnesyl diphosphate synthase, were determined on basal, squalestatin 1-inducible, and phenobarbital-inducible CYP2B expression in primary cultured rat hepatocytes. Alendronate treatment alone had no effect on CYP2B or CYP3A mRNA expression in the hepatocyte cultures, but alendronate co-treatment completely suppressed squalestatin 1-mediated CYP2B mRNA induction at concentrations (60 and 100 μM) that effectively inhibited cellular farnesyl diphosphate synthase activity, as assessed by reductions of squalestatin 1-mediated FPP accumulation, and that were not toxic to the cells, as indicated by a lack of effect on MTT activity. Alendronate co-treatment also partially suppressed phenobarbital-inducible CYP2B expression, and this suppressive effect was attenuated by additional co-treatment with the upstream pathway inhibitor, pravastatin. These findings demonstrate that squalestatin 1-mediated CYP2B induction cannot occur in the absence of FPP biosynthesis, but also indicate that one or more upstream isoprenoids, possibly isopentenyl pyrophosphate and/or dimethylallyl pyrophosphate, function to antagonize the CYP2B induction process. PMID:18617600

  12. Quantitative measurement of the L-type pentose phosphate cycle with [2-14C]glucose and [5-14C]glucose in isolated hepatocytes.

    PubMed Central

    Longenecker, J P; Williams, J F

    1980-01-01

    1. Investigations of the mechanism of the non-oxidative segment of the pentose phosphate cycle in isolatd hepatocytes by prediction-labelling studies following the metabolism of [2-14C]-, [5-14C]- and [4,5,6-14C]glucose are reported. The 14C distribution patterns in glucose 6-phosphate show that the reactions of the L-type pentose pathway in hepatocytes. 2. Estimates of the quantitative contribution of the L-type pentose cycle are the exclusive form of the pentose cycle to glucose metabolism have been made. The contribution of the L-type pentose cycle to the metabolism of glucose lies between 22 and 30% in isolated hepatocytes. 3. The distribution of 14C in the carbon atoms of glucose 6-phosphate following the metabolism of [4,5,6-14C]- and [2-14C]glucose indicate that gluconeogenesis from triose phosphate and non-oxidative formation of pentose 5-phosphate do not contribute significantly to randomization of 14C in isolated hepatocytes. The transaldolase exchange reaction between fructose 6-phosphate and glyceraldehyde 3-phosphate is very active in these cells. PMID:7470039

  13. Changes in number of alpha-adrenergic receptor subtypes in hepatocytes from rats fed 3'-methyl-4-dimethylaminoazobenzene.

    PubMed

    Miyamoto, K; Sanae, F; Kohei, K; Nomura, M; Koshiura, R

    1990-01-01

    Changes in numbers of alpha 1- and alpha 2-adrenergic receptors in the plasma membranes of hepatocytes from female Donryu rats given feed containing 0.06% of the carcinogen 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB), were examined. alpha 1-Adrenergic receptors, measured in terms of [3H]prazosin binding, decreased to half of the control 2 weeks after the start of this diet, then gradually decreased for the next 22 weeks. alpha 2-Adrenergic receptors, measured in terms of [3H]clonidine binding, transiently increased 3-fold over the control at 2 weeks. These changes in the early period of the 3'-MeDAB diet intake may be related to hepatocarcinogenesis.

  14. Biokinetics of chlorpromazine in primary rat and human hepatocytes and human HepaRG cells after repeated exposure.

    PubMed

    Broeders, Jessica J W; Parmentier, Céline; Truisi, Germaine L; Jossé, Rozenn; Alexandre, Eliane; Savary, Camille C; Hewitt, Philip G; Mueller, Stefan O; Guillouzo, André; Richert, Lysiane; van Eijkeren, Jan C H; Hermens, Joop L M; Blaauboer, Bas J

    2015-12-25

    Since drug induced liver injury is difficult to predict in animal models, more representative tests are needed to better evaluate these effects in humans. Existing in vitro systems hold great potential to detect hepatotoxicity of pharmaceuticals. In this study, the in vitro biokinetics of the model hepatotoxicant chlorpromazine (CPZ) were evaluated in three different liver cell systems after repeated exposure in order to incorporate repeated-dose testing into an in vitro assay. Primary rat and human hepatocytes, cultured in sandwich configuration and the human HepaRG cell line were treated daily with CPZ for 14 days. Samples were taken from medium, cells and well plastic at specific time points after the first and last exposure. The samples were analysed by HPLC-UV to determine the amount of CPZ in these samples. Based on cytotoxicity assays, the three models were tested at 1-2 μM CPZ, while the primary rat hepatocytes and the HepaRG cell line were in addition exposed to a higher concentration of 15-20 μM. Overall, the mass balance of CPZ decreased in the course of 24 h, indicating the metabolism of the compound within the cells. The largest decrease in parent compound was seen in the primary cultures; in the HepaRG cell cultures the mass balance only decreased to 50%. CPZ accumulated in the cells during the 14-day repeated exposure. Possible explanations for the accumulation of CPZ are a decrease in metabolism over time, inhibition of efflux transporters or binding to phospholipids. The biokinetics of CPZ differed between the three liver cell models and were influenced by specific cell properties as well as culture conditions. These results support the conclusion that in vitro biokinetics data are necessary to better interpret chemical-induced cytotoxicity data.

  15. Repeated intravenous injection of recombinant human hepatocyte growth factor ameliorates liver cirrhosis but causes albuminuria in rats.

    PubMed

    Kusumoto, Kazunori; Ido, Akio; Moriuchi, Akihiro; Katsura, Toshiya; Kim, Ildeok; Takahama, Yuka; Numata, Masatsugu; Kodama, Mayumi; Hasuike, Satoru; Nagata, Kenji; Uto, Hirofumi; Inui, Ken-Ichi; Tsubouchi, Hirohito

    2006-03-01

    Hepatocyte growth factor (HGF) is a promising agent for the treatment of liver cirrhosis because of its mitogenic and anti-fibrotic effects. We investigated the effect of recombinant human HGF (rh-HGF) on cirrhosis development; its pharmacokinetics and nephrotoxicity in rats with liver cirrhosis induced by 4-week treatment with dimethylnitrosamine (DMN). rh-HGF (0.3 mg/kg) was intravenously administered to rats once a day for 4 weeks in parallel with DMN treatment or twice a day for the last 2 weeks of DMN treatment. Repeated doses of rh-HGF increased the liver weight and serum albumin, and reduced serum ALT. The development of hepatic fibrosis was inhibited more efficiently by extended low-dose treatment with rh-HGF. In cirrhotic rats, serum levels of rh-HGF increased and clearance was decreased, leading to an increase in the area under the plasma-concentration time curve and a decrease in the steady-state volume of distribution. Repeated doses of rh-HGF led to increased urinary albumin excretion, but no rh-HGF-treated animals developed increased serum creatinine levels. Urinary albumin excretion returned to baseline after the cessation of rh-HGF. These results suggest that extended treatment with rh-HGF is required for the attenuation of cirrhosis, and repeated doses of rh-HGF cause adverse effects in extra-hepatic organs. These issues must be resolved before the widespread application of rh-HGF in the treatment of liver cirrhosis.

  16. Enhancement of Gastric Ulcer Healing and Angiogenesis by Hepatocyte Growth Factor Gene Mediated by Attenuated Salmonella in Rats

    PubMed Central

    2017-01-01

    The present study developed an oral hepatocyte growth factor (HGF) gene therapy strategy for gastric ulcers treatment. An attenuated Salmonella typhimurium that stably expressed high HGF (named as TPH) was constructed, and the antiulcerogenic effect of TPH was evaluated in a rat model of gastric ulcers that created by acetic acid subserosal injection. From day 5 after injection, TPH (1 × 109 cfu), vehicle (TP, 1 × 109 cfu), or sodium bicarbonate (model control) was administered orally every alternate day for three times. Then ulcer size was measured at day 21 after ulcer induction. The ulcer area in TPH-treated group was 10.56 ± 3.30 mm2, which was smaller when compared with those in the TP-treated and model control groups (43.47 ± 4.18 and 56.25 ± 6.38 mm2, respectively). A higher level of reepithelialization was found in TPH-treated group and the crawling length of gastric epithelial cells was significantly longer than in the other two groups (P < 0.05). The microvessel density in the ulcer granulation tissues of the TPH-treated rats was 39.9 vessels/mm2, which was greater than in the TP-treated and model control rats, with a significant statistical difference. These results suggest that TPH treatment significantly accelerates the healing of gastric ulcers via stimulating proliferation of gastric epithelial cells and enhancing angiogenesis on gastric ulcer site. PMID:28049228

  17. An evaluation of the CYP1A induction potential of pantoprazole in primary rat hepatocytes: a comparison with other proton pump inhibitors.

    PubMed

    Masubuchi, N; Okazaki, O

    1997-11-06

    The ability of pantoprazole to affect the induction of cytochrome P450 (CYP) 1A subfamily was evaluated and compared with two other proton pump inhibitors, omeprazole and lansoprazole, in primary cultured hepatocytes from female Sprague-Dawley rats. The hepatocytes were cultured for 2 days, followed by treatment for 2 days with the proton pump inhibitors at 2, 5 and 10 microM, concentrations that are similar to plasma concentrations found in rats in vivo. The CYP1A inducer 3-methylcholanthrene (at 1 microM) was also evaluated as a positive control. Induction potentials of these chemicals for CYP1A were determined by 7-ethoxyresorufin O-deethylase activity and isozyme contents. The results showed that for CYP1A induction, the rank ordering in induction potential was consistently lansoprazole > omeprazole > pantoprazole. The results are consistent with the existing rat in vivo data, i.e. pantoprazole has lower CYP1A induction potential than omeprazole and lansoprazole.

  18. Recombinant human hepatocyte growth factor (HGF), but not rat HGF, elicits glomerular injury and albuminuria in normal rats via an immune complex-dependent mechanism.

    PubMed

    Mizuno, Shinya; Ikebuchi, Fumie; Fukuta, Kazuhiro; Kato, Takashi; Matsumoto, Kunio; Adachi, Kiichi; Abe, Tetsushi; Nakamura, Toshikazu

    2011-03-01

    1. Hepatocyte growth factor (HGF) has the therapeutic potential to improve renal fibrosis and proteinuria in rodents with chronic kidney disease. In contrast, long-term administration of human HGF to normal rats reportedly elicits proteinuria. Thus, the role of HGF during proteinuria remains contentious. The aim of the present study was to demonstrate that human HGF is antigenic to rodents and that immune complex formation causes proteinuria. 2. We administered either human or rat HGF to normal rats for 28 days. Albuminuria was evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The renal phenotypes of the two HGF treatments were examined using histological techniques. 3. Administration of human HGF (1 mg/kg per day, i.v.) to rats led to severe albuminuria and glomerular hypertrophy in association with increased blood levels of anti-human HGF IgG and IgG deposition in mesangial areas. Furthermore, an immune complex between human HGF and anti-human HGF IgG stimulated the production of proteinuric cytokines (including transforming growth factor-β) in rat cultured mesangial cells. In contrast, treatment of healthy rats with rat HGF for 4 weeks caused neither mesangial IgG deposition nor elevated anti-HGF IgG in the blood. Overall, rat HGF did not provoke albuminuria. 4. We conclude that human HGF produces pseudotoxic effects in normal rat kidneys via an immune complex-mediated pathway, whereas syngenic HGF is safe due to less deposition of glomerular IgG. Our results affirm the safety of the repeated use of syngenic HGF for the treatment of chronic organ diseases, such as renal fibrosis and liver cirrhosis.

  19. Cobalt induces oxidative stress in isolated liver mitochondria responsible for permeability transition and intrinsic apoptosis in hepatocyte primary cultures.

    PubMed

    Battaglia, Valentina; Compagnone, Alessandra; Bandino, Andrea; Bragadin, Marcantonio; Rossi, Carlo Alberto; Zanetti, Filippo; Colombatto, Sebastiano; Grillo, Maria Angelica; Toninello, Antonio

    2009-03-01

    It is well established that cobalt mediates the occurrence of oxidative stress which contributes to cell toxicity and death. However, the mechanisms of these effects are not fully understood. This investigation aimed at establishing if cobalt acts as an inducer of mitochondrial-mediated apoptosis and at clarifying the mechanism of this process. Cobalt, in the ionized species Co(2+), is able to induce the phenomenon of mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) with the opening of the transition pore. In fact, Co(2+) induces mitochondrial swelling, which is prevented by cyclosporin A and other typical MPT inhibitors such as Ca(2+) transport inhibitors and bongkrekic acid, as well as anti-oxidant agents. In parallel with mitochondrial swelling, Co(2+) also induces the collapse of electrical membrane potential. However in this case, cyclosporine A and the other MPT inhibitors (except ruthenium red and EGTA) only partially prevent DeltaPsi drop, suggesting that Co(2+) also has a proton leakage effect on the inner mitochondrial membrane. MPT induction is due to oxidative stress, as a result of generation by Co(2+) of the highly damaging hydroxyl radical, with the oxidation of sulfhydryl groups, glutathione and pyridine nucleotides. Co(2+) also induces the release of the pro-apoptotic factors, cytochrome c and AIF. Incubation of rat hepatocyte primary cultures with Co(2+) results in apoptosis induction with caspase activation and increased level of expression of HIF-1alpha. All these observations allow us to state that, in the presence of calcium, Co(2+) is an inducer of apoptosis triggered by mitochondrial oxidative stress.

  20. Changes in ascorbic acid content in primary cultured rat hepatocytes exposed to 2,2'-azobis (2-amidinopropane) dihydrochloride, a radical initiator.

    PubMed

    Sasakii, K; Kimura, K; Kitaguchi, Y; Onoue, T; Ogura, H; Aoyagi, Y

    2001-08-01

    Changes in ascorbate content in primary cultured rat hepatocytes exposed to oxidative stress derived from water soluble radical initiator 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) were examined. Cells were exposed to 0.05 and 5 mg/ml of AAPH as 'mild' and severe' oxidative stresses, respectively. Lipid peroxidation in hepatocytes was induced by 'severe' oxidative stress, but not by 'mild' oxidative stress. Ascorbate decreased at 6 hr after administration of both mild' and severe' oxidative stresses, and recovered to the control level after a further 6 hr. In cells treated with 'severe oxidative stress, however, total ascorbate (reduced form plus oxidized form) had increased 24 hr after administration. These results indicated that consumption alone did not account for the increase of ascorbate in hepatocytes under oxidative stress.

  1. Effect of berberine on expression of hepatocyte nuclear factor-4alpha in rats with fructose-induced insulin resistance.

    PubMed

    Gao, Zhiqiang; Leng, Sanhua; Lu, Fuer; Xie, Meijuan; Xu, Lijun; Wang, Kaifu

    2008-06-01

    The effects of berberine on the expression of hepatocyte nuclear factor-4alpha (HNF-4alpha) in liver of rats with fructose-induced insulin resistance and the molecular mechanism of berberine preventing insulin resistance were investigated. The experimental animals were divided into two groups of 16 animals each. The control group received a control routine diet containing 60% carbohydrate, and the study group a high-fructose diet containing 60% fructose as the sole source of carbohydrate. At the end of 6 weeks these were each subdivided into two groups. One was administered with berberine [187.5 mg/(kg x d) in 5 g/L carboxymethyl cellulose] by intragastric intubation and the other group was treated with a vehicle (5 g/L carboxymethyl cellulose). The rats were fed on the same dietary regimen for the next 4 weeks. After the experimental period of 10 weeks, plasma glucose, insulin and triglyceride levels were measured. HOMA insulin resistance index (HOMA-IR) was assayed. Immunohistochemistry, semiquantitative RT-PCR and western blot were used to detect the expression of HNF-4alpha in liver. Compared with control diet, fructose feeding induced hyperinsulinemia, HOMA-IR and increased triglyceride (all P<0.01). Berberine prevented the rise in plasma insulin (P<0.01), HOMA-IR (P<0.01) and triglyceride (P<0.05) in the fructose-fed rats. No change in plasma glucose was seen among these groups. The mRNA and protein expression of HNF-4alpha was decreased in the fructose-fed rats, but berberine could promote its expression. It was concluded that berberine could prevent fructose-induced insulin resistance in rats possibly by promoting the expression HNF-4alpha in liver.

  2. Induction of foci of altered, γ-glutamyltranspeptidase-positive hepatocytes in carcinogen-treated rats fed a choline-deficient diet

    PubMed Central

    Sells, M. A.; Katyal, S. L.; Sell, S.; Shinozuka, H.; Lombardi, B.

    1979-01-01

    A series of experiments was performed to investigate whether, after exposure of rats to a chemical hepatocarcinogen, feeding a choline-deficient (CD) diet would promote the proliferation of initiated liver cells, and their evolution to foci of altered γ-glutamyltranspeptidase (GGT)-positive hepatocytes, without subjecting the animals to further experimental manipulations. Diethylnitrosamine (DEN), in single doses of 15-150 mg/kg body weight, was injected into male, Sprague-Dawley rats, either intact or 18 h after a partial hepatectomy (PH). The animals were then fed either a CD or a choline-supplemented (CS) diet for 2-8 weeks. Emergence in the liver of foci of altered, GGT+ hepatocytes was studied by histological and histochemical techniques. Foci, in varying numbers, developed in the liver of all rats fed the CD diet. The number of foci induced was larger when DEN was administered after PH rather than to intact rats. Foci developed in none of the livers of rats fed the CS diet, except in one experiment in which 30 mg DEN/kg body weight was injected after a PH. In all cases, foci of altered, GGT+ hepatocytes were shown to be α-foetoprotein after immunofluorescence staining of liver sections. It is concluded that feeding a CD diet exerts a strong promoting action on the proliferation and further evolution of liver cells initiated by a chemical carcinogen, providing the basis for a new and efficient procedure for the induction of foci of altered hepatocytes in rat liver. ImagesFig. 2Fig. 3Fig. 4Fig. 5 PMID:89859

  3. Treatment of surgically induced acute liver failure with transplantation of highly differentiated immortalized human hepatocytes.

    PubMed

    Kobayashi, N; Miyazaki, M; Fukaya, K; Inoue, Y; Sakaguchi, M; Noguchi, H; Matsumura, T; Watanabe, T; Totsugawa, T; Tanaka, N; Namba, M

    2000-01-01

    Primary human hepatocytes are an ideal source of hepatic function in bioartficial liver (BAL), but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, primary human fetal hepatocytes were immortalized using simian virus 40 large T antigen. One of the immortal cell lines, OUMS-29, showed highly differentiated liver functions. Intrasplenic transplantation of OUMS-29 cells protected 90% hepatectomized rats from hyperammonemia and significantly prolonged their survival. Essentially unlimited availability of OUMS-29 cells supports their clinical use for BAL treatment.

  4. Cytotoxicity of the redox cycling compound diquat in isolated hepatocytes: involvement of hydrogen peroxide and transition metals.

    PubMed

    Sandy, M S; Moldeus, P; Ross, D; Smith, M T

    1987-11-15

    Diquat is a hepatotoxin whose toxicity in vivo and in vitro is mediated by redox cycling and greatly enhanced by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. The mechanism by which redox cycling mediates diquat cytotoxicity is unclear, however. Here, we have attempted to examine the roles of three potential products of redox cycling, namely superoxide anion radical (O2-.), hydrogen peroxide (H2O2), and hydroxyl radical (.OH), in the toxicity of diquat to BCNU-treated isolated hepatocytes. Addition of high concentrations of catalase, but not superoxide dismutase, to the incubations provided some protection against the toxic effect of diquat, but much better protection was observed when catalase was added in combination with the iron chelator desferrioxamine. Addition of desferrioxamine alone also provided considerable protection, whereas the addition of copper ions enhanced diquat cytotoxicity. Taken together, these results indicate that both H2O2 and the transition metals iron and copper could play major roles in the cytotoxicity of diquat. The role of O2-. remains less clear, however, but studies with diethylenetriaminepentaacetic acid indicate that O2-. is unlikely to significantly contribute to the reduction of Fe3+ to Fe2+. The hydroxyl radical or a related species seems the most likely ultimate toxic product of the H2O2/Fe2+ interaction, but hydroxyl radical scavengers afforded only minimal protection.

  5. Acetate represents a major product of heptanoate and octanoate beta-oxidation in hepatocytes isolated from neonatal piglets.

    PubMed

    Lin, X; Adams, S H; Odle, J

    1996-08-15

    An experiment was conducted to explore the nature of the radiolabel distribution in acid-soluble products (ASPs) resulting from the oxidation of [1-14C]C7:0 or C8:0 by isolated piglet hepatocytes. The differences between odd and even chain-length and the impacts of valproate and malonate upon the rate of beta-oxidation and ASP characteristics were tested. A minor amount of fatty acid carboxyl carbon (< or = 10% of organic acids identified by radio-HPLC) accumulated in ketone bodies regardless of chain-length or inhibitor used. In all cases, acetate represented the major reservoir of carboxyl carbon, accounting for 60-70% of radiolabel in identified organic acids. Cells given [1-14C]C7:0 accumulated 85% more carboxyl carbon in Krebs cycle intermediates when compared with C8:0, while accumulation in acetate was unaffected. The results are consistent with the hypothesis that anaplerosis from odd-carbon fatty acids affects the oxidative fate of fatty acid carbon. The piglet appears unique in that non-ketogenic routes of fatty acid carbon flow (i.e. acetogenesis) predominate in the liver of this species.

  6. Na(+)-Ca sup 2+ exchange in cultured rat hepatocytes: Evidence against a role in cytosolic Ca sup 2+ regulation or signaling

    SciTech Connect

    Lidofsky, S.D.; Xie, M.H.; Scharschmidt, B.F. )

    1990-07-01

    Plasma membrane Na(+)-Ca2+ exchange contributes importantly to the regulation of cytosolic Ca2+ concentration ((Ca2+)i) in excitable cells. Despite extensive study in excitable tissues, the role of this transporter in the regulation of (Ca2+)i in hepatocytes is unknown, and conflicting information has been reported regarding the presence of Na(+)-Ca2+ exchange in hepatocyte plasma membrane vesicles. We have therefore assessed the role of Na(+)-dependent Ca2+ transport in the regulation of (Ca2+)i in rat hepatocytes in primary culture under basal conditions and after exposure to vasopressin, a hormone that elevates (Ca2+)i. Ca2+ efflux, measured using 45Ca, did not differ in the presence or absence of extracellular Na+, either under basal conditions or in response to vasopressin. (Ca2+)i, measured using the Ca2(+)-sensitive dye fura-2, was not altered by transient or prolonged exposure to Na(+)-free media or by exposure to ouabain in concentrations sufficient to produce a five-fold elevation in intracellular Na+ concentration. The (Ca2+)i response to vasopressin was also unaffected by Na+ removal or ouabain. By contrast, in cultured rat cardiac myocytes, cells that possess Na(+)-Ca2+ exchange, transient or prolonged Na+ removal as well as ouabain exposure produced greater than fivefold increases in (Ca2+)i compared with controls. We conclude that Na(+)-Ca2+ exchange does not contribute to the regulation of (Ca2+)i in hepatocytes.

  7. Comparison of 2,2-bis(bromomethyl)-1,3-propanediol induced genotoxicity in UROtsa cells and primary rat hepatocytes: relevance of metabolism and oxidative stress.

    PubMed

    Kong, Weixi; Gu, Pengfei; Knudsen, Gabriel A; Sipes, I Glenn

    2013-10-09

    2,2-Bis(bromomethyl)-1,3-propanediol (BMP) is a brominated flame retardant used in urethane foams and polyester resins. In a two year dietary study, BMP caused neoplastic lesions at multiple sites including the urinary bladder of both rats and mice. However, liver was not a target tissue. We previously reported that BMP elicited oxidative DNA damage in a human uroepithelial cell line (UROtsa). The present in vitro study investigated the susceptibility of target (UROtsa cells) and non-target cells (primary rat hepatocytes) to BMP-induced genotoxicity. In contrast to hepatocytes, BMP exhibited greater genotoxic potential in UROtsa cells as evidenced by the concentration dependent increase in DNA strand breaks and DNA binding. Total content of intracellular GSH quantified in UROtsa cells (2.7±1.0nmol/mg protein) was 4 fold lower than that in hepatocytes (10.7±0.3nmol/mg protein). HPLC analysis indicated BMP was not metabolized and/or consumed in UROtsa cells at any of the concentrations tested (10-250μM) but was extensively converted to a mono-glucuronide in hepatocytes. These results demonstrate that a target cell line such as UROtsa cells are more susceptible to BMP-induced DNA damage when compared to non-target cells. This increased susceptibility may relate to the deficiency of antioxidant and/or metabolic capabilities in UROtsa cells.

  8. Olanzapine modulation of hepatic oxidative stress and inflammation in socially isolated rats.

    PubMed

    Todorović, Nevena; Tomanović, Nada; Gass, Peter; Filipović, Dragana

    2016-01-01

    Olanzapine, an atypical antipsychotic, is efficient in stress associated psychiatric diseases, but its effect on the liver, a primary organ for drug activation and detoxification, still remains unclear. The effect of olanzapine administration (7.5mg/kg/day), on rat hepatic glutathione (GSH)-dependent defense and proinflammatory cytokines following 6weeks of chronic social isolation (CSIS), which causes depressive- and anxiety-like behavior in adult male Wistar rats, was investigated. The subcellular distribution of nuclear factor-κB (NF-κB), cytosolic inducible nitric oxide synthase (iNOS) protein levels and hepatic histological alterations were also determined. Decreased GSH content and glutathione reductase activity associated with increased catalase and glutathione S-transferase activity following CSIS indicated hepatic oxidative stress. Moreover, CSIS caused NF-κB nuclear translocation and the concomitant increase in iNOS together with increase in interleukin-1beta and tumor necrosis factor alpha protein levels, but no effect on interleukin-6. Olanzapine treatment suppressed NF-κB activation and iNOS expression and caused modulation of GSH-dependent defense systems but failed to reverse CSIS-induced increase in hepatic proinflammatory cytokines. Portal inflammation, focal hepatocyte necrosis and an increased number of Kupffer cells in CSIS rats (vehicle- or olanzapine-treated) were found. Olanzapine-treated socially reared rats showed portal inflammation and focal hepatocyte necrosis. Data suggest that CSIS compromised GSH-dependent defense, triggered a proinflammatory response and histological alterations in rat liver. Olanzapine treatment partially reversed the alterations in hepatic GSH-dependent defense, but showed no anti-inflammatory effect suggesting that it may provide protective effect against hepatic CSIS-induced oxidative stress, but not against inflammation.

  9. Omega-3 improves glucose tolerance but increases lipid peroxidation and DNA damage in hepatocytes of fructose-fed rats.

    PubMed

    de Castro, Gabriela Salim Ferreira; dos Santos, Raquel Alves; Portari, Guilherme Vannucchi; Jordão, Alceu Afonso; Vannucchi, Helio

    2012-04-01

    The high consumption of fructose is linked to the increase in various characteristics of the metabolic syndrome. Fish oil is beneficial for the treatment of these comorbidities, such as insulin resistance, dyslipidemia, and hepatic steatosis. The objective of this study was to evaluate the consequences of the administration of fish oil concomitant to fructose ingestion during the experiment (45 days) and during the final 15 days in high-fructose-fed rats. Male Wistar rats were divided into 5 groups: control; those receiving 10% fish oil (FO); those receiving 60% fructose (Fr); those receiving 60% fructose and 10% fish oil for 45 days (FrFO); and those receiving fructose plus soybean oil for 30 days and fish oil for the final 15 days of the study (FrFO15). There was an increase in triacylglycerol, serum total cholesterol, and hepatic volume in the Fr group. The FO and FrFO groups experienced an increase in lipid peroxidation and a decrease in serum reduced glutathione. The FrFO group suffered greater hepatic injury, with increased alanine aminotransferase levels and DNA damage. Marked n-3 incorporation occurred in the groups receiving fish oil, favoring a better response to the oral glucose tolerance test. Fructose induced comorbidities of the metabolic syndrome, and the use of fish oil promoted a better glucose tolerance, although it was accompanied by more hepatocyte damage.

  10. Inhibition of bile salt transport by drugs associated with liver injury in primary hepatocytes from human, monkey, dog, rat, and mouse.

    PubMed

    Zhang, Jie; He, Kan; Cai, Lining; Chen, Yu-Chuan; Yang, Yifan; Shi, Qin; Woolf, Thomas F; Ge, Weigong; Guo, Lei; Borlak, Jürgen; Tong, Weida

    2016-08-05

    Interference of bile salt transport is one of the underlying mechanisms for drug-induced liver injury (DILI). We developed a novel bile salt transport activity assay involving in situ biosynthesis of bile salts from their precursors in primary human, monkey, dog, rat, and mouse hepatocytes in suspension as well as LC-MS/MS determination of extracellular bile salts transported out of hepatocytes. Glycine- and taurine-conjugated bile acids were rapidly formed in hepatocytes and effectively transported into the extracellular medium. The bile salt formation and transport activities were time‒ and bile-acid-concentration‒dependent in primary human hepatocytes. The transport activity was inhibited by the bile salt export pump (BSEP) inhibitors ketoconazole, saquinavir, cyclosporine, and troglitazone. The assay was used to test 86 drugs for their potential to inhibit bile salt transport activity in human hepatocytes, which included 35 drugs associated with severe DILI (sDILI) and 51 with non-severe DILI (non-sDILI). Approximately 60% of the sDILI drugs showed potent inhibition (with IC50 values <50 μM), but only about 20% of the non-sDILI drugs showed this strength of inhibition in primary human hepatocytes and these drugs are associated only with cholestatic and mixed hepatocellular cholestatic (mixed) injuries. The sDILI drugs, which did not show substantial inhibition of bile salt transport activity, are likely to be associated with immune-mediated liver injury. Twenty-four drugs were also tested in monkey, dog, rat and mouse hepatocytes. Species differences in potency were observed with mouse being less sensitive than other species to inhibition of bile salt transport. In summary, a novel assay has been developed using hepatocytes in suspension from human and animal species that can be used to assess the potential for drugs and/or drug-derived metabolites to inhibit bile salt transport and/or formation activity. Drugs causing sDILI, except those by immune

  11. Therapeutic effect of hepatocyte growth factor-secreting mesenchymal stem cells in a rat model of liver fibrosis

    PubMed Central

    Kim, Myung-Deok; Kim, Sung-Soo; Cha, Hyun-Young; Jang, Seung-Hun; Chang, Da-Young; Kim, Wookhwan; Suh-Kim, Haeyoung; Lee, Jae-Ho

    2014-01-01

    Bone marrow-derived mesenchymal stromal cells (MSCs) have been reported to be beneficial for the treatment of liver fibrosis. Here, we investigated the use of genetically engineered MSCs that overexpress hepatocyte growth factor (HGF) as a means to improve their therapeutic effect in liver fibrosis. Liver fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. HGF-secreting MSCs (MSCs/HGF) were prepared by transducing MSCs with an adenovirus carrying HGF-encoding cDNA. MSCs or MSCs/HGF were injected directly into the spleen of fibrotic rats. Tissue fibrosis was assessed by histological analysis 12 days after stem cell injection. Although treatment with MSCs reduced fibrosis, treatment with MSCs/HGF produced a more significant reduction and was associated with elevated HGF levels in the portal vein. Collagen levels in the liver extract were decreased after MSC/HGF therapy, suggesting recovery from fibrosis. Furthermore, liver function was improved in animals receiving MSCs/HGF, indicating that MSC/HGF therapy resulted not only in reduction of liver fibrosis but also in improvement of hepatocyte function. Assessment of cell and biochemical parameters revealed that mRNA levels of the fibrogenic cytokines PDGF-bb and TGF-β1 were significantly decreased after MSC/HGF therapy. Subsequent to the decrease in collagen, expression of matrix metalloprotease-9 (MMP-9), MMP-13, MMP-14 and urokinase-type plasminogen activator was augmented following MSC/HGF, whereas tissue inhibitor of metalloprotease-1 (TIMP-1) expression was reduced. In conclusion, therapy with MSCs/HGF resulted in an improved therapeutic effect compared with MSCs alone, probably because of the anti-fibrotic activity of HGF. Thus, MSC/HGF represents a promising approach toward a cell therapy for liver fibrosis. PMID:25145391

  12. Changes in alpha 2- and beta-adrenoceptors in hepatocytes from rats during treatment with 3'-methyl-4-dimethylaminoazobenzene.

    PubMed

    Sanae, F; Kohei, K; Nomura, M; Miyamoto, K

    1992-05-01

    A 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) containing diet was given to 6 weeks old female Donryu rats, and the number of adrenoceptors and the response of adenylate cyclase in the hepatocytes were measured. The treatment with 3'-MeDAB led to rapid increases in [125I]iodocyanopindolol ([125I]ICYP)- and [3H]clonidine-binding sites to hepatic membranes without significant changes in the Kd values. The number or beta-adrenoceptors defined by [125I]ICYP binding sites was increased with a biphagic mode. The [3H]clonidine binding reached a peak 2 weeks after the start of the carcinogen diet and then began a slow descent. The alpha 2-adrenoceptor was defined by [3H]clonidine binding being selectively inhibited by an alpha 2-antagonist, yohimbine, but not by an alpha 1-antagonist, prazosin, or a beta-antagonist propranolol. Catecholamine responsiveness to adenylate cyclase in hepatocytes also increased during treatment with 3'-MeDAB. However, the efficacy of norepinephrine (NE) in activating cyclase was lower than that of isoproterenol (IPN) during 4 to 8 weeks of the carcinogen diet. The difference between the efficacies of IPN and NE resulted from inhibiting adenylate cyclase through alpha 2-adrenoceptors by NE. Therefore, we noticed that the increasing pattern of the number of beta-adrenoceptors did not always parallel IPN-stimulated adenylate cyclase activity and that the increase in the number of alpha 2-adrenoceptors preceded the difference between the efficacies of IPN and NE in activating adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Extra virgin olive oil phenols down-regulate lipid synthesis in primary-cultured rat-hepatocytes.

    PubMed

    Priore, Paola; Siculella, Luisa; Gnoni, Gabriele Vincenzo

    2014-07-01

    Hydroxytyrosol, tyrosol, and oleuropein, the main phenols present in extra virgin olive oil, have been reported to exert several biochemical and pharmacological effects. Here, we investigated the short-term effects of these compounds on lipid synthesis in primary-cultured rat-liver cells. Hydroxytyrosol, tyrosol and oleuropein inhibited both de novo fatty acid and cholesterol syntheses without an effect on cell viability. The inhibitory effect of individual compounds was already evident within 2 h of 25 μM phenol addition to the hepatocytes. The degree of cholesterogenesis reduction was similar for all phenol treatments (-25/30%), while fatty acid synthesis showed the following order of inhibition: hydroxytyrosol (-49%) = oleuropein (-48%) > tyrosol (-30%). A phenol-induced reduction of triglyceride synthesis was also detected. To clarify the lipid-lowering mechanism of these compounds, their influence on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase and fatty acid synthase), triglyceride synthesis (diacylglycerol acyltransferase) and cholesterogenesis (3-hydroxy-3-methyl-glutaryl-CoA reductase) was investigated in situ by using digitonin-permeabilized hepatocytes. Acetyl-CoA carboxylase, diacylglycerol acyltransferase and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities were reduced after 2 h of 25 μM phenol treatment. No change in fatty acid synthase activity was observed. Acetyl-CoA carboxylase inhibition (hydroxytyrosol, -41%, = oleuropein, -38%, > tyrosol, -17%) appears to be mediated by phosphorylation of AMP-activated protein kinase. These findings suggest that a decrease in hepatic lipid synthesis may represent a potential mechanism underlying the reported hypolipidemic effect of phenols of extra virgin olive oil.

  14. Use of Isolated Trout Hepatocytes to Predict Measured Hepatic Clearance and Whole-animal Bioconcentration Factors for Six Polyaromatic Hydrocarbons

    EPA Science Inventory

    Hepatic metabolism is an important determinant of chemical bioaccumulation in fish. Consequently, measured in vitro hepatic metabolism may improve model predictions of bioaccumulation. In this study, fresh and cryopreserved trout hepatocytes were used to measure in vitro intrin...

  15. The activation and differential signalling of the growth hormone receptor induced by pGH or anti-idiotypic monoclonal antibodies in primary rat hepatocytes.

    PubMed

    Li, Wei; Lan, Hainan; Liu, Huimin; Fu, Zhiling; Yang, Yanhong; Han, Weiwei; Guo, Feng; Liu, Yu; Zhang, Hui; Liu, Jingsheng; Zheng, Xin

    2013-08-25

    In this report, we have developed a panel of monoclonal anti-idiotypic antibodies to pGH by immunising BALB/c mice with a purified monoclonal anti-pGH antibody (1A3), among which one mAb, termed CG-8F, was selected for further characterisation. We found that CG-8F behaved as a typical Ab2β, not only conformationally competing with pGH for 1A3 but also exhibiting recognition for GHR in a rat hepatocyte model. We next examined the resulting signal transduction pathways triggered by this antibody in rat hepatocytes and found that both pGH and CG-8F could trigger the JAK2-STAT1/3/5-mediated signal transduction pathway. Furthermore, the phosphorylation kinetics of pSTAT1/3/5 induced by either pGH or CG-8F were remarkably similar in the dose-response and time course rat hepatocyte experiments. In contrast, only pGH, but not CG-8F, was capable of inducing ERK phosphorylation. Further experimental studies indicated that the two functional binding sites on CG-8F are required for GHR activation. This study partially reveals the mechanism of action of GH anti-idiotypic antibodies and also indicates that monoclonal anti-idiotypic antibodies represent an effective way to produce GH mimics, suggesting that it is possible to produce signal-specific cytokine agonists using an anti-idiotypic antibody approach.

  16. Effects of insulin, dexamethasone and cytokines on {alpha}{sub 1}-acid glycoprotein gene expression in primary cultures of normal rat hepatocytes

    SciTech Connect

    Barraud, B.; Balavoine, S.; Feldmann, G.; Lardeux, B.

    1996-04-01

    While the effects of insulin, dexamethasone and cytokines on {alpha}{sub 1}-acid glycoprotein gene expression have been investigated in various hepatoma cell lines, the individual and combined effects of these components on the expression of this gene have been rarely studied in cultured normal rat hepatocytes. In this cell model, we have shown that mRNA levels of {alpha}{sub 1}-acid glycoprotein were not decreased at least during the first 24 h of culture under basal conditions. During these short-term cultures, the expression of {alpha}{sub 1}-acid glycoprotein in normal hepatocytes showed a high degree of responsiveness to dexamethasone alone (20-fold increase) and to dexamethasone associated with various cytokines (interleukin-1{beta}, interleukin-6 and tumor necrosis factor {alpha}) with a 40 to 100-fold increase depending on the cytokine. Insulin alone did not modify {alpha}{sub 1}-acid glycoprotein mRNA; however, this hormone exerted a positive effect (about 50% increase) in the presence of dexamethasone or dexamethasone with cytokines. These results indicate that the regulation of {alpha}{sub 1}-acid glycoprotein in cultured normal rat hepatocytes presents major differences when compared to reported observations in rat hepatoma cell lines. 49 refs., 2 figs., 2 tabs.

  17. Quantitative analysis of intermediary metabolism in rat hepatocytes incubated in the presence and absence of ethanol with a substrate mixture including ketoleucine.

    PubMed

    Baranyai, J M; Blum, J J

    1989-02-15

    Hepatocytes isolated from livers of fed rats were incubated with a mixture of glucose (10 mM), ribose (1.0 mM), acetate (1.25 mM), alanine (3.5 mM), glutamate (2.0 mM), aspartate (2.0 mM), 4-methyl-2-oxovaleric acid (ketoleucine) (3.0 mM), and, in paired flasks, 10 mM-ethanol. One substrate was 14C-radiolabelled in any given incubation. Incorporation of 14C into glucose, glycogen, CO2, lactate, alanine, aspartate, glutamate, acetate, urea, lipid glycerol, fatty acids and the 1- and 2,3,4-positions of ketone bodies was measured after 20 and 40 min of incubation under quasi-steady-state conditions. Data were analysed with the aid of a realistic structural metabolic model. In each of the four conditions examined, there were approx. 77 label incorporation measurements and several measurements of changes in metabolite concentrations. The considerable excess of measurements over the 37 independent flux parameters allowed for a stringent test of the model. A satisfactory fit to these data was obtained for each condition. There were large bidirectional fluxes along the gluconeogenic/glycolytic pathways, with net gluconeogenesis. Rates of ureagenesis, oxygen consumption and ketogenesis were high under all four conditions studied. Oxygen utilization was accurately predicted by three of the four models. There was complete equilibration between mitochondrial and cytosolic pools of acetate and of CO2, but for several of the metabolic conditions, two incompletely equilibrated pools of mitochondrial acetyl-CoA and oxaloacetate were required. Ketoleucine was utilized at a rate comparable to that reported by others in perfused liver and entered the mitochondrial pool of acetyl-CoA directly associated with ketone body formation. Ethanol, which was metabolized at rates comparable to those in vivo, caused relatively few changes in overall flux patterns. Several effects related to the increased NADH/NAD+ ratio were observed. Pyruvate dehydrogenase was completely inhibited and the ratio

  18. The effect of thiazolidin carboxylic acid (ThCA) on the redox equilibrium maintaining in the rat hepatocyte with streptozotocin-induced diabetes.

    PubMed

    Bartoc, R; Oancia, M; Pirvănescu, L

    1978-01-01

    The variation of the activities of glutathion peroxidase, glutathion reductase, glucose-6-phosphate dehydrogenase, as well as of the concentrations of lipid peroxides and -SH groups of nonproteic nature, was followed up in the hepatocyte of normal rats, of those with, streptozotocin-induced-diabetes, and of diabetic rats treated with thiazolidin carboxylic (ThCA) acid. Free peroxides and glutathion peroxidase were increased in the diabetic animals as against the normals, whereas glutathion reductase, glucose-6-phosphate dehydrogenase and the -SH groups of nonproteic nature had lower values. A return to normal of these parameters was noticed in the animals treated with ThCA.

  19. Parallel increase of dolichol pathway and nucleotide pyrophosphatases in rat hepatocytes by dexamethasone

    SciTech Connect

    Sarkar, M.; Mookerjea, S.

    1986-05-01

    Incorporation of (/sup 14/C)-mannose to dolichol phosphate mannose, dolichol pyrophosphate oligosaccharide and N-linked glycoproteins in cultured hepatocytes was increased by dexamethasone. Nucleotide pyrophosphatases are now measured to investigate possible control of glycosylation by the nucleotide sugar pools. Dexamethasone caused about 2 fold increase of UDP-GlcNAc and GDP-Man pyrophosphatase activity which is evident as early as 4 hr and increased up to 12 hr of incubation. The K/sub m/ for UDP-GlcNAc and GDP-Man were respectively 0.43 mM and 0.47 mM in homogenate membrane and the values remained unchanged by dexamethasone treatment. However the V/sub max/ of the enzymes were increased with both UDP-GlcNAc and GDP-Man. The broad pH optima of the enzymes (pH 8 to 10) indicated their alkaline nature. Mixing experiments of the cell homogenates from control and dexamethasone treated cells showed that UDP-GlcNAc and GDP-Man pyrophosphatase activities were additive which ruled out the possibility of presence of any activator or removal of any inhibitor due to dexamethasone. The parallel increase of nucleotide pyrophosphatase and dolichol linked pathway by dexamethasone does not support the possibility that stimulation of glycoprotein synthesis by dexamethasone is mediated by transfer of nucleotide sugars towards dolichol saccharides.

  20. Prolonged rest period enables the detection of micronucleated hepatocytes in the liver of young adult rats after a single dose of diethylnitrosamine or mitomycin C.

    PubMed

    Shimada, Keisuke; Yamamoto, Mika; Takashima, Miyuki; Seki, Jiro; Miyamae, Yoichi; Wakata, Akihiro

    2015-09-01

    A repeated-dose micronucleus assay utilizing young adult rat hepatocytes was recently developed to evaluate the genotoxicity. In this assay, accumulation of micronucleated hepatocytes (MNHEPs) induced by repeated dosing of genotoxic chemicals is considered to be a key factor in the detection of micronuclei induction. Then, we hypothesized that the period following chemical exposure enable the detection of MNHEP induction in young adult rats, namely that MNHEPs can be generated from chromosomally damaged cells and accumulate following initiation of chemical exposure until sampling. We therefore measured MNHEP induction at 2 or 4 weeks after a single oral administration of 12.5, 50, or 100mg/kg of diethylnitrosamine (DEN) or an intraperitoneal administration of 0.5, 1.0, or 2.0mg/kg of mitomycin C (MMC) to young adult rats. Results showed a statistically significant, dose-dependent increase in the numbers of MNHEPs in DEN- or MMC-treated rats, indicating that prolonged rest period following a single dose of a genotoxic chemical enables the detection of MNHEP induction in the liver of young adult rats. From these results, a single oral administration of 50mg/kg of DEN with a 2- or 4- week rest period can be used as a positive control in repeated-dose liver micronucleus assays. This procedure is superior in terms of labor saving and animal welfare to repeated dosing of DEN.

  1. Nitric oxide inhibits specific enzymes in the Krebs cycle and the respiratory chain of rat hepatocyte mitochondria

    SciTech Connect

    Stadler, J.; Billiar, T.R.; Curran, R.D.; Kim, R.; Simmons, R.L. )

    1990-02-26

    Nitric oxide (NO) is a highly-reactive molecule produced from L-arginine as recently described. In macrophages and tumor cells, NO inhibits specific mitochondrial enzymes presumably by attacking their intrinsic 4Fe-4S centers. The susceptible enzymes include aconitase of the Krebs cycle and oxidoreductase (complex II) of the electron transport chain. The authors have recently demonstrated that hepatocytes (HC) produce NO in large amounts in response to endotoxin and inflammatory cytokines. To determine whether HC suffer a similar enzyme inhibition, the authors exposed rat HC to increasing concentrations of NO solutions for 5 minutes. The activity of aconitase, complex 1, complex 2, and complex 4 (cytochrome oxidase) was determined by measuring O{sub 2} consumption after addition of enzyme-specific substrates. An NO concentration-dependent inhibition of aconitase, complex 1, and complex 2 was measured. After exposure to 0.6 mM solution, the activity of aconitase was blocked to non-measurable values while complex 1 was reduced to 11 + 8%, and complex 2 to 36 + 2% of the activity of control HC. Complex 4 of the respiratory chain remained intact at 100 + 8%. These data indicate that HC, like other cell types, are susceptible to inhibition of important steps of energy production by NO. As NO is produced in response to septic stimuli, this mechanism may play a role in the metabolic dysfunction of HC in sepsis.

  2. Inhibition of cholesterol biosynthesis by a water-soluble garlic extract in primary cultures of rat hepatocytes.

    PubMed

    Gebhardt, R

    1991-08-01

    Cultured rat hepatocytes continually synthesize cholesterol form radiolabeled acetate during a 24 h incubation period and export it, presumably as VLDL (very low density lipoprotein) to the culture medium. Mevastatin inhibits cholesterol biosynthesis by 90%. Incubation of the cultures with water-soluble extracts of garlic powder (Kwai, Sapec) diminish cholesterol biosynthesis (20-25%) as well as its export into the medium (30-35%). The IC50-value is 90 micrograms/ml. Between about 0.25 and 10 mg/ml the average maximal inhibition amounts to about 23%. Cytotoxicity of the extracts is apparent at concentrations above 125 mg/ml only. Pure alliin alone, or after incubation with alliinase (conversion to allicin) in concentrations corresponding to its content in the extracts does not exert any inhibition. Replacement of 14C-acetate by 14C-mevalonate omits the inhibitory effect. The activity of HMGCoA (hydroxymethylglutaryl-CoA) reductase is significantly reduced by garlic extracts at 50 micrograms/ml. At higher concentrations fatty acid synthetase, cholesterol 7 alpha-hydroxylase and cholesterol acyltransferase are slightly inhibited. Fatty acid synthetase is the only one of these enzymes which is inhibited by alliin at very high concentrations. These results demonstrate that water-soluble garlic extracts diminish hepatic cholesterol biosynthesis, thus contributing to the reduction of blood cholesterol. The main target site seems to be HMGCoA-reductase. The actual active principle(s) is still unknown. Alliin, however, does not seem to be of major significance.

  3. Validation of a high-throughput in vitro alkaline elution/rat hepatocyte assay for DNA damage.

    PubMed

    Gealy, Robert; Wright-Bourque, Jennifer L; Kraynak, Andrew R; McKelvey, Troy W; Barnum, John E; Storer, Richard D

    2007-04-20

    In vitro alkaline elution is a sensitive and specific short term assay which measures DNA strand breakage in a mammalian test system (primary rat hepatocytes). This lab has previously demonstrated the performance of the assay with known genotoxic and non-genotoxic compounds. The methodology employed has relatively low sample throughput and is labor-intensive, requiring a great deal of manual processing of samples in a format that is not amenable to automation. Here, we present an automated version of the assay. This high-throughput alkaline elution assay (HT-AE) was made possible through 3 key developments: (1) DNA quantitation using PicoGreen and OliGreen fluorescent DNA binding dyes; (2) design and implementation of a custom automation system; and (3) reducing the assay to a 96-well plate format. The assay can now be run with 5-50mg of test compound. HT-AE was validated in a similar manner as the original assay, including assessment of non-genotoxic and non-carcinogenic compounds and evaluation of cytotoxicity to avoid confounding effects of toxicity-associated DNA degradation. The validation test results from compounds of known genotoxic potential were used to set appropriate criteria to classify alkaline elution results for genotoxicity.

  4. Early effects of Escherichia coli endotoxin infusion on vasopressin-stimulated breakdown and metabolism of inositol lipids in rat hepatocytes

    SciTech Connect

    Rodriguez de Turco, E.B.; Spitzer, J.A.

    1988-08-30

    The turnover of vasopressin-stimulated 32P-phosphoinositides and 32P-phosphatidic acid and accumulation of (2-3H)-inositol phosphates were examined in hepatocytes from rats infused i.v. with saline and E. coli endotoxin for 3 hrs. Within 60s of VP stimulation the decrease in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate labeling as well as the increased uptake of 32P into phosphatidic acid were similar in both groups. However, at a later time (300s) the 32P-phosphatidylinositol turnover was greatly decreased concomitantly with a higher labeling of phosphatidic acid. The accumulation of (2-3H)-inositol phosphates in ET-cells was significantly decreased both at 30s and 600s after VP addition. The distribution of (2-3H)-inositol labeling accumulated in the different inositol phosphate fractions over the first 30s of VP stimulation showed a tendency to lower accumulation of inositol trisphosphate, and a significantly lower accumulation of inositol bisphosphate simultaneously with a higher labeling of the inositol tetrakisphosphate fraction. These observations reflect an early effect of ET-infusion on VP-stimulated inositol lipid turnover and on the subsequent metabolism of the released inositol phosphates.

  5. Effect of proteasome inhibition on toxicity and CYP3A23 induction in cultured rat hepatocytes: Comparison with arsenite

    SciTech Connect

    Noreault-Conti, Trisha L.; Jacobs, Judith M.; Trask, Heidi W.; Wrighton, Steven A.; Sinclair, Jacqueline F.; Nichols, Ralph C. . E-mail: ralph.c.nichols@dartmouth.edu

    2006-12-15

    Previous work in our laboratory has shown that acute exposure of primary rat hepatocyte cultures to non-toxic concentrations of arsenite causes major decreases in the DEX-mediated induction of CYP3A23 protein, with minor decreases in CYP3A23 mRNA. To elucidate the mechanism for these effects of arsenite, the effects of arsenite and proteasome inhibition, separately and in combination, on induction of CYP3A23 protein were compared. The proteasome inhibitor, MG132, inhibited proteasome activity, but also decreased CYP3A23 mRNA and protein. Lactacystin, another proteasome inhibitor, decreased CYP3A23 protein without affecting CYP3A23 mRNA at a concentration that effectively inhibited proteasome activity. This result, suggesting that the action of lactacystin is similar to arsenite and was post-transcriptional, was confirmed by the finding that lactacystin decreased association of DEX-induced CYP3A23 mRNA with polyribosomes. Both MG132 and lactacystin inhibited total protein synthesis, but did not affect MTT reduction. Arsenite had no effect on ubiquitination of proteins, nor did arsenite significantly affect proteasomal activity. These results suggest that arsenite and lactacystin act by similar mechanisms to inhibit translation of CYP3A23.

  6. Hepatocyte Growth Factor Mediates the Antifibrogenic Action of Ocimum bacilicum Essential Oil against CCl4-Induced Liver Fibrosis in Rats.

    PubMed

    Ogaly, Hanan A; Eltablawy, Nadia A; El-Behairy, Adel M; El-Hindi, Hatim; Abd-Elsalam, Reham M

    2015-07-23

    The current investigation aimed to evaluate the antifibrogenic potential of Ocimum basilicum essential oil (OBE) and further to explore some of its underlying mechanisms. Three groups of rats were used: group I (control), group II (CCl4 model) and group III (OBE-treated) received CCl4 and OBE 2 weeks after the start of CCl4 administration. Oxidative damage was assessed by the measurement of MDA, NO, SOD, CAT, GSH and total antioxidant capacity (TAC). Liver fibrosis was assessed histopathologically by Masson's trichrome staining and α-smooth muscle actin (α-SMA) immunostaining. Expression of hepatocyte growth factor (HGF) and cytochrome P450 (CYP2EI isoform) was estimated using real-time PCR and immunohistochemistry. OBE successfully attenuated liver injury, as shown by histopathology, decreased serum transaminases and improved oxidative status of the liver. Reduced collagen deposition and α-SMA immuopositive cells indicated an abrogation of hepatic stellate cell activation by OBE. Furthermore, OBE was highly effective in stimulating HGF mRNA and protein expression and inhibiting CCl4-induced CYP2E1 down-regulation. The mechanism of antifibrogenic action of OBE is hypothesized to proceed via scavenging free radicals and activating liver regeneration by induction of HGF. These data suggest the use of OBE as a complementary treatment in liver fibrosis.

  7. Preserving low perfusion during surgical liver blood inflow control prevents hepatic microcirculatory dysfunction and irreversible hepatocyte injury in rats.

    PubMed

    Li, Chong-Hui; Chen, Yong-Wei; Chen, Yong-Liang; Yao, Li-Bin; Ge, Xin-Lan; Pan, Ke; Zhang, Ai-Qun; Dong, Jia-Hong

    2015-09-24

    Hepatic ischaemia/reperfusion (I/R) injury is of primary concern during liver surgery. We propose a new approach for preserving low liver blood perfusion during hepatectomy either by occlusion of the portal vein (OPV) while preserving hepatic artery flow or occlusion of the hepatic artery while limiting portal vein (LPV) flow to reduce I/R injury. The effects of this approach on liver I/R injury were investigated. Rats were randomly assigned into 4 groups: sham operation, occlusion of the portal triad (OPT), OPV and LPV. The 7-day survival rate was significantly improved in the OPV and LPV groups compared with the OPT group. Microcirculatory liver blood flow recovered rapidly after reperfusion in the OPV and LPV groups but decreased further in the OPT group. The OPV and LPV groups also showed much lower ALT and AST levels, Suzuki scores, inflammatory gene expression levels, and parenchymal necrosis compared with the OPT group. An imbalance between the expression of vasoconstriction and vasodilation genes was observed in the OPT group but not in the OPV or LPV group. Therefore, preserving low liver blood perfusion by either the OPV or LPV methods during liver surgery is very effective for preventing hepatic microcirculatory dysfunction and hepatocyte injury.

  8. Hepatocyte growth factor modification promotes the amelioration effects of human umbilical cord mesenchymal stem cells on rat acute kidney injury.

    PubMed

    Chen, Yuan; Qian, Hui; Zhu, Wei; Zhang, Xu; Yan, Yongmin; Ye, Shengqin; Peng, Xiujuan; Li, Wei; Xu, Wenrong

    2011-01-01

    Human umbilical cord-derived mesenchymal stem cells (hucMSCs) are particularly attractive cells for cellular and gene therapy in acute kidney injury (AKI). Adenovirus-mediated gene therapy has been limited by immune reaction and target genes selection. However, in the present study, we investigated the therapeutic effects of hepatocyte growth factor modified hucMSCs (HGF-hucMSCs) in ischemia/reperfusion-induced AKI rat models. In vivo animal models were generated by subjecting to 60 min of bilateral renal injury by clamping the renal pedicles and then introduced HGF-hucMSCs via the left carotid artery. Our results revealed that serum creatinine and urea nitrogen levels decreased to the baseline more quickly in HGF-hucMSCs-treated group than that in hucMSCs- or green fluorescent protein-hucMSCs-treated groups at 72 h after injury. The percent of proliferating cell nuclear antigen-positive cells in HGF-hucMSCs-treated group was higher than that in the hucMSCs or green fluorescent protein-hucMSCs-treated groups. Moreover, injured renal tissues treated with HGF-hucMSCs also exhibited less hyperemia and renal tubule cast during the recovery process. Immunohistochemistry and living body imaging confirmed that HGF-hucMSCs localize to areas of renal injury. Real-time polymerase chain reaction result showed that HGF-hucMSCs also inhibited caspase-3 and interleukin-1β mRNA expression in injured renal tissues. Western blot also showed HGF-hucMSCs-treated groups had lower expression of interleukin-1β. Terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate (dUTP) nick end labeling method indicated that HGF-hucMSCs-treated group had the least apoptosis cells. In conclusion, our findings suggest that HGF modification promotes the amelioration of ischemia/reperfusion-induced rat renal injury via antiapoptotic and antiinflammatory mechanisms; thus, providing a novel therapeutic application for hucMSCs in AKI.

  9. MPTP and MPTP analogs induced cell death in cultured rat hepatocytes involving the formation of pyridinium metabolites.

    PubMed

    Singh, Y; Swanson, E; Sokoloski, E; Kutty, R K; Krishna, G

    1988-11-01

    MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) which has been shown to produce a Parkinson-like syndrome in humans and monkeys also causes cell death in cultures of rat hepatocytes. Treatment of cells with MPTP or its metabolite MPP+ (1-methyl-4-phenyl pyridinium ion), resulted in leakage of lactic acid dehydrogenase and 14C-labeled adenine nucleotides, as well as marked depletion of ATP and glutathione. Deprenyl, a specific inhibitor of monoamine oxidase-B, the enzyme catalyzing the oxidation of MPTP into MPP+, blocked the lethal effect of MPTP, but gave no protection from MPP+-induced cell death. The 4'-fluoro and 4'-chloro analogs of MPTP evoked toxicities similar to that of the parent compound, while N-butyl-PTP, 4'-amino-MPTP, and 2'-methyl-MPTP were relatively less toxic. N-Acetylamino-MPTP was found virtually nontoxic. The cell death produced by these analogs was also associated with leakage of [14C]adenine nucleotides, which is an indicator of loss of ATP from cells. All these compounds except the N-acetylamino analog were converted to corresponding pyridinium metabolites by liver cells when analyzed by high-pressure liquid chromatography and plasma desorption mass spectrometry. MPTP and its analogs also served as substrates for rat liver mitochondrial monoamine oxidase to varying degrees. Toxicity of various analogs, with the noticeable exception of 2'-methyl-MPTP, was inhibited by deprenyl. These findings indicate that the conversion of MPTP and its analogs to corresponding pyridinium metabolites is essential for the expression of toxicity.

  10. Differentiation-Promoting Medium Additives for Hepatocyte Cultivation and Cryopreservation.

    PubMed

    Gouliarmou, Varvara; Pelkonen, Olavi; Coecke, Sandra

    2015-01-01

    Isolated primary hepatocytes are considered as the reference system for in vitro hepatic methods. Following the isolation of primary hepatocytes from liver tissue, an unfavorable process named dedifferentiation is initiated leading to the attenuation of the hepatocellular phenotype both at the morphological and functional level. Freshly isolated hepatocytes can be used immediately or can be cryopreserved for future purposes. Currently, a number of antidedifferentiation strategies exist to extend the life span of isolated hepatocytes. The addition of differentiation-promoting compounds to the hepatocyte culture medium is the oldest and simplest antidedifferentiation approach applied. In the present chapter, the most commonly used medium additives for cultivation and cryopreservation of primary hepatocytes are reviewed.

  11. HEPATIC ENZYME INDUCERS INCREASE THYROXINE (T4) CATABOLISM IN HUMAN AND RAT HEPATOCYTES

    EPA Science Inventory

    Nuclear receptor agonists such as 3-methylcholantrene (3-MC), phenobarbital (PB), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and, pregnenolone-16a-carbonitrile (PCN) decrease serum thyroxine (T4) concentrations in rats. It appears that this decrease occurs through the induction...

  12. Effects of hepatic enzyme inducers on thyroxine (T4) catabolism in primary rat hepatocytes

    EPA Science Inventory

    Nuclear receptor agonists such as phenobarbital (PB), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and 3-methylcholantrene (3-MC) decrease circulating thyroxine (T4) concentrations in rats. It is suspected that this decrease occurs through the induction of hepatic metabolizing en...

  13. PCB-153 AND BDE-47 INCREASE THYROXINE T4) CATABOLISM IN RAT AND HUMAN HEPATOCYTES

    EPA Science Inventory

    Previous studies demonstrate that in vivo exposure to 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) decrease serum thyroxine (T4) levels in rats. This decrease is thought to occur through the induction of hepatic metabolizing enzymes ...

  14. THYROXINE (T4) CATABOLISM IN HUMAN AND RAT HEPATOCYTES INCREASES FOLLOWING EXPOSURE TO HEPATIC ENZYME INDUCERS

    EPA Science Inventory

    Nuclear receptor agonists phenobarbital (PB), 3-methylcholanthrene (3MC), pregnenolone-16a-carbonitrile (PCN), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and 2,2' ,4,4'-tetrabromodiphenyl ether (BDE 47) decrease serum thyroxine (T4) in rats. This decrease is thought to occur th...

  15. Source-Related Effects of Wastewater on Transcription Factor (AhR, CAR and PXR)-Mediated Induction of Gene Expression in Cultured Rat Hepatocytes and Their Association with the Prevalence of Antimicrobial-Resistant Escherichia coli

    PubMed Central

    Guruge, Keerthi S.; Yamanaka, Noriko; Sonobe, Miyuki; Fujizono, Wataru; Yoshioka, Miyako; Akiba, Masato; Yamamoto, Takehisa; Joshua, Derrick I.; Balakrishna, Keshava; Yamashita, Nobuyoshi; Kannan, Kurunthachalam; Tsutsui, Toshiyuki

    2015-01-01

    Extracts of wastewater collected from 4 sewage treatment plants (STPs) receiving effluents from different sources in South India were investigated for their levels of transcription factor-mediated gene induction in primary cultured rat hepatocytes. In addition, the relation between gene induction levels and the prevalence of antimicrobial-resistant Escherichia coli (E. coli) in wastewater was examined. STP-3, which treats only hospital wastewater, exhibited significantly greater induction potency of all 6 drug metabolizing cytochrome P450 (CYP) genes examined, CYP1A1, 1A2, 1B1, 2B15, 3A1, and 3A2, whereas the wastewater at STP-1, which exclusively receives domestic sewage, showed significantly diminished levels of induction of 3 CYP genes when compared to the levels of CYP induction at STP-2, which receives mixed wastewater. Samples collected during the monsoon season showed a significantly altered gene induction capacity compared to that of samples from the pre-monsoon period. The data suggest that the toxicity of wastewater in STPs was not significantly diminished during the treatment process. The chemical-gene interaction data predicted that a vast number of chemicals present in the wastewater would stimulate the genes studied in the rat hepatocytes. The multivariable logistic regression analysis demonstrated that the prevalence of isolates resistant to cefotaxime, imipenem and streptomycin was significantly correlated with the levels of induction of at least three CYP-isozymes in STP wastewater. In addition, the resistance of isolates in treatment plants was not altered by the treatment steps, whereas the sampling season did have an impact on the resistance to specific antimicrobials. The identification of receptor-mediated gene regulation capacities offers important data not limited to the (synergistic) physiological role of chemicals in biological systems but may provide new insight into the link between the effects of known/unknown drugs and prevalence of

  16. Source-Related Effects of Wastewater on Transcription Factor (AhR, CAR and PXR)-Mediated Induction of Gene Expression in Cultured Rat Hepatocytes and Their Association with the Prevalence of Antimicrobial-Resistant Escherichia coli.

    PubMed

    Guruge, Keerthi S; Yamanaka, Noriko; Sonobe, Miyuki; Fujizono, Wataru; Yoshioka, Miyako; Akiba, Masato; Yamamoto, Takehisa; Joshua, Derrick I; Balakrishna, Keshava; Yamashita, Nobuyoshi; Kannan, Kurunthachalam; Tsutsui, Toshiyuki

    2015-01-01

    Extracts of wastewater collected from 4 sewage treatment plants (STPs) receiving effluents from different sources in South India were investigated for their levels of transcription factor-mediated gene induction in primary cultured rat hepatocytes. In addition, the relation between gene induction levels and the prevalence of antimicrobial-resistant Escherichia coli (E. coli) in wastewater was examined. STP-3, which treats only hospital wastewater, exhibited significantly greater induction potency of all 6 drug metabolizing cytochrome P450 (CYP) genes examined, CYP1A1, 1A2, 1B1, 2B15, 3A1, and 3A2, whereas the wastewater at STP-1, which exclusively receives domestic sewage, showed significantly diminished levels of induction of 3 CYP genes when compared to the levels of CYP induction at STP-2, which receives mixed wastewater. Samples collected during the monsoon season showed a significantly altered gene induction capacity compared to that of samples from the pre-monsoon period. The data suggest that the toxicity of wastewater in STPs was not significantly diminished during the treatment process. The chemical-gene interaction data predicted that a vast number of chemicals present in the wastewater would stimulate the genes studied in the rat hepatocytes. The multivariable logistic regression analysis demonstrated that the prevalence of isolates resistant to cefotaxime, imipenem and streptomycin was significantly correlated with the levels of induction of at least three CYP-isozymes in STP wastewater. In addition, the resistance of isolates in treatment plants was not altered by the treatment steps, whereas the sampling season did have an impact on the resistance to specific antimicrobials. The identification of receptor-mediated gene regulation capacities offers important data not limited to the (synergistic) physiological role of chemicals in biological systems but may provide new insight into the link between the effects of known/unknown drugs and prevalence of

  17. In vivo transfer of hepatocyte growth factor gene accelerates proliferation of hepatic oval cells in a 2-acetylaminofluorene/partial hepatectomy model in rats.

    PubMed

    Shiota, G; Kunisada, T; Oyama, K; Udagawa, A; Nomi, T; Tanaka, K; Tsutsumi, A; Isono, M; Nakamura, T; Hamada, H; Sakatani, T; Sell, S; Sato, K; Ito, H; Kawasaki, H

    2000-03-31

    To clarify the effect of hepatocyte growth factor (HGF) on proliferation of hepatic oval cells, we transferred HGF gene into liver of the Solt-Farber rat model. Male Fisher 344 rats were infected with a recombinant adenovirus carrying the cDNA for HGF (pAxCAHGF) from tail vein. HGF mRNA showed its peak at 4 days, and diminished thereafter. The total and proliferating cell nuclear antigen-positive hepatic oval cells were significantly elevated in HGF-transferred rats, in which stem cell factor and c-kit mRNA increased at each time point. Our results suggest that in vivo transfer of the HGF gene into liver accelerates proliferation of hepatic oval cells in the Solt-Farber model in rats.

  18. The use of primary rat hepatocytes to achieve metabolic activation of promutagens in the Chinese hamster ovary/hypoxantine-guanine phosphoribosyl transferase mutational assay

    SciTech Connect

    Bermudez, E.; Couch, D.B.; Tillery, D.

    1982-01-01

    A method is described in which primary rat hepatocytes have been cocultured with chinese hamster ovary (CHO) cells to provide metabolic activation of promutgens in the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) mutational assay. Single cell hepatocyte suspensions were prepared from male Fisher-344 rats using the in situ collagenase perfusion technique. Hepatocytes were allowed to attach for 1.5 hours in tissue culture dishes containing an approximately equal number of CHO cells in log growth. The cocultures were exposed to promutagens for up to 20 hours in serum-free medium. The survival and 6-thioguanine-resistant fraction of treated CHO cells were then determined as in the standard CHO/HGPRT assay. Aflatoxin B/sub 1/ (AFB/sub 1/) 7,12-dimethylbenz(a)anthracene (DMBA) and benzo(a)pyrene (B(a)P) were found to produce increases in the mutant fractions of treated CHO cells as a function of concentration. The time required for optimum expression of the mutant phenotype following exposure to DMBA and AFB/sub 1/ was approximately 8 days. Primary cell-mediated mutagenesis may be useful in elucidating methobolic pathways important in the production and detoxification of genotoxic products in vivo.

  19. Interferon-alpha preserves erythrocyte and hepatocyte ATPase activities from liver damage induced by prolonged bile duct ligation in the rat.

    PubMed

    Muriel, P

    1995-01-01

    Interferons have been used to treat chronic hepatitis owing to their antiviral properties. However, now interferons are recognized to inhibit collagen production. Because fibrosis has been associated with liver damage and dysfunction, the effects of interferon-alpha 2b on biliary obstruction-induced cirrhosis were investigated. Obstructive jaundice was induced in male Wistar rats (ca. 200 g) by double ligation and division of the common bile duct. Control rats were sham operated. Interferon-alpha 2b (IFN-alpha; 1000 000 IU per rat) was administered subcutaneously daily after surgery. The animals were sacrificed after 4 weeks of bile duct ligation (BDL) or sham operation. Bilirubins and serum enzyme activities of alkaline phosphatase and gamma-glutamyl transpeptidase (determined as markers of liver damage) increased several-fold after BDL. Erythrocyte and hepatocyte plasma membrane Na+/K+- and Ca2+-ATPase activities decreased significantly in the BDL group. Administration of IFN-alpha to BDL rats resulted in a partial normalization of serum markers of liver damage. The normal activity of both ATPases on erythrocyte and hepatocyte plasma membranes was completely preserved by IFN-alpha. It is concluded that interferons possess interesting hepatoprotective effects not related to their antiviral properties but probably associated with their antifibrogenic effect.

  20. Long-Term Selenium-Deficient Diet Induces Liver Damage by Altering Hepatocyte Ultrastructure and MMP1/3 and TIMP1/3 Expression in Growing Rats.

    PubMed

    Han, Jing; Liang, Hua; Yi, Jianhua; Tan, Wuhong; He, Shulan; Wang, Sen; Li, Feng; Wu, Xiaofang; Ma, Jing; Shi, Xiaowei; Guo, Xiong; Bai, Chuanyi

    2017-02-01

    The effects of selenium (Se)-deficient diet on the liver were evaluated by using growing rats which were fed with normal and Se-deficient diets, respectively, for 109 days. The results showed that rats fed with Se-deficient diet led to a decrease in Se concentration in the liver, particularly among male rats from the low-Se group. This causes alterations to the ultrastructure of hepatocytes with condensed chromatin and swelling mitochondria observed after low Se intake. Meanwhile, pathological changes and increased fibrosis in hepatic periportal were detected by hematoxylin and eosin and Masson's trichrome staining in low-Se group. Furthermore, through immunohistochemistry (IHC) staining, higher expressions of metalloproteinases (MMP1/3) and their tissue inhibitors of metalloproteinases (TIMP1/3) were observed in the hepatic periportal of rats from the low-Se group. However, higher expressions of MMP1/3 and lower expressions of TIMP1/3 were detected in hepatic central vein and hepatic sinusoid. In addition, upregulated expressions of MMP1/3 and downregulated expressions of TIMP1/3 at the messenger RNA (mRNA) and protein levels also appeared to be relevant to low Se intake. In conclusion, Se-deficient diet could cause low Se concentration in the liver, alterations of hepatocyte ultrastructure, differential expressions of MMP1/3 and TIMP1/3 as well as fibrosis in the liver hepatic periportal.

  1. Aroclor 1254 pretreatment effects on DNA repair in rat hepatocytes elicited by in vivo or in vitro exposure to various chemicals

    SciTech Connect

    Kornbrust, D.; Dietz, D.

    1985-01-01

    Inducers of liver mixed function oxidase (MFO) activities have profound effects on the genotoxicity of substances that undergo metabolic activation by the MFO system. The polychlorinated biphenyl mixture Aroclor 1254 is a broad-spectrum inducer of liver MFO activities that has been employed as a pretreatment to augment the metabolic activation capabilities of rat liver fractions used in a number of short-term tests for genotoxicity, including the Ames Salmonella/bacterial mutagenicity assay. The present study was designed to characterize the effects of Aroclor pretreatment of rats on the DNA repair responses elicited by various chemicals in the in vitro hepatocyte primary culture/DNA repair (HPC/DR) assay as well as the in vivo/in vitro HPC/DR assay. The amount of DNA repair produced in vitro by diethylnitrosamine (DEN), benzo(a)pyrene (B(a)P0, 3-methylcholanthrene (3-MC), 2-acetylaminofluorene (2-AAF), o-aminoazotoluene (o-AT), and aflatoxin B1 (AFB1) was significantly greater in hepatocytes derived from Aroclor-pretreated rats than in control rat hepatocytes. The pretreatment-related potentiation of DNA repair observed for 6 out of 12 compounds tested in vitro was considered to be due to enhanced metabolic activation. These results suggested that pretreatment with Aroclor may increase the sensitivity of the in vitro HPC/DR assay to certain compounds. In contrast, Aroclor pretreatment had little effect on the amount of hepatocellular DNA repair elicited by in vivo administration of DMN, DEN, o-AT, 2-AFF, 3-MC, or AFB1, which indicated that this pretreatment regimen may have little utility for improving the sensitivity of the in vivo/in vitro HPC/DR assay.

  2. Cytochemical Changes in Hepatocytes of Rats with Endotoxemia and Sepsis: Localization of Fibronectin, Calcium, and Enzymes

    DTIC Science & Technology

    1988-01-01

    magnification x 62,990. 5 N. t% 4A 0.. ’V .I f e o. 3 . 1m ~ .;.. ,~, &<: -- ~~ -. FigureS6. Calcium o~calization in the hepa- * *~ $ S tocytes of LPS...1985) reported that intravenous administration of E . coli LPS to portation of plasma fibronectin into the basal plasma membrane rats significantly...murinehepatitiscaused thesis inhibition. Anm X Ma)d Sit 3861O37 p by frog virus 3 . J Infect Dis 149:621 Gold L. Pearlsyce E 1980oin h8Fibmn.etin

  3. Synergistic acceleration of thyroid hormone degradation by phenobarbital and the PPAR{alpha} agonist WY14643 in rat hepatocytes

    SciTech Connect

    Wieneke, N.; Neuschaefer-Rube, F.; Bode, L.M.; Kuna, M.; Andres, J.; Carnevali, L.C.; Hirsch-Ernst, K.I.; Pueschel, G.P.

    2009-10-01

    Energy balance is maintained by controlling both energy intake and energy expenditure. Thyroid hormones play a crucial role in regulating energy expenditure. Their levels are adjusted by a tight feedback-controlled regulation of thyroid hormone production/incretion and by their hepatic metabolism. Thyroid hormone degradation has previously been shown to be enhanced by treatment with phenobarbital or other antiepileptic drugs due to a CAR-dependent induction of phase II enzymes of xenobiotic metabolism. We have recently shown, that PPAR{alpha} agonists synergize with phenobarbital to induce another prototypical CAR target gene, CYP2B1. Therefore, it was tested whether a PPAR{alpha} agonist could enhance the phenobarbital-dependent acceleration of thyroid hormone elimination. In primary cultures of rat hepatocytes the apparent half-life of T3 was reduced after induction with a combination of phenobarbital and the PPAR{alpha} agonist WY14643 to a larger extent than after induction with either compound alone. The synergistic reduction of the half-life could be attributed to a synergistic induction of CAR and the CAR target genes that code for enzymes and transporters involved in the hepatic elimination of T3, such as OATP1A1, OATP1A3, UGT1A3 and UGT1A10. The PPAR{alpha}-dependent CAR induction and the subsequent induction of T3-eliminating enzymes might be of physiological significance for the fasting-induced reduction in energy expenditure by fatty acids as natural PPAR{alpha} ligands. The synergism of the PPAR{alpha} agonist WY14643 and phenobarbital in inducing thyroid hormone breakdown might serve as a paradigm for the synergistic disruption of endocrine control by other combinations of xenobiotics.

  4. Apoptosis induced by tumor necrosis factor-alpha in rat hepatocyte cell lines expressing hepatitis B virus.

    PubMed Central

    Guilhot, S.; Miller, T.; Cornman, G.; Isom, H. C.

    1996-01-01

    Three well differentiated SV40-immortalized rat hepatocyte cell lines, CWSV1, CWSV2, and CWSV14, and Hepatitis B Virus (HBV)-producing cell lines derived from them were examined for sensitivity to tumor necrosis factor (TNF)-alpha. CWSV1, CWSV2, and CWSV14 cells were co-transfected with a DNA construct containing a dimer of the HBV genome and the neo gene and selected in G418 to generate stable cell lines. Characterization of these cell lines indicated that they contain integrated HBV DNA, contain low molecular weight HBV DNA compatible with the presence of HBV replication intermediates, express HBV transcripts, and produce HBV proteins. The viability of CWSV1, CWSV2, and CWSV2 cells was not significantly altered when they were treated with TNF-alpha at concentrations as high as 20,000 U/ml. The HBV-expressing CWSV1 cell line, SV1di36, and the HBV-expressing CWSV14 cell line, SV14di208, were also not killed when treated with TNF-alpha. However, the HBV-expressing CWSV2 cell line, SV2di366, was extensively killed when treated with TNF-alpha at concentrations ranging from 200 to 20,000 U/ml. Analysis of several different HBV-producing CWSV2 cell lines indicated that TNF-alpha killing depended upon the level of HBV expression. The TNF-alpha-induced cell killing in high HBV-producing CWSV2 cell lines was accompanied by the presence of an oligonucleosomal DNA ladder characteristic of apoptosis. Images Figure 2 Figure 3 Figure 4 Figure 6 Figure 9 Figure 10 Figure 11 PMID:8774135

  5. Studies on responsiveness of hepatoma cells to catecholamines. IV. Lack of adrenergic activation of phosphorylase in rat ascites hepatoma cells.

    PubMed

    Miyamoto, K; Yanaoka, T; Sanae, F; Wakusawa, S; Koshiura, R

    1986-10-01

    Glycogen phosphorylase a activity in 7 rat ascites hepatoma cell lines treated with adrenergic agents, phenylephrine, epinephrine and isoproterenol, was investigated as compared with that in freshly isolated rat hepatocytes. Basal phosphorylase activities in hepatoma cells except AH7974 cells were lower than that in hepatocytes. Phosphorylase in hepatoma cells was not activated by any of the agents, while the enzyme activity in hepatocytes was clearly increased in a dose- and time-dependent manner. Phosphorylase in hepatocytes was sensitive to glucagon, but it was found to be insensitive to glucagon in all hepatoma cells. The present results suggest that rat ascites hepatoma cells may escape the glycogenolytic regulation by catecholamines and glucagon.

  6. Formation of human hepatocyte-like cells with different cellular phenotypes by human umbilical cord blood-derived cells in the human-rat chimeras

    SciTech Connect

    Sun, Yan; Xiao, Dong; Zhang, Ruo-Shuang; Cui, Guang-Hui; Wang, Xin-Hua; Chen, Xi-Gu . E-mail: xiguchen1516@yahoo.com.cn

    2007-06-15

    We took advantage of the proliferative and permissive environment of the developing pre-immune fetus to develop a noninjury human-rat xenograft small animal model, in which the in utero transplantation of low-density mononuclear cells (MNCs) from human umbilical cord blood (hUCB) into fetal rats at 9-11 days of gestation led to the formation of human hepatocyte-like cells (hHLCs) with different cellular phenotypes, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), cytokeratin 19 (CK19), cytokeratin 8 (CK8), cytokeratin 18 (CK18), and albumin (Alb), and with some animals exhibiting levels as high as 10.7% of donor-derived human cells in the recipient liver. More interestingly, donor-derived human cells stained positively for CD34 and CD45 in the liver of 2-month-old rat. Human hepatic differentiation appeared to partially follow the process of hepatic ontogeny, as evidenced by the expression of AFP gene at an early stage and albumin gene at a later stage. Human hepatocytes generated in this model retained functional properties of normal hepatocytes. In this xenogeneic system, the engrafted donor-derived human cells persisted in the recipient liver for at least 6 months after birth. Taken together, these findings suggest that the donor-derived human cells with different cellular phenotypes are found in the recipient liver and hHLCs hold biological activity. This humanized small animal model, which offers an in vivo environment more closely resembling the situations in human, provides an invaluable approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future.

  7. Hep-G2 cells and primary rat hepatocytes differ in their response to inhibitors of HMG-CoA reductase.

    PubMed

    Shaw, M K; Newton, R S; Sliskovic, D R; Roth, B D; Ferguson, E; Krause, B R

    1990-07-31

    CI-981, a novel synthetic inhibitor of HMG-CoA reductase, was previously reported to be highly liver-selective using an ex vivo approach. In order to determine liver-selectivity at the cellular level, CI-981 was evaluated in cell culture and compared to lovastatin, pravastatin, fluvastatin and BMY-21950. Using human cell lines, none of the compounds tested showed liver-selectivity, i.e. strong inhibition of cholesterol synthesis in Hep-G2 cells (liver model) but weak inhibition in human fibroblasts (peripheral cell model). In contrast, all drugs tested produced equal and potent inhibition of sterol synthesis in primary cultures of rat hepatocytes, and CI-981, pravastatin and BMY-21950 were more than 100-fold more potent in rat hepatocytes compared to human fibroblasts. Since all compounds were also equally potent at inhibiting sterol synthesis in a rat subcellular system and in vivo, the data suggest that the use of Hep-G2 cells may not be the cell system of choice in which to study inhibition of hepatic cholesterogenesis or to demonstrate liver selectivity of inhibitors of HMG-CoA reductase.

  8. Antioxidative and apoptotic properties of polyphenolic extracts from edible part of artichoke (Cynara scolymus L.) on cultured rat hepatocytes and on human hepatoma cells.

    PubMed

    Miccadei, Stefania; Di Venere, Donato; Cardinali, Angela; Romano, Ferdinando; Durazzo, Alessandra; Foddai, Maria Stella; Fraioli, Rocco; Mobarhan, Sohrab; Maiani, Giuseppe

    2008-01-01

    Cultured rat hepatocytes and human hepatoma HepG2 cells were used to evaluate the hepatoprotective properties of polyphenolic extracts from the edible part of artichoke (AE). The hepatocytes were exposed to H2O2generated in situ by glucose oxidase and were treated with either AE, or pure chlorogenic acid (ChA) or with the well known antioxidant, N, N'-diphenyl-p-phenilenediamine (DPPD). Addition of glucose oxidase to the culture medium caused depletion of intracellular glutathione (GSH) content, accumulation of malondialdehyde (MDA) in the cultures, as a lipid peroxidation indicator, and cell death. These results demonstrated that AE protected cells from the oxidative stress caused by glucose oxidase, comparable to DPPD. Furthermore, AE, as well as ChA, prevented the loss of total GSH and the accumulation of MDA. Treatment of HepG2 cells for 24 h with AE reduced cell viability in a dose-dependent manner, however, ChA had no prominent effects on the cell death rate. Similarly, AE rather than ChA induced apoptosis, measured by flow cytometric analysis of annexin and by activation of caspase-3, in HepG2 cells. Our findings indicate that AE had a marked antioxidative potential that protects hepatocytes from an oxidative stress. Furthermore, AE reduced cell viability and had an apoptotic activity on a human liver cancer cell line.

  9. Hepatoprotective Activity of Water Extracts from Chaga Medicinal Mushroom, Inonotus obliquus (Higher Basidiomycetes) Against Tert-Butyl Hydroperoxide-Induced Oxidative Liver Injury in Primary Cultured Rat Hepatocytes.

    PubMed

    Hong, Ki Bae; Noh, Dong Ouk; Park, Yooheon; Suh, Hyung Joo

    2015-01-01

    We examined the hepatoprotective activity of Inonotus obliquus water extract (IO-W) against tert-butyl hydroperoxide (t-BHP)-induced oxidative liver injury in the primary cultured rat hepatocyte. The 50% radical scavenging concentrations (SC50s) of IO-W for radical-scavenging activity against 2,2'-azino-bis-(3-ethylbenzothi- azoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) were 5.19 mg/mL and 0.39 mg/mL, respectively. IO-W pretreatment to the primary cultured hepatocytes significantly (p<0.05) protected the cells from t-BHP-induced cytotoxic injury even at a low concentration of IO-W (10 µg/mL). The cellular leakage of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH), as well as malondialdehyde (MDA) formation caused by t-BHP were significantly (p<0.05) suppressed by IO-W pretreatment (>100 µg/ mL). In conclusion, this study demonstrates that IO-W exhibited hepatoprotective activity against t-BHP-induced oxidative liver injury in the primary cultured hepatocyte probably via its abilities of quenching free radicals, inhibiting the leakage of ALT, AST, and LDH, and decreasing MDA formation.

  10. Hepatocyte Polarity

    PubMed Central

    Treyer, Aleksandr; Müsch, Anne

    2013-01-01

    Hepatocytes, like other epithelia, are situated at the interface between the organism’s exterior and the underlying internal milieu and organize the vectorial exchange of macromolecules between these two spaces. To mediate this function, epithelial cells, including hepatocytes, are polarized with distinct luminal domains that are separated by tight junctions from lateral domains engaged in cell-cell adhesion and from basal domains that interact with the underlying extracellular matrix. Despite these universal principles, hepatocytes distinguish themselves from other nonstriated epithelia by their multipolar organization. Each hepatocyte participates in multiple, narrow lumina, the bile canaliculi, and has multiple basal surfaces that face the endothelial lining. Hepatocytes also differ in the mechanism of luminal protein trafficking from other epithelia studied. They lack polarized protein secretion to the luminal domain and target single-spanning and glycosylphosphatidylinositol-anchored bile canalicular membrane proteins via transcytosis from the basolateral domain. We compare this unique hepatic polarity phenotype with that of the more common columnar epithelial organization and review our current knowledge of the signaling mechanisms and the organization of polarized protein trafficking that govern the establishment and maintenance of hepatic polarity. The serine/threonine kinase LKB1, which is activated by the bile acid taurocholate and, in turn, activates adenosine monophosphate kinase-related kinases including AMPK1/2 and Par1 paralogues has emerged as a key determinant of hepatic polarity. We propose that the absence of a hepatocyte basal lamina and differences in cell-cell adhesion signaling that determine the positioning of tight junctions are two crucial determinants for the distinct hepatic and columnar polarity phenotypes. PMID:23720287

  11. Attenuated Lead Induced Apoptosis in Rat Hepatocytes in the Presence of Lycopersicon Esculentum.

    PubMed

    Ahmadi Ashtiani, Hamidreza; Khaki, Arash; Ejtemaei Mehr, Shahram; Anjarani, Soghra; Dadgarnejad, Manochehr; Alebouyeh, Mahmoud; Rastegar, Hossein

    2016-04-01

    Lead (Pb), has, for decades, being known for its adverse effects on various body organs and systems. In the present study, the damage of Pb on the Liver tissue apoptosis was investigated, and Lycopersicon esculentum as an antioxidants source was administered orally to prevent the adverse effects of Pb. Eighteen Wistar rats, randomized into three groups (n=6), were used for this study. Animals in Group A served as the control and were drinking distilled water. Animals in Groups B and C were drinking 1%Lead acetate (LA). Group C animals were, in addition to drinking LA, treated with 1.5 ml/day of Lycopersicon esculentum. Treatments were for three months. The obtained results showed that lead acetate caused significant reductions in the liver weight, plasma and tissue superoxide dismutase and catalase activity, but a significant increase in plasma and tissue malondialdehyde concentration but Lycopersicon esculentum have an inhibitory effect on LA liver adverse effect. So, it can be concluded that Lycopersicon esculentum have a significant protective effect on liver lead acetate adverse effects as well as, lead acetate-induced oxidative stress.

  12. Intracellular and transcellular transport of secretory and membrane proteins in the rat hepatocyte

    SciTech Connect

    Sztul, E.S.

    1984-01-01

    The intra- and transcellular transport of hepatic secretory and membrane proteins was studied in rats in vivo using (/sup 3/H)fucose and (/sup 35/S)cyteine as metabolic precursors. Incorporated radioactivity in plasma, bile, and liver subcellular fractions was measured and the labeled proteins of the Golgi complex, bile and plasma were separated by SDS-PAGE and identified by fluorography. /sup 3/H-radioactivity in Golgi fractions peaked at 10 min post injection (p.i.) and then declined concomitantly with the appearance of labeled glycoproteins in plasma. Maximal secretion of secretory fucoproteins from the Golgi complex occurred between 10 and 20 min p.i. In contrast, the clearance of labeled proteins from Golgi membrane subfractions occurred past 30 min p.i., indicating that membrane proteins leave the Golgi complex at least 10 min later than the bulk of content proteins. A major 80K form of Secretory Component (SC) was identified in the bile by precipitation with an anti IgA antibody. A comparative study of kinetics of transport of /sup 35/S-labeled SC and /sup 35/S-labeled albumin showed that albumin peaked in bile at approx.45 min p.i., whereas the SC peak occurred at 80 min p.i., suggesting that the transit time differs for plasma and membrane proteins which are delivered to the bile canaliculus (BC).

  13. Subtoxic Alterations in Hepatocyte-Derived Exosomes: An Early Step in Drug-Induced Liver Injury?

    PubMed

    Holman, Natalie S; Mosedale, Merrie; Wolf, Kristina K; LeCluyse, Edward L; Watkins, Paul B

    2016-06-01

    Drug-induced liver injury (DILI) is a significant clinical and economic problem in the United States, yet the mechanisms that underlie DILI remain poorly understood. Recent evidence suggests that signaling molecules released by stressed hepatocytes can trigger immune responses that may be common across DILI mechanisms. Extracellular vesicles released by hepatocytes, principally hepatocyte-derived exosomes (HDEs), may constitute one such signal. To examine HDE alterations as a function of drug-induced stress, this work utilized prototypical hepatotoxicant acetaminophen (APAP) in male Sprague-Dawley (SD) rats, SD rat hepatocytes, and primary human hepatocytes. HDE were isolated using ExoQuick precipitation reagent and analyzed by quantification of the liver-specific RNAs albumin and microRNA-122 (miR-122). In vivo, significant elevations in circulating exosomal albumin mRNA were observed at subtoxic APAP exposures. Significant increases in exosomal albumin mRNA were also observed in primary rat hepatocytes at subtoxic APAP concentrations. In primary human hepatocytes, APAP elicited increases in both exosomal albumin mRNA and exosomal miR-122 without overt cytotoxicity. However, the number of HDE produced in vitro in response to APAP did not increase with exosomal RNA quantity. We conclude that significant drug-induced alterations in the liver-specific RNA content of HDE occur at subtoxic APAP exposures in vivo and in vitro, and that these changes appear to reflect selective packaging rather than changes in exosome number. The current findings demonstrate that translationally relevant HDE alterations occur in the absence of overt hepatocellular toxicity, and support the hypothesis that HDE released by stressed hepatocytes may mediate early immune responses in DILI.

  14. Signal Transduction Mechanism for Serotonin 5-HT2B Receptor-Mediated DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes.

    PubMed

    Naito, Kota; Tanaka, Chizuru; Mitsuhashi, Manami; Moteki, Hajime; Kimura, Mitsutoshi; Natsume, Hideshi; Ogihara, Masahiko

    2016-01-01

    The involvement of serotonin (5-hydroxytryptamine; 5-HT) and the 5-HT2 receptor subtypes in the induction of DNA synthesis and proliferation was investigated in primary cultures of adult rat hepatocytes to elucidate the intracellular signal transduction mechanisms. Hepatocyte parenchymal cells maintained in a serum-free, defined medium, synthesized DNA and proliferated in the presence of 5-HT or a selective 5-HT2B receptor agonist, BW723C86, but not in the presence of 5-HT2A, or 5-HT2C receptor agonists (TCB-2 and CP809101, respectively), in a time- and dose-dependent manner. A selective 5-HT2B receptor antagonist, LY272015 (10(-7) M), and a specific phospholipase C (PLC) inhibitor, U-73122 (10(-6) M), as well as specific inhibitors of growth-related signal transducers-including AG1478, LY294002, PD98059, and rapamycin-completely inhibited 5-HT (10(-6) M)- or BW723C86 (10(-6) M)-induced hepatocyte DNA synthesis and proliferation. Both 5-HT and BW723C86 were shown to significantly stimulate the phosphorylation of epidermal growth factor (EGF)/transforming growth factor (TGF)-α receptor tyrosine kinase (p175 kDa) and extracellular signal-regulated kinase (ERK) 2 on Western blot analysis. These results suggest that the proliferative mechanism of activating 5-HT is mediated mainly through 5-HT2B receptor-stimulated Gq/PLC and EGF/TGF-α-receptor/phosphatidylinositol 3-kinase (PI3K)/ERK2/mammalian target of rapamycin (mTOR) signaling pathways in primary cultured hepatocytes.

  15. Desaturation and chain elongation of essential fatty acids in isolated liver cells from rat and rainbow trout

    SciTech Connect

    Hagve, T.A.; Christophersen, B.O.; Dannevig, B.H.

    1986-03-01

    Isolated hepatocytes from rainbow trout and rat were incubated with /sup 14/C-labeled linoleic acid, linolenic acid, dihomogammalinolenic acid or eicosapentaenoic acid. The most striking difference in the desaturase activity was the lower level of delta 5 desaturase in trout than in rat. No delta 4 desaturation of 22:4(n-6) to 22:5(n-6) was observed in either of the two species, while the conversion of 22:5(n-3) to 22:6(n-3) was significant in both groups and highest in rainbow trout. The chain-elongating activity was remarkably similar in the two species, except for the dead-end elongation which was distinctly more important in fish.

  16. Hormonally produced changes in caeruloplasmin synthesis and secretion in primary cultured rat hepatocytes. Relationship to hepatic copper metabolism.

    PubMed Central

    Weiner, A L; Cousins, R J

    1983-01-01

    Hormonally produced changes in the synthesis and secretion of the serum copper-containing protein caeruloplasmin were studied in primary cultures of rat liver parenchymal cells isolated by the collagenase-perfusion technique. A rabbit antibody directed against rat caeruloplasmin was used to immunoprecipitate labelled caeruloplasmin. Isolated liver cells synthesized and secreted caeruloplasmin over a period of 3 days. Synthesis and secretion of this protein was enhanced when cells were treated with dexamethasone. The accumulation of copper was also moderately enhanced with glucocorticoid treatment. Inclusion of adrenaline in the culture medium resulted in elevated incorporation of copper into newly synthesized caeruloplasmin as well as an increase in 64Cu-labelled caeruloplasmin in the culture medium. However, adrenaline did not seem to increase the secretion of 3H-labelled protein, despite the elevation in secreted 64Cu-caeruloplasmin. This may be due to a large increase in the intracellular pool of 64Cu caused by enhanced accumulation of this metal when adrenaline is included in the incubation medium. Enhanced copper accumulation was also seen when cells were treated with glucagon. Adrenaline-stimulated accumulation of 64Cu could be inhibited by including phenoxybenzamine, an alpha-adrenergic blocker, in the culture medium. Elevation of extracellular copper caused enhancement in the detection of labelled caeruloplasmin in the medium of cultured cells, probably owing to the ability of this metal to stabilize the protein. Images Fig. 1. PMID:6882374

  17. Rat hepatocarcinogenesis induced by N-nitrosodiethylamine and N-nitrosomorpholine continuously administered at low doses. From basophilic areas of hepatocytes to hepatocellular tumors.

    PubMed Central

    Cortinovis, C.; Klimek, F.; Nogueira, E.

    1991-01-01

    The development of hepatocellular tumors was investigated with histological, histochemical, and morphometrical methods in male Sprague-Dawley rats continuously administered N-nitrosodiethylamine (DEN) or N-nitrosomorpholine (NNM) in the drinking water at low doses (0.5 mg DEN/100 ml; 1 mg NNM/100 ml). Groups of control, DEN-, and NNM-treated rats were investigated at 5-week intervals. Similar results were obtained in DEN- and NNM-treated rats. Two types of areas composed of basophilic or glycogenotic hepatocytes were observed preceding the appearance of hepatocellular adenomas and carcinomas. Besides their cytologic differences, the basophilic and glycogenotic areas induced displayed distinct histochemical features. Both types of areas were detected simultaneously and increased in parallel with time to a similar incidence, but basophilic areas reached larger sizes than the glycogenotic ones. Furthermore, each type of area, which clustered around and along efferent veins, was differently linked to tumorigenesis. Basophilic areas frequently developed into basophilic adenomas and trabecular carcinomas through a characteristic sequence. Early basophilic areas consisted of hepatocytes with lamellar cytoplasmic hyperbasophilia and exhibited the normal laminar liver structure. With time, an increasing number of basophilic areas also contained hepatocytes with powdered diffuse hyperbasophilia, which frequently were arranged in thick trabeculae, showed abundant mitotic figures, and invaded efferent veins. Neither such signs of malignancy nor conversion into basophilic areas or tumors could be established for areas of clear and acidophilic glycogenotic hepatocytes. However, a few small glycogenotic adenomas probably developed from glycogenotic areas. Our data thus underline the central role of basophilic areas for hepatocarcinogenesis. Moreover, taking into account the data from other experiments, it seems likely that although glycogenotic areas may be associated with the

  18. Protein and mRNA expression of Shh, Smo and Gli1 and inhibition by cyclopamine in hepatocytes of rats with chronic fluorosis.

    PubMed

    Zhao, Lina; Yu, Yanni; Deng, Chaonan

    2014-03-03

    In order to investigate the Sonic hedgehog (Shh) signaling pathway and the effect of cyclopamine in rat hepatocytes with chronic fluorosis, 48 Wistar rats were randomly divided into 4 groups. The control group was provided with tap water in which the fluorine concentration was <1mg/L, while the remaining three groups were provided with water containing sodium fluoride (NaF) at a concentration of 50mg/L. After 6 months, the blocking and blocking control groups were injected intraperitoneally once every 2 days for 6 days with 10mg/kg cyclopamine or dimethyl sulfoxide, respectively. The urinary and skeletal fluoride contents were determined by the ion selective electrode method. Levels of aspartate transaminase (AST), alanine transaminase (ALT), total protein (TP) and albumin (Alb) in the serum were determined by using autobiochemical machine. Histological changes in liver tissue were evaluated with Hematoxylin & Eeosin (H&E) staining using light microscopy. The protein and mRNA expression of Shh, Smo and Gli1 in hepatocytes of experimental animals was determined by immunohistochemistry (IHC), Western blotting (Wb) and Real-time quantitative PCR (RT-qPCR). Fluoride content of the urine and bone was increased in the fluorosis and blocking groups compared to those in the control group (P<0.05), while fluoride content in the blocking group was decreased compared to the fluorosis and blocking control groups (P<0.05). The expression of Shh, Smo and Gli1 at the mRNA and protein levels was significantly increased in hepatocytes from the fluorosis and blocking control groups compared with the control group, and expression in the blocking group was lower than that of the fluorosis and blocking control groups. The difference between any two groups was considered to be statistically significant (P<0.05). Taken together, our study indicates that the expression of Shh, Smo and Gli1 at the protein and mRNA level in hepatocytes of rats with chronic fluorosis can be increased by

  19. DHA down-regulates phenobarbital-induced cytochrome P450 2B1 gene expression in rat primary hepatocytes by attenuating CAR translocation

    SciTech Connect

    Li, C.-C.; Lii, C.-K.; Liu, K.-L.; Yang, J.-J.; Chen, H.-W.

    2007-12-15

    The constitutive androstane receptor (CAR) plays an important role in regulating the expression of detoxifying enzymes, including cytochrome P450 2B (CYP 2B). Phenobarbital (PB) induction of human CYP 2B6 and mouse CYP 2b10 has been shown to be mediated by CAR. Our previous study showed that PB-induced CYP 2B1 expression in rat primary hepatocytes is down-regulated by both n-6 and n-3 polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA); however, the mechanism for this down-regulation by DHA was previously unknown. The objective of the present study was to determine whether change in CAR translocation is involved in the down-regulation by n-6 and n-3 PUFAs of PB-induced CYP 2B1 expression in rat primary hepatocytes. We used 100 {mu}M arachidonic acid, linoleic acid, eicosapentaenoic acid, and DHA to test this hypothesis. PB triggered the translocation of CAR from the cytosol into the nucleus in a dose-dependent and time-dependent manner in our hepatocyte system, and the CAR distribution in rat primary hepatocytes was significantly affected by DHA. DHA treatment decreased PB-inducible accumulation of CAR in the nuclear fraction and increased it in the cytosolic fraction in a dose-dependent manner. The down-regulation of CYP 2B1 expression by DHA occurred in a dose-dependent manner, and a similar pattern was found for the nuclear accumulation of CAR. The results of immunoprecipitation showed a CAR/RXR heterodimer bound to nuclear receptor binding site 1 (NR-1) of the PB-responsive enhancer module (PBREM) of the CYP 2B1gene. The EMSA results showed that PB-induced CAR binding to NR-1 was attenuated by DHA. Taken together, these results suggest that attenuation of CAR translocation and decreased subsequent binding to NR-1 are involved in DHA's down-regulation of PB-induced CYP 2B1 expression.

  20. Hypocholesterolemic effects of Kluyveromyces marxianus M3 isolated from Tibetan mushrooms on diet-induced hypercholesterolemia in rat

    PubMed Central

    Xie, Yuanhong; Zhang, Hongxing; Liu, Hui; Xiong, Lixia; Gao, Xiuzhi; Jia, Hui; Lian, Zhengxing; Tong, Nengsheng; Han, Tao

    2015-01-01

    To investigate the effects of Kluyveromyces marxianus M3 isolated from Tibetan mushrooms on diet-induced hypercholesterolemia in rats, female Wistar rats were fed a high-cholesterol diet (HCD) for 28 d to generate hyperlipidemic models. Hyperlipidemic rats were assigned to four groups, which were individually treated with three different dosages of K. marxianus M3+HCD or physiological saline+HCD via oral gavage for 28 d. The total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels in the serum and liver of the rats were measured using commercially available enzyme kits. In addition, the liver morphology was also examined using hematoxylin and eosin staining and optical microscopy. According to our results, the serum and liver TC, TG, LDL-C levels and atherogenic index (AI) were significantly decreased in rats orally administered K. marxianus M3 (p <0.01), and the HDL-C levels and anti atherogenic index (AAI) were significantly increased (p <0.01) compared to the control group. Moreover, K. marxianus M3 treatment also reduced the build-up of lipid droplets in the liver and exhibited normal hepatocytes, suggesting a protective effect of K. marxianus M3 in hyperlipidemic rats. PMID:26273253

  1. Location of rRNA transcription to the nucleolar components: disappearance of the fibrillar centers in nucleoli of regenerating rat hepatocytes.

    PubMed

    Montanaro, Lorenzo; Govoni, Marzia; Orrico, Catia; Treré, Davide; Derenzini, Massimo

    2011-01-01

    The precise location of rDNA transcription to the components of mammalian cell nucleolus is still debated. This was due to the fact that all the molecules necessary for rRNA synthesis are located in two of the three components, the fibrillar centers (FCs) and the dense fibrillar component (DFC), which together with the granular component (GC) are considered to be constantly present in mammalian cell nucleoli. In the present study we demonstrated that in nucleoli of many regenerating rat hepatocytes at 15 h after partial hepatectomy the FCs were no longer present, only the DFC and the GC being detected. At this time of regeneration the rRNA transcriptional activity was three fold that of resting hepatocytes, while the synthesis of DNA was not yet significantly increased, indicating that these nucleolar changes were due to the rRNA synthesis up-regulation. The DFC appeared to be organized in numerous, small, roundish tufts of fibrils. The silver staining procedure for AgNOR proteins, which are associated with the ribosomal genes, selectively and homogeneously stained these fibrillar tufts. Immuno-gold visualization of the Upstream Binding Factor (UBF), which is associated with the promoter region and the transcribed portion of the rRNA 45S gene, demonstrated that UBF was selectively located in the fibrillar tufts. We concluded that in proliferating rat hepatocytes the increased synthesis of rRNA induced an activation of the rRNA transcription machinery located in the fibrillar centers which, by becoming associated with the ribonucleoprotein transcripts, assumed the morphological pattern of the DFC.

  2. Glutathione depletion by valproic acid in sandwich-cultured rat hepatocytes: Role of biotransformation and temporal relationship with onset of toxicity

    SciTech Connect

    Kiang, Tony K.L.; Teng Xiaowei; Surendradoss, Jayakumar; Karagiozov, Stoyan; Abbott, Frank S.; Chang, Thomas K.H.

    2011-05-01

    The present study was conducted in sandwich-cultured rat hepatocytes to investigate the chemical basis of glutathione (GSH) depletion by valproic acid (VPA) and evaluate the role of GSH depletion in VPA toxicity. Among the synthetic metabolites of VPA investigated, 4-ene-VPA and (E)-2,4-diene-VPA decreased cellular levels of total GSH, but only (E)-2,4-diene-VPA was more effective and more potent than the parent drug. The in situ generated, cytochrome P450-dependent 4-ene-VPA did not contribute to GSH depletion by VPA, as suggested by the experiment with a cytochrome P450 inhibitor, 1-aminobenzotriazole, to decrease the formation of this metabolite. In support of a role for metabolites, alpha-F-VPA and octanoic acid, which do not undergo biotransformation to form a 2,4-diene metabolite, CoA ester, or glucuronide, did not deplete GSH. A time course experiment showed that GSH depletion did not occur prior to the increase in 2',7'-dichlorofluorescein (a marker of oxidative stress), the decrease in [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (WST-1) product formation (a marker of cell viability), or the increase in lactate dehydrogenase (LDH) release (a marker of necrosis) in VPA-treated hepatocytes. In conclusion, the cytochrome P450-mediated 4-ene-VPA pathway does not play a role in the in situ depletion of GSH by VPA, and GSH depletion is not an initiating event in VPA toxicity in sandwich-cultured rat hepatocytes.

  3. Curcumin regulates the metabolism of low density lipoproteins by improving the C-to-U RNA editing efficiency of apolipoprotein B in primary rat hepatocytes.

    PubMed

    Tian, Nan; Li, Xiaoling; Luo, Yang; Han, Ziwu; Li, Zhaohui; Fan, Chunlei

    2014-01-01

    There are two isoforms of apolipoprotein B (apoB) in mammals: apoB-100 and apoB-48. The latter is generated by C-to-U editing of apoB mRNA, catalyzed by the apolipoprotein B mRNA editing enzyme, namely, catalytic polypeptide 1 (APOBEC-1). Lipid particles containing apoB-48 are cleared from the plasma more rapidly than those containing apoB-100 and thus do not contribute to plaque formation in the arterial wall. In the present study, we analyzed whether curcumin is capable of regulating lipid metabolism by improving the level of apoB mRNA editing. The cytotoxicity of curcumin in hepatocytes was determined using the 3-(4,5-dimethylthiazol‑2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the levels of APOBEC-1 mRNA and protein were analyzed by real-time quantitative polymerase chain reaction (qRT-PCR) and western blotting. The efficiency of apoB mRNA editing was determined by reverse transcription PCR (RT-PCR) products and cloning sequencing analysis. We demonstrated that curcumin concentrations up to 70 µM had no significant cytotoxic effects on primary rat hepatocytes at 24 h. At 15 µM, curcumin significantly increased the expression of APOBEC-1 mRNA and protein, and increased the editing level of apoB mRNA from 3.13 to 7.53%. At 25 µM, curcumin reduced the expression of APOBEC-1; however, it did not affect the apoB mRNA editing level. Our data suggested that curcumin at a concentration of 15 µM raised the level of apoB-48 and reduced the level of apoB-100 by increasing the expression of APOBEC-1 in primary rat hepatocytes; therefore, curcumin may be a novel preventative therapy for atherosclerosis.

  4. Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats.

    PubMed

    Yin, Li; Sun, Mingzeng; Ilic, Zoran; Leffert, Hyam L; Sell, Stewart

    2002-02-01

    Liver progenitor cells (LPCs) cloned from adult rat livers following allyl alcohol injury express hematopoietic stem cell and early hepatic lineage markers when cultured on feeder layers; under these conditions, neither mature hepatocyte nor bile duct, Ito, stellate, Kupffer cell, or macrophage markers are detected. These phenotypes have remained stable without aneuploidy or morphological transformation after more than 100 population doublings. When cultured without feeder layers, the early lineage markers disappear, and mature hepatocyte markers are expressed; mature hepatocytic differentiation and cell size are also augmented by polypeptide and steroidal growth factors. In contrast to hepatocytic potential, duct-like structures and biliary epithelial markers are expressed on Matrigel. Because they were derived without carcinogens or mutagens, these bipotential LPC lines provide novel tools for models of cellular plasticity and hepatocarcinogenesis, as well as lines for use in cellular transplantation, gene therapy, and bioreactor construction.

  5. The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.

    PubMed

    Banerjee, Sudip; Melnyk, Stepan B; Krager, Kimberly J; Aykin-Burns, Nukhet; Letzig, Lynda G; James, Laura P; Hinson, Jack A

    2015-12-01

    3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.

  6. Metabolism of medium- and long-chain fatty acids by isolated hepatocytes from small-for-gestational-age (SGA) and appropriate for-gestational-age (AGA) piglets

    SciTech Connect

    Odle, J.; Benevenga, N.J.; Crenshaw, T.D. )

    1990-02-26

    Hepatocytes were isolated from full-term, SGA and AGA piglets at 6 or 48 hours postpartum and were incubated with 1 mM (1-{sup 14}C)-octanoate (C8), -nonanoate (C9) or-oleate (C18:1). The cells oxidized (natom 1-C/(h 10{sup 6} cells)) C9 to Co{sub 2} (12.5) and acid soluble products (28.9) faster than C8 (10.9, 20.6, respectively), and both were oxidized faster than C18:1 (3.9, 9.9) regardless of the piglet age or weight. Oleate accumulated in lipid products 8-fold faster than C8 and C9. No differences between cells from SGA and AGA piglets were detected. Recovery of 1-C in CO{sub 2} was 48% higher in incubations with cells from 48 hours old than from 6 hour old piglets. This increase was attributable to a 70% higher oxygen consumption by 48 hour old cells. Theoretical oxygen consumption rates were computed from the fatty acid flux data and compared to measured oxygen consumption. hepatocytes from SGA and AGA piglets were equally capable of satisfying more that 57% of their energy needs from fatty acid oxidation. The oxygen consumption attributable to C9 metabolism was 30% higher than observed for C8 and C18:1. All fatty acids apparently spared endogenous fuels to a greater degree in 6 hour than in 48 hour piglets.

  7. Genistein as a potential inducer of the anti-atherogenic enzyme paraoxonase-1: studies in cultured hepatocytes in vitro and in rat liver in vivo

    PubMed Central

    Schrader, Charlotte; Ernst, Insa M A; Sinnecker, Heike; Soukup, Sebastian T; Kulling, Sabine E; Rimbach, Gerald

    2012-01-01

    A number of cardioprotective effects, including the reduced oxidation of the low-density lipoprotein (LDL) particles, have been attributed to dietary soy isoflavones. Paraoxonase 1 (PON1), an enzyme mainly synthesized in the liver, may exhibit anti-atherogenic activity by protecting LDL from oxidation. Thus, dietary and pharmacological inducers of PON1 may decrease cardiovascular disease risk. Using a luciferase reporter gene assay we screened different flavonoids for their ability to induce PON1 in Huh7 hepatocytes in culture. Genistein was the most potent flavonoid with regard to its PON1-inducing activity, followed by daidzein, luteolin, isorhamnetin and quercetin. Other flavonoids such as naringenin, cyanidin, malvidin and catechin showed only little or no PON1-inducing activity. Genistein-mediated PON1 transactivation was partly inhibited by the oestrogen-receptor antagonist fulvestrant as well as by the aryl hydrocarbon receptor antagonist 7-ketocholesterol. In contrast to genistein, the conjugated genistein metabolites genistein-7-glucuronide, genistein-7-sulfate and genistein-7,4′-disulfate were only weak inducers of PON1 transactivation. Accordingly, dietary genistein supplementation (2 g/kg diet over three weeks) in growing rats did not increase hepatic PON1 mRNA and protein levels as well as plasma PON1 activity. Thus, genistein may be a PON1 inducer in cultured hepatocytes in vitro, but not in rats in vivo. PMID:22304296

  8. Fusarium moniliforme extract fed before a single dose of diethylnitrosamine increases the numbers of placental glutathione S-transferase positive hepatocytes in rat liver

    SciTech Connect

    Lebepe, S.; Hendrich, S. )

    1991-03-11

    The carcinogenic potential of an alcohol:water (1:1) extract of Fusarium moniliforme (FUSX), containing 20 ppm fumonisin B{sub 1} was assayed. Groups of six 5-week-old female F344/N rats were fed a semipurified diet, with and without FUSX. A dose of initiating agent, diethylnitrosamine, was given orally. Placental glutathione S-transferase-positive (PGST(+)) hepatocytes were detected by immunohistochemistry and counted on 5 frozen hepatic sections/rat, as an endpoint to assess early stages of carcinogenesis. FUSX had significant co-initiating activity. Fusarium moniliforme infection of feed has been shown to promote hepatocarcinogenesis, and may pose a cocarcinogenic risk even during short-term, low-level exposure.

  9. Assessment of the role of in situ generated (E)-2,4-diene-valproic acid in the toxicity of valproic acid and (E)-2-ene-valproic acid in sandwich-cultured rat hepatocytes

    SciTech Connect

    Surendradoss, Jayakumar; Chang, Thomas K.H.; Abbott, Frank S.

    2012-11-01

    Valproic acid (VPA) undergoes cytochrome P450-mediated desaturation to form 4-ene-VPA, which subsequently yields (E)-2,4-diene-VPA by β-oxidation. Another biotransformation pathway involves β-oxidation of VPA to form (E)-2-ene-VPA, which also generates (E)-2,4-diene-VPA by cytochrome P450-mediated desaturation. Although the synthetic form of (E)-2,4-diene-VPA is more hepatotoxic than VPA as shown in various experimental models, there is no conclusive evidence to implicate the in situ generated (E)-2,4-diene-VPA in VPA hepatotoxicity. The present study investigated the effects of modulating the in situ formation of (E)-2,4-diene-VPA on markers of oxidative stress (formation of 2′,7′-dichlorofluorescein; DCF), steatosis (accumulation of BODIPY 558/568 C{sub 12}), necrosis (release of lactate dehydrogenase; LDH), and on cellular total glutathione (GSH) levels in sandwich-cultured rat hepatocytes treated with VPA or (E)-2-ene-VPA. Treatment with either of these chemicals alone increased each of the toxicity endpoints. In VPA-treated hepatocytes, (E)-2,4-diene-VPA was detected only at trace levels, even after phenobarbital (PB) pretreatment and there was no effect on the toxicity of VPA. Furthermore, pretreatment with a cytochrome P450 enzyme inhibitor, 1-aminobenzotriazole (1-ABT), did not influence the extent of VPA toxicity in both PB-pretreated and vehicle-pretreated hepatocytes. However, in (E)-2-ene-VPA-treated hepatocytes, PB pretreatment greatly enhanced the levels of (E)-2,4-diene-VPA and this was accompanied by a further enhancement of the effects of (E)-2-ene-VPA on DCF formation, BODIPY accumulation, LDH release, and GSH depletion. Pretreatment with 1-ABT reduced the concentrations of (E)-2,4-diene-VPA and the extent of (E)-2-ene-VPA toxicity; however, this occurred in PB-pretreated hepatocytes, but not in control hepatocytes. In conclusion, in situ generated (E)-2,4-diene-VPA is not responsible for the hepatocyte toxicity of VPA, whereas it

  10. Vanadate-induced toxicity towards isolated perfused rat livers: the role of lipid peroxidation.

    PubMed

    Younes, M; Strubelt, O

    1991-02-11

    The toxic potential of sodium orthovanadate towards isolated perfused rat livers was investigated at a dose of 2 mmol/l. In livers from fasted rats, vanadate led to a release of cytosolic (glutamate-pyruvate-transaminase (GPT) and lactate dehydrogenase (LDH] and mitochondrial (glutamate dehydrogenase (GLDH] enzymes, an accumulation of calcium in the liver, a marked depletion of hepatic glutathione and an enhanced release of it into the perfusate, as well as an augmented formation and release of thiobarbituric acid-reactive material by the liver. Furthermore, a marked inhibition of oxygen consumption was observed. Vanadate-induced vasoconstriction resulted in a progressive decrease in perfusate flow rate. Control experiments with similarly reduced flow rates led to a comparable reduction in oxygen consumption. GPT and LDH release and hepatic glutathione depletion were also evident, though to a lesser extent than in the presence of vanadate, but no increase in GLDH release, in tissue calcium content or TBA-reactive material in the liver or the perfusate were observed. Thus, indirect toxic effects due to a reduced flow rate contribute only partly to vanadate hepatotoxicity and do not affect mitochondrial integrity. Omission of calcium from the perfusate did not prevent hepatotoxic responses to vanadate, although less calcium was present in the treated livers than in the control organs, indicating that calcium influx is not involved in vanadate-induced hepatotoxicity in the intact organ, in contrast to isolated hepatocytes. Feeding the animals, resulting in an activation of anaerobic energy conservation reactions, strongly attenuated vanadate hepatotoxicity indicating that the energetic status of the liver is the main target of vanadate. Superoxide dismutase did not affect the hepatotoxic responses of livers from fasted rats towards vanadate, while allopurinol and deferrioxamine inhibited lipid peroxidation and hepatotoxicity due to vanadate. The strong correlation

  11. Proton leak in hepatocytes and liver mitochondria from archosaurs (crocodiles) and allometric relationships for ectotherms.

    PubMed

    Hulbert, A J; Else, P L; Manolis, S C; Brand, M D

    2002-07-01

    It has previously been shown that mitochondrial proton conductance decreases with increasing body mass in mammals and is lower in a 250-g lizard than the laboratory rat. To examine whether mitochondrial proton conductance is extremely low in very large reptiles, hepatocytes and mitochondria were prepared from saltwater crocodiles ( Crocodylus porosus) and freshwater crocodiles ( Crocodylus johnstoni). Respiration rates of hepatocytes and liver mitochondria were measured at 37 degrees C and compared with values obtained for rat or previously measured for other species. Respiration rates of hepatocytes from either species of crocodile were similar to those reported for lizards and approximately one fifth of the rates measured using cells from mammals (rat and sheep). Ten-to-thirty percent of crocodile hepatocyte respiration was used to drive mitochondrial proton leak, similar to the proportion in other species. Respiration rates of crocodile liver mitochondria were similar to those of mammalian species. Proton leak rate in isolated liver mitochondria was measured as a function of membrane potential. Contrary to our prediction, the mitochondrial proton conductance of liver mitochondria from crocodiles was greater than that of liver mitochondria from lizards and was similar to that of rats. The acyl composition of liver mitochondrial phospholipids from the crocodiles was more similar to that in mitochondria from rats than in mitochondria from lizards. The relatively high mitochondrial proton conductance was associated with a relatively small liver, which seems to be characteristic of crocodilians. Comparison of data from a number of diverse ectothermic species suggested that hepatocyte respiration rate may decrease with body mass, with an allometric exponent of about -0.2, similar to the exponent in mammalian hepatocytes. However, unlike mammals, liver mitochondrial proton conductance in ectotherms showed no allometric relationship with body size.

  12. Simultaneous induction of non-neoplastic and neoplastic lesions with highly proliferative hepatocytes following dietary exposure of rats to tocotrienol for 2 years.

    PubMed

    Tasaki, Masako; Umemura, Takashi; Kijima, Aki; Inoue, Tomoki; Okamura, Toshiya; Kuroiwa, Yuichi; Ishii, Yuji; Nishikawa, Akiyoshi

    2009-11-01

    It was recently shown that 1-year chronic exposure of rats to tocotrienol (TT) induced highly proliferative liver lesions, nodular hepatocellular hyperplasia (NHH), and independently increased the number of glutathione S-transferase placental form (GST-P)-positive hepatocytes. Focusing attention on the pathological intrinsic property of NHH, a 104-week carcinogenicity study was performed in male and female Wistar Hannover rats given TT at concentrations of 0, 0.4 or 2% in the diet. The high-dose level was adjusted to 1% in both sexes from week 51 because the survival rate of the high-dose males dropped to 42% by week 50. At necropsy, multiple cyst-like nodules were observed, as in the chronic study, but were further enlarged in size, which consequently formed a protuberant surface with a partly pedunculated shape in the liver at the high dose in both sexes. Unlike the chronic study, NHH was not always accompanied by spongiosis, and instead angiectasis was prominent in some nodules. However, several findings in the affected hepatocytes such as minimal atypia, no GST-P immunoreactivity and heterogeneous proliferation, implied that NHH did not harbor neoplastic characteristics from increased exposure despite sustained high cell proliferation. On the other hand, in the high-dose females, the incidence of hepatocellular adenomas was significantly higher than in the control. There was no TT treatment-related tumor induction in any other organs besides the liver. Thus, the overall data clearly suggested that NHH is successively enlarged by further long-term exposure to TT, but does not become neoplastic. In contrast, TT induces low levels of hepatocellular adenomas in female rats.

  13. Nuclear translocation of UDCA by the glucocorticoid receptor is required to reduce TGF-beta1-induced apoptosis in rat hepatocytes.

    PubMed

    Solá, Susana; Amaral, Joana D; Castro, Rui E; Ramalho, Rita M; Borralho, Pedro M; Kren, Betsy T; Tanaka, Hirotoshi; Steer, Cifford J; Rodrigues, Cecília M P

    2005-10-01

    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 beta1 (TGF-beta1)-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 anti-apoptotic function of UDCA. Our results with short interference RNA (siRNA) technology confirmed that UDCA significantly reduces TGF-beta1-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-beta1-induced apoptosis. In co-transfection experiments with GR response element reporter and overexpression constructs, UDCA did not enhance the transactivation of GR with TGF-beta1. Finally, using a fluorescently 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 anti-apoptotic mechanism(s) of UDCA and suggest that GR is crucial for the nuclear translocation of this bile acid for reducing apoptosis.

  14. Mitochondrial permeability transition in rat hepatocytes after anoxia/reoxygenation: role of Ca2+-dependent mitochondrial formation of reactive oxygen species.

    PubMed

    Kim, Jae-Sung; Wang, Jin-Hee; Lemasters, John J

    2012-04-01

    Onset of the mitochondrial permeability transition (MPT) is the penultimate event leading to lethal cellular ischemia-reperfusion injury, but the mechanisms precipitating the MPT after reperfusion remain unclear. Here, we investigated the role of mitochondrial free Ca(2+) and reactive oxygen species (ROS) in pH- and MPT-dependent reperfusion injury to hepatocytes. Cultured rat hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 4 h and then reoxygenated at pH 7.4 to simulate ischemia-reperfusion. Some cells were loaded with the Ca(2+) chelators, BAPTA/AM and 2-[(2-bis-[carboxymethyl]aono-5-methoxyphenyl)-methyl-6-methoxy-8-bis[carboxymethyl]aminoquinoline, either by a cold loading protocol for intramitochondrial loading or by warm incubation for cytosolic loading. Cell death was assessed by propidium iodide fluorometry and immunoblotting. Mitochondrial Ca(2+), inner membrane permeability, membrane potential, and ROS formation were monitored with Rhod-2, calcein, tetramethylrhodamine methylester, and dihydrodichlorofluorescein, respectively. Necrotic cell death increased after reoxygenation. Necrosis was blocked by 1 μM cyclosporin A, an MPT inhibitor, and by reoxygenation at pH 6.2. Confocal imaging of Rhod-2, calcein, and dichlorofluorescein revealed that an increase of mitochondrial Ca(2+) and ROS preceded onset of the MPT after reoxygenation. Intramitochondrial Ca(2+) chelation, but not cytosolic Ca(2+) chelation, prevented ROS formation and subsequent necrotic and apoptotic cell death. Reoxygenation with the antioxidants, desferal or diphenylphenylenediamine, also suppressed MPT-mediated cell death. However, inhibition of cytosolic ROS by apocynin or diphenyleneiodonium chloride failed to prevent reoxygenation-induced cell death. In conclusion, Ca(2+)-dependent mitochondrial ROS formation is the molecular signal culminating in onset of the MPT after reoxygenation of anoxic hepatocytes, leading to cell death.

  15. [Electrophysiological characteristics of the isolated muscle spindle in rats].

    PubMed

    Zhao, Xue-Hong; Fan, Xiao-Li; Song, Xin-Ai; Shi, Lei

    2011-06-25

    The aim of this study was to observe the electrophysiological characteristics of the isolated rat muscle spindle. The muscle spindle was isolated from rat soleus and the afferent discharge of the isolated muscle spindle was recorded by air-gap technique. In the basic physiological salt solution, the spontaneous impulses of muscle spindle were at a lower level with irregular intervals. The mean frequency of afferents was (51.78 ± 25.63) impulses/1 000 s (n = 13). The muscle spindle afferents were significantly increased and maintained over time by the addition of certain amino acids during the observation. The number of the action potential recorded per 1 000 s was 200-1 000 [mean: (687.62 ± 312.56) impulses/1 000 s, n = 17]. In addition to the typical propagated action potential, a large number of abortive spikes were observed. The results indicate that the activities of isolated muscle spindles in rats can be well maintained by the addition of certain amino acids. The results initially establish and provide the possibility for further research conducted in isolated rat muscle spindles.

  16. Characteristics of intracellular Ca2+ signals consisting of two successive peaks in hepatocytes during liver regeneration after 70% partial hepatectomy in rats

    PubMed Central

    Taira, Zenei; Ueda, Yukari; Monmasu, Hiroshi; Yamase, Daisuke; Miyake, Sayaka; Shiraishi, Maya

    2016-01-01

    Two specific signals for regulating liver regeneration were found after 70% partial hepatectomy (PH) in rats. The first finding was a sustained increasing signal of intracellular Ca2+ ([Ca2+]i) in hepatocytes, consisting of two successive peaks with the first narrow peak at 1 hour and the second broad peak increasing by day 3 and then returning to normal by day 4. The second finding was an abnormal peak in the restoring ratio (Rr) curve of liver regeneration after 70% PH at day 4, where the Rr exceeded 100% temporarily, returned to a lower level, and then proceeded to a termination phase of liver regeneration. For 4 days around the two successive [Ca2+]i peaks and abnormal peak, various physiological activities were induced to promote liver regeneration after 70% PH. mRNA expression of genes encoding Ca2+-binding proteins S100A4 and calpain was induced between the two Ca2+ peaks. Hepatocytes underwent synchronous cell proliferation as the liver was restored from 30% to 70% at day 4, and significant expression of VEGF mRNA at around day 4 promoted angiogenesis to remodel the sinusoidal system. Cytochrome P450 activity levels in microsomes and alanine aminotransferase values at 24 hours after CCl4 administration were decreased after 70% PH, which recovered transiently to the control level at day 4, returned to the decreased level, and then slowly recovered by day 10. Thus, these results indicate that day 4 is important during liver regeneration after 70% PH. PMID:27757054

  17. Drug biokinetic and toxicity assessments in rat and human primary hepatocytes and HepaRG cells within the EU-funded Predict-IV project.

    PubMed

    Mueller, Stefan O; Guillouzo, André; Hewitt, Philip G; Richert, Lysiane

    2015-12-25

    The overall aim of Predict-IV (EU-funded collaborative project #202222) was to develop improved testing strategies for drug safety in the late discovery phase. One major focus was the prediction of hepatotoxicity as liver remains one of the major organ leading to failure in drug development, drug withdrawal and has a poor predictivity from animal experiments. In this overview we describe the use and applicability of the three cell models employed, i.e., primary rat hepatocytes, primary human hepatocytes and the human HepaRG cell line, using four model compounds, chlorpromazine, ibuprofen, cyclosporine A and amiodarone. This overview described the data generated on mode of action of liver toxicity after long-term repeat-dosing. Moreover we have quantified parent compound and its distribution in various in vitro compartments, which allowed us to develop biokinetic models where we could derive real exposure concentrations in vitro. In conclusion, the complex data set enables quantitative measurements that proved the concept that we can define human relevant free and toxic exposure levels in vitro. Further compounds have to be analyzed in a broader concentration range to fully exploit these promising results for improved prediction of hepatotoxicity and hazard assessment for humans.

  18. Strategies for immortalization of primary hepatocytes

    PubMed Central

    Eva, Ramboer; Bram, De Craene; Joery, De Kock; Tamara, Vanhaecke; Geert, Berx; Vera, Rogiers; Mathieu, Vinken

    2014-01-01

    The liver has the unique capacity to regenerate in response to a damaging event. Liver regeneration is hereby largely driven by hepatocyte proliferation, which in turn relies on cell cycling. The hepatocyte cell cycle is a complex process that is tightly regulated by several well-established mechanisms. In vitro, isolated hepatocytes do not longer retain this proliferative capacity. However, in vitro cell growth can be boosted by immortalization of hepatocytes. Well-defined immortalization genes can be artificially overexpressed in hepatocytes or the cells can be conditionally immortalized leading to controlled cell proliferation. This paper discusses the current immortalization techniques and provides a state-of-the-art overview of the actually available immortalized hepatocyte-derived cell lines and their applications. PMID:24911463

  19. Three Dimensional Primary Hepatocyte Culture

    NASA Technical Reports Server (NTRS)

    Yoffe, Boris

    1998-01-01

    Our results demonstrated for the first time the feasibility of culturing PHH in microgravity bioreactors that exceeded the longest period obtained using other methods. Within the first week of culture, isolated hepatocytes started to form aggregates, which continuously increased in size (up to 1 cm) and macroscopically appeared as a multidimensional tissue-like assembly. To improve oxygenation and nutrition within the spheroids we performed experiments with the biodegradable nonwoven fiber-based polymers made from PolyGlycolic Acid (PGA). It has been shown that PGA scaffolds stimulate isolated cells to regenerate tissue with defined sizes and shapes and are currently being studied for various tissue-engineering applications. Our data demonstrated that culturing hepatocytes in the presence of PGA scaffolds resulted in more efficient cell assembly and formations of larger cell spheroids (up to 3 cm in length, see figure). The histology of cell aggregates cultured with PGA showed polymer fibers with attached hepatocytes. We initiated experiments to co-culture primary human hepatocytes with human microvascular endothelial cells in the bioreactor. The presence of endothelial cells in co-cultures were established by immunohistochemistry using anti-CD34 monoclonal Ab. Our preliminary data demonstrated that cultures of purified hepatocytes with human microvascular endothelial cells exhibited better growth and expressed higher levels of albumin MRNA for a longer period of time than cultures of ppfified, primary human hepatocytes cultured alone. We also evaluated microsomal deethylation activity of hepatocytes cultured in the presence of endothelial cells.In summary, we have established liver cell culture, which mimicked the structure and function of the parent tissue.

  20. Long live the liver: immunohistochemical and stereological study of hepatocytes, liver sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells of male and female rats throughout ageing.

    PubMed

    Marcos, Ricardo; Correia-Gomes, Carla

    2016-12-01

    Male/female differences in enzyme activity and gene expression in the liver are known to be attenuated with ageing. Nevertheless, the effect of ageing on liver structure and quantitative cell morphology remains unknown. Male and female Wistar rats aged 2, 6, 12 and 18 months were examined by means of stereological techniques and immunohistochemical tagging of hepatocytes (HEP), liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC) and hepatic stellate cells (HSC) in order to assess the total number and number per gram of these cells throughout life. The mean cell volume of HEP and HSC, the lobular position and the collagen content of the liver were also evaluated with stereological techniques. The number per gram of HSC was similar for both genders and was maintained throughout ageing. The mean volume of HSC was also conserved but differences in the cell body and lobular location were observed. Statistically significant gender differences in HEP were noted in young rats (females had smaller and more binucleated HEP) but were attenuated with ageing. The same occurred for KC and LSEC, since the higher number per gram in young females disappeared in older animals. Liver collagen increased with ageing but only in males. Thus, the numbers of these four cell types are related throughout ageing, with well-defined cell ratios. The shape and lobular position of HSC change with ageing in both males and females. Gender dimorphism in HEP, KC and LSEC of young rat liver disappears with ageing.

  1. Identification and characterization of hepatocyte-specific regulatory regions of the rat pyruvate kinase L gene. The synergistic effect of multiple elements.

    PubMed

    Yamada, K; Noguchi, T; Matsuda, T; Takenaka, M; Monaci, P; Nicosia, A; Tanaka, T

    1990-11-15

    The rat pyruvate kinase L (PKL) gene produces the L- and R-type isozymes by alternative transcription that is regulated in a tissue-specific manner. To investigate which DNA elements are involved in hepatocyte-specific expression of the L-type isozyme, we performed transient DNA transfer experiments with PKL/chloramphenicol acetyltransferase fusion genes. We found three positive regulatory regions required for expression of the L-type isozyme in adult rat hepatocytes by functional analyses of a series of 5' and internal deletion constructs of the fusion genes. These regions, designated as PKL-I, PKL-II, and PKL-III, were located between nucleotides -76 and -94, -126 and -149, and -150 and -170, respectively. PKL-I showed enhancer-like activity alone, whereas PKL-II and PKL-III did not have any independent effect. Combinations of L-I + L-II and L-II + L-III, but not of L-I + L-III, showed synergistic enhancer activities when oriented in the same direction. The inclusion of all three elements oriented in the same direction had the maximum synergistic effect, indicating that these elements function as a unit. This unit enhanced expression from heterologous as well as homologous promoters in a manner that was independent of its orientation and position relative to the cap site. The activity of the unit was not detected in HeLa cells or K562 erythroleukemia cells, suggesting that this unit possessed cell-type specificity. PKL-I consists of a palindrome sequence 5'-CTGGTTATACTTTAACCAG-3', which contain a sequence homologous to the LF-B1-binding site. PKL-II contains the sequence 5'-TTCCTGGACTCTGGCCCCCAGTGT-3', which is similar to that of the LF-A1-binding site. PKL-III contains a palindrome sequence 5'-CCACGGGGCACTCCCGTGG-3', which include a sequence homologous to the binding site of the adenovirus major late transcription factor. Gel retardation assay indicated that the different trans-acting factors interacted with three elements and that the transacting protein bound

  2. The Ras/Rac1/Cdc42/SEK/JNK/c-Jun Cascade Is a Key Pathway by Which Agonists Stimulate DNA Synthesis in Primary Cultures of Rat Hepatocytes

    PubMed Central

    Auer, Kelly L.; Contessa, Joseph; Brenz-Verca, Stefano; Pirola, Luciano; Rusconi, Sandro; Cooper, Geoffrey; Abo, Arie; Wymann, Matthias P.; Davis, Roger J.; Birrer, Michael; Dent, Paul

    1998-01-01

    The ability of signaling via the JNK (c-Jun NH2-terminal kinase)/stress-activated protein kinase cascade to stimulate or inhibit DNA synthesis in primary cultures of adult rat hepatocytes was examined. Treatment of hepatocytes with media containing hyperosmotic glucose (75 mM final), tumor necrosis factor α (TNFα, 1 ng/ml final), and hepatocyte growth factor (HGF, 1 ng/ml final) caused activation of JNK1. Glucose, TNFα, or HGF treatments increased phosphorylation of c-Jun at serine 63 in the transactivation domain and stimulated hepatocyte DNA synthesis. Infection of hepatocytes with poly-l-lysine–coated adenoviruses coupled to constructs to express either dominant negatives Ras N17, Rac1 N17, Cdc42 N17, SEK1−, or JNK1− blunted the abilities of glucose, TNFα, or HGF to increase JNK1 activity, to increase phosphorylation of c-Jun at serine 63, and to stimulate DNA synthesis. Furthermore, infection of hepatocytes by a recombinant adenovirus expressing a dominant-negative c-Jun mutant (TAM67) also blunted the abilities of glucose, TNFα, and HGF to stimulate DNA synthesis. These data demonstrate that multiple agonists stimulate DNA synthesis in primary cultures of hepatocytes via a Ras/Rac1/Cdc42/SEK/JNK/c-Jun pathway. Glucose and HGF treatments reduced glycogen synthase kinase 3 (GSK3) activity and increased c-Jun DNA binding. Co-infection of hepatocytes with recombinant adenoviruses to express dominant- negative forms of PI3 kinase (p110α/p110γ) increased basal GSK3 activity, blocked the abilities of glucose and HGF treatments to inhibit GSK3 activity, and reduced basal c-Jun DNA binding. However, expression of dominant-negative PI3 kinase (p110α/p110γ) neither significantly blunted the abilities of glucose and HGF treatments to increase c-Jun DNA binding, nor inhibited the ability of these agonists to stimulate DNA synthesis. These data suggest that signaling by the JNK/stress-activated protein kinase cascade, rather than by the PI3 kinase cascade

  3. Isolation and characterization of hepatic mast cells from cholestatic rats

    PubMed Central

    Hargrove, Laura; Graf-Eaton, Allyson; Kennedy, Lindsey; Demieville, Jennifer; Owens, Jennifer; Hodges, Kyle; Ladd, Brittany; Francis, Heather

    2016-01-01

    Mast cells (MCs) are immune cells that release histamine and other mediators. MC number increases after bile duct ligation (BDL) and blocking mast cell-derived histamine decrease biliary proliferation. We aimed to isolate and characterize MCs from cholestatic livers. Rats were subjected to BDL starting at 6 hrs and up to 14 days. MC infiltration was evaluated by toluidine blue. BDL rats were perfused using standard collagenase perfusion. Following enzymatic digestion, tissue was passed through a fine gauge needle. Suspensions were incubated with MAb AA4, washed and incubated with goat anti-mouse coated Dynal® beads. MCs were stained with toluidine blue, and in isolated MCs, the expression of FCεRI and MC proteases was measured. The expression of histidine decarboxylase, histamine receptors, VEGF-receptors and TIE 1 and 2 was evaluated by qPCR. Histamine and VEGF-A secretion was measured in MC supernatants. MC purity was evaluated by CK-19, CK-8, albumin, VAP-1 and α-SMA expression. In vitro, cholangiocytes and HSCs were treated with isolated MC supernatants from BDL rats treated with either NaCl or cromolyn sodium (to block MC histamine release) and biliary proliferation and hepatic fibrosis were measured. MCs infiltrate the liver and surround bile ducts starting at day 2. We isolated a virtually pure preparation of mature, functional MCs. TEM images reveal distinct secretory granules and isolated MCs secrete histamine. MCs express FCεRI, chymase, tryptase, RMCPI and RMCPII, but were virtually void of other cell markers. Biliary proliferation and fibrosis increased following treatment with MC supernatants from BDL rats + NaCl and these parameters decreased in cells treated with MC supernatants from BDL + cromolyn sodium. In conclusion, we have isolated and characterized MCs from cholestatic livers. MCs regulate cholestatic liver injury and hepatic fibrosis. This tool provides a better understanding of the paracrine influence of mast cells on biliary

  4. Early social isolation augments alcohol consumption in rats.

    PubMed

    Lesscher, Heidi M B; Spoelder, Marcia; Rotte, Marthe D; Janssen, Martijn J; Hesseling, Peter; Lozeman-van't Klooster, José G; Baars, Annemarie M; Vanderschuren, Louk J M J

    2015-10-01

    There is a considerable degree of individual vulnerability for alcohol use disorder (AUD) as only a subpopulation of individuals who regularly consume alcohol develop AUD. It is therefore very important to understand the factors and mechanisms that contribute towards the individual risk for AUD. In this respect, social influences, in particular during development, may be relevant for AUD as disruptions in early social experiences are associated with an increased risk for AUD. Social play, the most prominent form of social behaviour shown by young mammals, is rewarding and considered to be important for social, emotional and cognitive development. Recent studies suggest that early social isolation, effectively depriving animals from social play, increases the risk for addictive behaviour. The aim of this study was therefore to explore the long-term consequences of early social isolation on alcohol consumption and motivation for alcohol. To this end, rats were socially isolated from postnatal days 21-42, followed by 4 weeks of social housing, and voluntary alcohol consumption and operant responding for alcohol were determined in adulthood. We observed enhanced levels of alcohol consumption in adulthood in previously isolated rats, whereas operant responding for alcohol was not altered. The impact of early social isolation was independent of the individual variation in alcohol consumption. These data indicate that social isolation, during a developmental period when social play is highly abundant, enhances the propensity to consume alcohol in adulthood. This implies that early social experience may be a protective factor against excessive alcohol use.

  5. ISOLATION OF RAT LIVER PLASMA MEMBRANES

    PubMed Central

    Touster, Oscar; Aronson, N. N.; Dulaney, John T.; Hendrickson, Herman

    1970-01-01

    Nucleotide pyrophosphatase and phosphodiesterase I of rat liver have been found to be localized primarily in cell particulates highly enriched with respect to the most commonly accepted plasma membrane marker, 5'-nucleotidase, and therefore should themselves be assigned a plasma membrane localization. The observation that plasma membranes sediment in isotonic sucrose with both nuclear and microsomal fractions was exploited to obtain plasma membrane preparations from each fraction. Both preparations are similar in chemical and enzymic composition. Moreover, the preparative method developed in this study appears to give the best combination of yield, purity, and reproducibility available. The question of the possible identity of nucleotide pyrophosphatase and phosphodiesterase I is considered, and evidence is presented suggesting that these activities may be manifestations of the same enzyme. PMID:5497542

  6. Depressive behavior induced by social isolation of predisposed female rats.

    PubMed

    Zanier-Gomes, Patrícia Helena; de Abreu Silva, Tomaz Eugênio; Zanetti, Guilherme Cia; Benati, Évelyn Raquel; Pinheiro, Nanci Mendes; Murta, Beatriz Martins Tavares; Crema, Virgínia Oliveira

    2015-11-01

    Depression is a mood disorder that is more prevalent in women and has been closely associated with chronic stress. Many models of depression have been suggested that consider different forms of stress. In fact, stress is present in the life of every human being, but only a few develop depression. Accordingly, it seems wrong to consider all stressed animals to be depressed, emphasizing the importance of predisposition for this mood disorder. Based on this finding, we evaluated a predisposition to depressive behavior of female rats on the forced swim test (FST), and the more immobile the animal was during the FST, the more predisposed to depression it was considered to be. Then, animals were subjected to the stress of social isolation for 21 days and were re-evaluated by the FST. The Predisposed/Isolated rats presented higher immobility times. Once all the rats had prior experience in the FST, we calculated an Index of Increase by Isolation, confirming the previous results. Based on this result, we considered the Predisposed/Isolated group as presenting depressive behavior ('Depressed') and the Nonpredisposed/Nonisolated group as the control group ('Nondepressed'). The animals were distributed into 4 new groups: Nondepressed/Vehicle, Nondepressed/Amitriptyline, Depressed/Vehicle, Depressed/Amitriptyline. After 21 days of treatment, only the Depressed/Vehicle group differed from the other 3 groups, demonstrating the efficacy of amitriptyline in treating the depressive behavior of the Depressed animals, validating the model. This study shows that conducting an FST prior to any manipulation can predict predisposition to depressive behavior in female rats and that the social isolation of predisposed animals for 21 days is effective in inducing depressive behavior. This behavior can be considered real depressive behavior because it takes into account predisposition, chronic mild stress, and the prevalent gender.

  7. Choline deficient diet enhances the initiating and promoting effects of methapyrilene hydrochloride in rat liver as assayed by the induction of gamma-glutamyltranspeptidase-positive hepatocyte foci.

    PubMed Central

    Perera, M. I.; Katyal, S. L.; Shinozuka, H.

    1987-01-01

    Earlier we demonstrated that short-term feeding of methapyrilene hydrochloride (MPH) and of a choline deficient (CD) diet to rats induced peroxidative damage of microsomal membrane lipids of liver cells. In the present study, we investigated whether a CD diet modifies the extent of MPH-induced lipid peroxidation and whether the modifications lead to changes in the initiating and promoting action of these agents using assays of the induction of gamma-glutamyltranspeptidase (GGT)-positive hepatocyte foci. Addition of 0.1% MPH to a CD diet enhanced the extent of microsomal lipid peroxidation induced by a CD diet alone. Feeding a choline supplemented (CS) or a CD diet containing 0.1% MPH for 2 weeks followed by 7 weeks promotion by a CD diet plus phenobarbital was ineffective in inducing GGT-positive foci. Feeding MPH in a CS or a CD diet for 4 weeks, however, resulted in the development of substantial numbers of GGT-positive foci. There was a 3 fold increase in the number of foci in rats initiated with a CD + MPH diet over that in rats initiated with a CS + MPH diet. 0.1% MPH in a CS diet or a CD diet exerted significant promotional effects on the induction of GGT-positive foci in rats initiated with a single injection of diethylnitrosamine. Addition of MPH to a CD diet was additive in inducing GGT-positive foci. The results suggest that lipid peroxidation of the liver may be involved in the carcinogenic and/or promoting effects of MPH and a CD diet. PMID:2893639

  8. Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes

    PubMed Central

    Hiriart, Marcia; Sanchez-Soto, Carmen; Diaz-Garcia, Carlos Manlio; Castanares, Diana T.; Avitia, Morena; Velasco, Myrian; Mas-Oliva, Jaime; Macias-Silva, Marina; González-Villalpando, Clicerio; Delgado-Coello, Blanca; Sosa-Garrocho, Marcela; Vidaltamayo, Román; Fuentes-Silva, Deyanira

    2014-01-01

    It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l−1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance. PMID:24995000

  9. Modeling fibrosis using fibroblasts isolated from scarred rat vocal folds

    PubMed Central

    Kishimoto, Yo; Kishimoto, Ayami Ohno; Ye, Shuyun; Kendziorski, Christina; Welham, Nathan V.

    2016-01-01

    Following injury, pathologically activated vocal fold fibroblasts (VFFs) can engage in disordered extracellular matrix (ECM) remodeling, leading to VF fibrosis and impaired voice function. Given the importance of scar VFFs to phenotypically appropriate in vitro modeling of VF fibrosis, we pursued detailed characterization of scar VFFs obtained from surgically injured rat VF mucosae, compared to those obtained from experimentally naïve, age-matched tissue. Scar VFFs initially exhibited a myofibroblast phenotype characterized by increased proliferation, increased Col1a1 transcription and collagen, type I synthesis, increased Acta2 transcription and α-smooth muscle actin synthesis, and enhanced contractile function. These features were most distinct at passage 1 (P1); we observed a coalescence of the scar and naïve VFF phenotypes at later passages. An empirical Bayes statistical analysis of the P1 cell transcriptome identified 421 genes that were differentially expressed by scar, compared to naïve, VFFs. These genes were primarily associated with the wound response, ECM regulation, and cell proliferation. Follow-up comparison of P1 scar VFFs and their in vivo tissue source showed substantial transcriptomic differences. Finally, P1 scar VFFs responded to treatment with hepatocyte growth factor and transforming growth factor-β3, two biologics with reported therapeutic value. Despite the practical limitations inherent to working with early passage cells, this experimental model is easily implemented in any suitably equipped laboratory and has the potential to improve the applicability of preclinical VF fibrosis research. PMID:27111284

  10. Strategies for short-term storage of hepatocytes for repeated clinical infusions.

    PubMed

    Jorns, Carl; Gramignoli, Roberto; Saliem, Mohammed; Zemack, Helen; Mörk, Lisa-Mari; Isaksson, Bengt; Nowak, Greg; Ericzon, Bo-Göran; Strom, Stephen; Ellis, Ewa

    2014-01-01

    Hepatocyte transplantation is an upcoming treatment for patients with metabolic liver diseases. Repeated cell infusions over 1-2 days improve clinical outcome. Isolated hepatocytes are usually cold stored in preservation solutions between repeated infusions. However, during cold storage isolated hepatocytes undergo cell death. We investigated if tissue preservation and repeated isolations are better than storage of isolated hepatocytes when cold preserving human hepatocytes. Liver tissue obtained from liver surgery or organ donors was divided into two pieces. Hepatocytes were isolated by collagenase digestion. Hepatocytes were analyzed directly after isolation (fresh) or after storage for 48 h at 4°C in University of Wisconsin solution (UW cells). Liver tissue from the same donor was stored at 4°C in UW and hepatocytes were isolated after 48 h (UW tissue cells). Hepatocyte viability and function was evaluated by trypan blue exclusion, plating efficiency, ammonia metabolism, CYP 1A1/2, 2C9, 3A7, and 3A4 activities, phase II conjugation, and apoptosis evaluation by TUNEL assay and caspase-3/7 activities. Hepatocytes stored in UW showed a significantly lower viability compared to fresh cells or hepatocytes isolated from tissue stored for 48 h (54% vs. 71% vs. 79%). Plating efficiency was significantly decreased for cells stored in UW (40%) compared to fresh and UW tissue cells (63% vs. 55%). No significant differences between UW cells and UW tissue cells could be shown for CYP activities or ammonia metabolism. Hepatocytes stored in UW showed a strong increase in TUNEL-positive cells, whereas TUNEL staining in cold-stored liver tissue and hepatocytes isolated after 48 h was unchanged. This observation was confirmed by increased caspase-3/7 activities in UW cells. Although preservation of isolated hepatocytes in UW maintains function, cold storage of liver tissue and repeated hepatocyte isolations is superior to cold storage of isolated hepatocytes in preserving

  11. Bixin protects hepatocytes against 1,2-dimethylhydrazine-induced genotoxicity but does not suppress DNA damage and pre-neoplastic lesions in the colon of Wistar rats.

    PubMed

    de Oliveira, Pollyanna Francielli; de Andrade, Kelly Jacqueline Barbosa; Paula, Marcela Cristina Ferreira; Oliveira Acésio, Nathália; da Silva Moraes, Thais; Borges, Priscilla Scalon Freitas; Barcelos, Gustavo Rafael Mazzaron; Tavares, Denise Crispim

    2014-01-01

    Bixin is a carotenoid found in the seeds of Bixa orellana L., a plant native to tropical America that is used in the food industry. The aim of this study was to investigate the effect of bixin on DNA damage and pre-neoplastic lesions induced by 1,2-dimethylhydrazine (DMH) in the liver and colon of Wistar rats. The animals received bixin at daily doses of 0.1, 1.0 and 10mg/kg body weight (bw) by gavage. For the assessment of DNA damage in hepatocytes and colon cells with the comet assay, the administration of bixin was for 7 days. The animals received a single subcutaneous injection of 25mg/kg bw of DMH, and were euthanized 4h later. For the evaluation of the frequency of aberrant crypt foci (ACF), the animals were treated with the different doses of bixin for 4 weeks. Four doses of 40mg/kg bw DMH, two doses in the first week and two doses in the second week, were administered and euthanasia occurred at 4 weeks after the beginning of treatment. Bixin reduced the frequency of DNA damage in hepatocytes at the highest two doses tested (1.0 and 10mg/kg bw). On the other hand, no differences in the frequency of DNA damage in colon cells were observed between animals treated with bixin plus DMH and those treated with DMH alone. In addition, the frequency of ACF did not differ significantly between the group treated with bixin plus DMH and the DMH group. The results suggest that bixin does not suppress the formation of ACF, indicating the absence of a protective effect against colon carcinogenesis.

  12. Fructose-induced increase in intracellular free Mg2+ ion concentration in rat hepatocytes: relation with the enzymes of glycogen metabolism.

    PubMed Central

    Gaussin, V; Gailly, P; Gillis, J M; Hue, L

    1997-01-01

    In rat hepatocytes subjected to a fructose load, ATP content decreased from 3.8 to 2.6 micromol/g of cells. Under these conditions, the intracellular free Mg2+ ion concentration,as measured with mag-fura 2, increased from 0.25 to 0.43 micromol/g of cells and 0.35 micromol of Mg2+ ions were released per g of cells in the extracellular medium. Therefore the increase in the intracellular free Mg2+ ion concentration was less than expected from the decrease in ATP, indicating that approx. 80% of the Mg2+ ions released from MgATP2- were buffered inside the cells. When this buffer capacity was challenged with an extra Mg2+ ion load by blocking the fructose-induced Mg2+ efflux, again approx. 80% of the extra Mg2+ ion load was buffered. The remaining 20% appearing as free Mg2+ions in fructose-treated hepatocytes could act as second messenger for enzymes having a Km for Mg2+ in the millimolar range. Fructose activated glycogen synthase and glycogen phosphorylase, although both the time course and the dose-dependence of activation were different. This was reflected in a stimulation of glycogen synthesis with concentrations of fructose below 5 mM. Indeed, activation of glycogen synthase reached a maximum at 30 min of incubation and was observed with small (5 mM or less) concentrations of fructose, whereas the activation of glycogen phosphorylase was almost immediate (within 5 min) and maximal with large doses of fructose. The fructose-induced activation of glycogen phosphorylase, but not that of glycogen synthase, could be related to an increase in free Mg2+ ion concentration. PMID:9307033

  13. Insulin and glucocorticoid dependence of hepatic gamma-glutamylcysteine synthetase and glutathione synthesis in the rat. Studies in cultured hepatocytes and in vivo.

    PubMed Central

    Lu, S C; Ge, J L; Kuhlenkamp, J; Kaplowitz, N

    1992-01-01

    We reported that glucagon and phenylephrine decrease hepatocyte GSH by inhibiting gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis (Lu, S.C., J. Kuhlenkamp, C. Garcia-Ruiz, and N. Kaplowitz. 1991. J. Clin. Invest. 88:260-269). In contrast, we have found that insulin (In, 1 microgram/ml) and hydrocortisone (HC, 50 nM) increased GSH of cultured hepatocytes up to 50-70% (earliest significant change at 6 h) with either methionine or cystine alone as the sole sulfur amino acid in the medium. The effect of In occurred independent of glucose concentration in the medium. Changes in steady-state cellular cysteine levels, cell volume, GSH efflux, or expression of gamma-glutamyl transpeptidase were excluded as possible mechanisms. Both hormones are known to induce cystine/glutamate transport, but this was excluded as the predominant mechanism since the induction in cystine uptake required a lag period of greater than 6 h, and the increase in cell GSH still occurred when cystine uptake was blocked. Assay of GSH synthesis in extracts of detergent-treated cells revealed that In and HC increased the activity of GCS by 45-65% (earliest significant change at 4 h) but not GSH synthetase. In and HC treatment increased the Vmax of GCS by 31-43% with no change in Km. Both the hormone-mediated increase in cell GSH and GCS activity were blocked with either cycloheximide or actinomycin D. Finally, when studied in vivo, streptozotocin-treated diabetic and adrenalectomized rats exhibited lower hepatic GSH levels and GCS activities than respective controls. Both of these abnormalities were prevented with hormone replacement. Thus, both in vitro and in vivo, In and glucocorticoids are required for normal expression of GCS. Images PMID:1353765

  14. Isolation rearing induced fear-like behavior without affecting learning abilities of Wistar rats.

    PubMed

    Molina-Hernandez, M; Tellez-Alcantara, P; Perez-Garcia, J

    2001-07-01

    1. Isolation-reared rats display fear-like behavior and depressive-like behavior in several behavioral tasks, suggesting that isolation rearing may model certain aspects of human psychopathologies. 2. After weaning (20 days old), male and female Wistar rats were isolation-reared during 20, 50 or 70 days. After that, they were tested in the elevated plus maze test, and in the open field test. Another group of isolation-reared rats (70 days of isolation) were tested in an auto-shaping task. 3. Isolation-reared rats displayed high levels of fear-like behavior in the elevated plus-maze test, and hyperlocomotion in the open field test. But, isolation-reared rats learned an auto-shaping task. 4. In conclusion, isolation rearing induced fear-like behavior, without affect learning abilities of rats.

  15. Hepatoprotective and Antioxidant Effects of Saponarin, Isolated from Gypsophila trichotoma Wend. on Paracetamol-Induced Liver Damage in Rats

    PubMed Central

    Vitcheva, Vessela; Kondeva-Burdina, Magdalena; Krasteva, Ilina; Manov, Vassil; Mitcheva, Mitka

    2013-01-01

    The hepatoprotective potential of saponarin, isolated from Gypsophila trichotoma, was evaluated in vitro/in vivo using a hepatotoxicity model of paracetamol-induced liver injury. In freshly isolated rat hepatocytes, paracetamol (100 μmol) led to a significant decrease in cell viability, increased LDH leakage, decreased levels of cellular GSH, and elevated MDA quantity. Saponarin (60–0.006 μg/mL) preincubation, however, significantly ameliorated paracetamol-induced hepatotoxicity in a concentration-dependent manner. The beneficial effect of saponarin was also observed in vivo. Rats were challenged with paracetamol alone (600 mg/kg, i.p.) and after 7-day pretreatment with saponarin (80 mg/kg, oral gavage). Paracetamol toxicity was evidenced by increase in MDA quantity and decrease in cell GSH levels and antioxidant defence system. No changes in phase I enzyme activities of AH and EMND and cytochrome P 450 quantity were detected. Saponarin pretreatment resulted in significant increase in cell antioxidant defence system and GSH levels and decrease in lipid peroxidation. The biochemical changes are in good correlation with the histopathological data. Protective activity of saponarin was similar to the activity of positive control silymarin. On the basis of these results, it can be concluded that saponarin exerts antioxidant and hepatoprotective activity against paracetamol liver injury in vitro/in vivo. PMID:23878818

  16. Isolation and characterization of alpha 1-antitrypsin in PAS-positive hepatic granules from rats with experimental alpha 1-antitrypsin deficiency.

    PubMed

    Bolmer, S; Kleinerman, J

    1986-05-01

    Chronic galactosamine (GalNH2) administration in rats decreases plasma alpha 1-antitrypsin (AAT) levels to 10-50% of control levels and induces the formation of diastase-resistant, PAS-positive granules, which contain AAT in hepatocytes. This report describes the isolation and purification of hepatic granule AAT by three different methods: solubilization with guanidine hydrochloride followed by gel filtration on Bio-gel A5M, extraction with methylamine and 2-chloroethanol, and solubilization with sodium dodecyl sulfate (SDS) followed by preparative SDS-polyacrylamide gel electrophoresis. All three methods yield a single protein which precipitates with anti-rat plasma AAT antibody, and which has an apparent molecular weight of 45,000 daltons, in contrast to the molecular weight of plasma AAT, 50,000 daltons. Unlike plasma AAT, granule AAT contains no sialic acid, galactose, or fucose. Moreover, granule AAT contains a reduced amount of N-acetylglucosamine and an increased amount of mannose, compared with plasma AAT. The carbohydrate content of granule AAT varies with the isolation procedure used. Granule AAT is susceptible to cleavage by endoglucosaminidase H, which indicates the presence of high-mannose type oligosaccharides. Comparison of the molecular weight, carbohydrate composition, isoelectric point, and endoglucosaminidase H sensitivity of granule AAT isolated from rats with GalNH2-induced AAT deficiency with granule AAT from PiZ humans extends the list of similarities between experimental GalNH2-induced AAT deficiency in rats by and genetically determined AAT deficiency in humans.

  17. A physiologically based biodynamic (PBBD) model for estragole DNA binding in rat liver based on in vitro kinetic data and estragole DNA adduct formation in primary hepatocytes

    SciTech Connect

    Paini, Alicia; Punt, Ans; Viton, Florian; Scholz, Gabriele; Delatour, Thierry; Marin-Kuan, Maricel; Schilter, Benoit; Bladeren, Peter J. van; Rietjens, Ivonne M.C.M.

    2010-05-15

    Estragole has been shown to be hepatocarcinogenic in rodent species at high-dose levels. Translation of these results into the likelihood of formation of DNA adducts, mutation, and ultimately cancer upon more realistic low-dose exposures remains a challenge. Recently we have developed physiologically based biokinetic (PBBK) models for rat and human predicting bioactivation of estragole. These PBBK models, however, predict only kinetic characteristics. The present study describes the extension of the PBBK model to a so-called physiologically based biodynamic (PBBD) model predicting in vivo DNA adduct formation of estragole in rat liver. This PBBD model was developed using in vitro data on DNA adduct formation in rat primary hepatocytes exposed to 1'-hydroxyestragole. The model was extended by linking the area under the curve for 1'-hydroxyestragole formation predicted by the PBBK model to the area under the curve for 1'-hydroxyestragole in the in vitro experiments. The outcome of the PBBD model revealed a linear increase in DNA adduct formation with increasing estragole doses up to 100 mg/kg bw. Although DNA adduct formation of genotoxic carcinogens is generally seen as a biomarker of exposure rather than a biomarker of response, the PBBD model now developed is one step closer to the ultimate toxic effect of estragole than the PBBK model described previously. Comparison of the PBBD model outcome to available data showed that the model adequately predicts the dose-dependent level of DNA adduct formation. The PBBD model predicts DNA adduct formation at low levels of exposure up to a dose level showing to cause cancer in rodent bioassays, providing a proof of principle for modeling a toxicodynamic in vivo endpoint on the basis of solely in vitro experimental data.

  18. Regulation of collagen production in freshly isolated cell populations from normal and cirrhotic rat liver: Effect of lactate

    SciTech Connect

    Cerbon-Ambriz, J.; Cerbon-Solorzano, J.; Rojkind, M. )

    1991-03-01

    Previous work has shown that lactic acid, and to a lesser extent pyruvic acid, is able to increase collagen synthesis significantly in liver slices of CCl4-treated rats but not normal rats. The purpose of this report is to document which cells in the cirrhotic liver are responsible for the lactate-stimulated increase in collagen synthesis. It was found that (a) incorporation of 3H-proline into protein-bound 3H-hydroxyproline is increased threefold to fourfold in hepatocytes from CCl4-treated rats as compared with normal rat hepatocytes; (b) neither the hepatocytes from normal nor those from CCl4-treated rats modify their collagen synthesizing capacity when 30 mmol/L lactic acid was added to the incubation medium; (c) nonparenchymal cells obtained from livers of CCl4-treated rats synthesize much less collagen than hepatocytes, but their synthesis is stimulated twofold by lactic acid; (d) from the different nonparenchymal cells, only fat-storing (Ito) cells increase collagen synthesis when lactic acid is present in the incubation medium. These results suggest that the increased lactic acid levels observed in patients with alcoholic hepatic cirrhosis may play an important role in the development of fibrosis by stimulating collagen production by fat-storing (Ito) cells.

  19. Death receptor and mitochondria-mediated hepatocyte apoptosis underlies liver dysfunction in rats exposed to organic pollutants from drinking water.

    PubMed

    Yang, Guanghong; Zhou, Zhiwei; Cen, Yanli; Gui, Xiaolin; Zeng, Qibing; Ao, Yunxia; Li, Qian; Wang, Shiran; Li, Jun; Zhang, Aihua

    2015-01-01

    Persistent organic pollutants in drinking water impose a substantial risk to the health of human beings, but the evidence for liver toxic effect and the underlying mechanism is scarce. This study aimed to examine the liver toxicity and elucidate the molecular mechanism of organic pollutants in drinking water in normal human liver cell line L02 cells and rats. The data showed that organic extraction from drinking water remarkably impaired rat liver function, evident from the increase in the serum level of alanine aminotransferase, aspartate aminotransferase, and cholinesterase, and decrease in the serum level of total protein and albumin. Organic extraction dose-dependently induced apoptotic cell death in rat liver and L02 cells. Administration of rats with organic extraction promoted death receptor signaling pathway through the increase in gene and protein expression level of Fas and FasL. Treatment of rats with organic extraction also induced mitochondria-mediated apoptosis via increasing the expression level of proapoptotic protein, Bax, but decreasing the expression level of antiapoptotic protein, Bcl-2, resulting in an upregulation of cytochrome c and activation of caspase cascade at both transcriptional and post-transcriptional levels. Moreover, organic extraction enhanced rat liver glutathione S-transferases activity and reactive oxygen species generation, and upregulated aryl hydrocarbon receptor and glutathione S-transferase A1 at both transcriptional and translational levels. Collectively, the results indicate that organic extraction from drinking water impairs liver function, with the involvement of death receptor and mitochondria-mediated apoptosis in rats. The results provide evidence and molecular mechanisms for organic pollutants in drinking water-induced liver dysfunction, which may help prevent and treat organic extraction-induced liver injury.

  20. Electrochemical sensing of hepatocyte viability.

    PubMed

    Tsai, Hweiyan; Tsai, Shang-heng; Ting, Wei-Jen; Hu, Chao-Chin; Fuh, C Bor

    2014-05-21

    We investigated the use of amperometric and chronoamperometric methods with a double mediator system and screen-printed electrodes (SPEs) for the electrochemical sensing of hepatocyte viability. Cell counts were determined based on measuring cellular respiration via interaction of electroactive redox mediators. The oxidation currents of chronoamperometric measurement were proportional to the concentrations of ferrocyanide which was produced via interaction of cellular respiration, succinate and ferricyanide. The integrated oxidation charges increased linearly with the density of the cultured primary rat hepatocytes over a range of 1 × 10(5) to 5 × 10(5) cells per well (slope = 1.98 (±0.08) μC per 10(5) cells; R(2) = 0.9969), and the detection limit was 7600 (±300) cells per well based on S/N = 3. Each density of cells was cultured in triple replicates and individual cell samples were evaluated. The results of the cytotoxic effect of the chronoamperometric method are comparable to those of the tetrazolium-based colorimetric assay. The chronoamperometric method with ferricyanide and succinate mediators is an efficient, alternative method for assessing the viability of primary hepatocytes which can be completed in 20 min. Succinate did not provide an efficient electron shuttle between cytosolic respiratory redox activity of cancer cells and extracellular ferricyanide, an effect that may be useful for distinguishing hepatocarcinoma cells from healthy hepatocytes.

  1. Distribution and processing of the polymeric immunoglobulin receptor in the rat hepatocyte: morphological and biochemical characterization of subcellular fractions.

    PubMed

    Solari, R; Racine, L; Tallichet, C; Kraehenbuhl, J P

    1986-01-01

    The transepithelial transport of polymeric immunoglobulins is an essential process in the mucosal immune system. Transport across the epithelial cells of mucous or exocrine glands is affected by an integral membrane glycoprotein receptor known as membrane secretory component (SCm) or as polymeric immunoglobulin receptor (pIgR). This receptor binds polymeric immunoglobulins at the basolateral cell surface and mediates their transcellular translocation and their release from the apical plasma membrane into external secretions. Release depends on cleavage of the membrane-anchoring domain of the receptor, resulting in liberation of polymeric immunoglobulin bound to the ectoplasmic domain of the receptor (secreted SC or SCs) into extracellular secretions. Using a monoclonal antibody directed against the cytoplasmic tail of the receptor and a polyclonal antibody directed against the secreted ectoplasmic domain, we have combined cell fractionation and Western blotting techniques to examine the fate of these receptor domains in the hepatocyte. In this study, we characterize biochemically and morphologically the various subcellular components separated by our fractionation scheme, and correlate this with biochemical analysis of the receptor in each fraction.

  2. Yes-associated protein/TEA domain family member and hepatocyte nuclear factor 4-alpha (HNF4α) repress reciprocally to regulate hepatocarcinogenesis in rats and mice.

    PubMed

    Cai, Wang-Yu; Lin, Ling-Yun; Hao, Han; Zhang, Sai-Man; Ma, Fei; Hong, Xin-Xin; Zhang, Hui; Liu, Qing-Feng; Ye, Guo-Dong; Sun, Guang-Bin; Liu, Yun-Jia; Li, Sheng-Nan; Xie, Yuan-Yuan; Cai, Jian-Chun; Li, Bo-An

    2017-04-01

    Great progress has been achieved in the study of Hippo signaling in regulating tumorigenesis; however, the downstream molecular events that mediate this process have not been completely defined. Moreover, regulation of Hippo signaling during tumorigenesis in hepatocellular carcinoma (HCC) remains largely unknown. In the present study, we systematically investigated the relationship between Yes-associated protein/TEA domain fa