The role of adrenergic agonists on glycogenolysis in rat hepatocyte cultures and possible involvement of NO.

Certain liver metabolic diseases point to the presence of disturbances in glycogen deposition. Epinephrine raises the cAMP level that activates protein kinase A leading to the activation of phosphorylase and glycogen breakdown. In the present report, we sought to investigate whether NO is produced during adrenoceptor agonist-induced glycogenolysis in rat hepatocytes in cultures. Isolated glycogen rich rat hepatocytes in cultures were used. NO production (NO(2)(-)) was assessed under the effect of adrenergic agonists and adrenergic agonist/antagonist pairs, dibutyryl cyclic AMP sodium-potassium salt (db-cAMP), NO synthase (NOS) inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME), aminoguanidine (AG) and the NO donor S-nitroso-N-acetyl penicillamine (SNAP). The inducible NO synthase (iNOS) mRNA was examined by the reverse transcription-polymerase chain reaction (RT-PCR). Glycogenolysis was quantified by glucose levels released into medium. The amount of glucose and NO(2)(-) released by hepatocytes was increased as a result of epinephrine, phenylephrine or db-cAMP treatments. The increase in glucose and NO(2)(-) released by epinephrine or phenylephrine was blocked or reduced by prazosin pretreatment and by NOS inhibitors aminoguanidine and L-NAME. iNOS gene expression was up-regulated by epinephrine. It can be concluded that glycogenolysis occurs through -adrenoceptor stimulation and a signaling cascade may involve NO production.

Urea synthesis and cyclosporin a biotransformation in a laboratory scale hepatocyte bioreactor model.

Inefficient oxygenation and build-up of waste products are inevitable in a conventional cell culture. The development of a perifusion method for isolated hepatocytes improves the process of oxygenation and helps in end-product removal. For the perifusion of cells, they must be immobilized to prepare a bioreactor model. The present work was directed to testing a hepatocyte bioreactor and maintaining tissue metabolizing activity for periods ranging from 24 to 72 h of continuous and intermittent perifusion and to test the ability of this system for cyclosporin A (CsA), biotransformation and urea synthesis as contrasted to hepatocyte in the culture. Hepatocytes were isolated, immobilized and perifused with William's E culture medium containing 1mM NH(4)Cl and CsA (20 microM). Hepatocytes in the culture were treated in the same way. CsA disappearance from the perifusion or culture media was determined by a HPLC method. Higher urea synthesis rate was achieved by cells in the continuously perifused bioreactor for 24 h compared to culture (0.5+/-0.05 mg h(-1) vs 0.33+/-0.03 mg h(-1), respectively). ALT leakage was lower in the bioreactor model (60 Ul(-1)) as compared to hepatocyte culture (125 Ul(-1)). The ability of hepatocytes in the bioreactor to metabolize CsA was very fast compared to hepatocytes in the culture during 24 h (95% vs 50%, respectively). The present data reveal the higher efficiency of hepatocytes in a bioreactor model as compared to hepatocyte culture. Further research is required in relation to better understanding and standardization of the culture conditions for immobilized and perifused hepatocytes. In addition, the cellular model described here inherits economic and ethical potentials.

Modulatory effect of cyclosporin A on tert-butyl hydroperoxide-induced oxidative damage in hepatocytes.

In the present work, we followed an in vitro protective action of cyclosporin A (CsA) against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in hepatocytes. Various parameters (cell viability, cytosolic calcium level, rhodamine 123 accumulation as indicator of mitochondrial membrane potential and alanine-aminotransferase leakage from cells) were measured as an index of cytotoxicity. Tert-butyl hydroperoxide (1 mM) significantly increased cytosolic Ca2+ and affected mitochondrial membrane potential. Pretreatment with cyclosporin A (0.5 microM) reduced t-BHP-induced cytosolic Ca2+ increase and ALT (alanine-aminotransferase) leakage, but had no protective effect on t-BHP-induced changes of mitochondrial membrane potential. Our data thus suggest that the mechanism of cytoprotection of CsA on the cytosolic Ca2+ changes and ALT leakage induced by t-BHP, does not directly correlate with protection of t-BHP-induced changes of mitochondrial membrane potential.

Cyclosporin A modifies cytoplasmic calcium levels in isolated hepatocytes exposed to oxidative stress due to tert-butyl hydroperoxide.

Within the framework of our studies on hypertension in various rat strains, we have examined the effect of cyclosporin A (CsA) on intracellular calcium signaling under conditions of oxidative stress. For these preliminary experiments, we have chosen isolated hepatocytes of normotensive rats as a model system for the study of the role of intracellular calcium. We used tert-butyl hydroperoxide (t-BHP, 1 mmol x l(-1)) as an prooxidant agent. When compared to the controls, we found increased levels of cytosolic free calcium concentration (Ca2+i) during 120 min incubation. The preincubation of hepatocytes with CsA in the concentration of 0.5 micromol x l(-1)] did not change the physiological level of cytosolic calcium. However, a dual action of CsA on elevated Ca2+i was observed during oxidative injury of hepatocytes: while in the first period of incubation CsA increased Ca2+i, CsA reduced the effect of t-BHP on Ca2+i during the next period of incubation. This indicates the ability of CsA to modify oxidative stress, but further studies are necessary to explain these findings.

Silymarin effects on intracellular calcuim and cytotoxicity: a study in perfused rat hepatocytes after oxidative stress injury.

The re-emergence of silymarin as a natural remedy for diseases of the liver and biliary tract necessitates reevaluation of the efficiency of this compound and its possible mode of action. The aim of this study was to investigate the potentials of silymarin on the amelioration of hepatic injuries. The possible mechanism(s) that contribute to the hepatoprotective effect of silymarin and the role played by intracellular calcium (Ca(2+)(i)) was investigated using tert-butyl hydroperoxide (TBH) and D-galactosamine (D-Gal) intoxication in a model of the isolated immobilized and perfused hepatocytes. Silymarin decreased lactate dehydrogenase (LDH) leakage, increased oxygen consumption, reduced the formation of lipid peroxides (malondialdehyde, MDA) in hepatocytes that were altered by TBH and increased urea synthesis in the perfusion medium. TBH treatment increased Ca(2+)(i) in hepatocytes significantly to a value of more than 600 nM and silymarin pre-treatment reduced the TBH-induced rise in Ca(2+)(i) and brought Ca(2+)(i) level to below 300 nM. Silymarin did not affect LD leakage or urea synthesis in D-Gal-injured cells. It is concluded that silymarin hepatoprotective effect under the present experimental conditions is due to the inhibition of lipid peroxidation and that the modulation of hepatocyte Ca(2+)(i) plays a pivotal role in a protective effect.

The effect of silibinin on experimental cyclosporine nephrotoxicity.

The immunosuppressive drug cyclosporine A (CsA), is metabolized by cytochrome P-450 IIIA. It causes acute reversible as well as chronic largely irreversible nephrotoxic effects. This effect is bases on vasoconstriction of the afferent and efferent glomerular arterioles which leads to a reduction in glomerular plasma flow and glomerular filtration rate. The mechanisms of the vasoconstriction are unclear with a number of different pathways under discussion. Silibinin is the main constituent of silymarin. Silibinin inhibits lipid peroxidation on hepatic microsomes and mitochondria of rats and is also able to reduce the activity of various monooxygenases. Cyclosporin-induced lipid peroxidation and affected cytochrome P-450 may even contribute to cyclosporine nephrotoxicity. We examined the possibility that silibinin had a protective effect as a result of its radical scavenging properties. Silibinin, 5 mg/kg BW i.p., was administered 30 min before cyclosporine application at dose of 30 mg/kg BW daily i.p. The biochemical parameters, total malondialdehyde (MDA) in whole blood and kidney homogenates and specific content of cytochrome P-450 in microsomal liver suspension were estimated. Three groups were studied: controls (con), cyclosporine alone (CsA), and cyclosporine plus silibinin (CsA + Sili). Creatinine was significantly increased after 2 weeks in both cyclosporine treated groups compared to controls (CsA 60.2 +/- 10.6 versus 45.8 +/- 10.4 mumol/L, p < 0.05; and CsA + Sili 72.0 +/- 8.3 versus 45.8 +/- 10.4 mumol/L, p < 0.001) and glomerular filtration rate (GFR) was significantly decreased (p < 0.0001) in the same groups. Total MDA was elevated only in CsA rats (2.26 +/- 0.35 mumol/L, p < 0.05) in comparison with controls (1.60 +/- 0.44 mumol/L, p < 0.05) and with rats treated by CsA + Sili (1.65 +/- 0.27 mumol/L, p < 0.05). The specific content of cytochrome P-450 in microsomal liver suspension was increased in group CsA + Sili (1.179 +/- 0.115 nmol/mg prot) compared to control group (0.775 +/- 0.086 nmol/mg prot., p < 0.05) and also CsA group (0.806 +/- 0.098 nmol/mg prot., p < 0.05). In conclusion, silibinin decreased cyclosporine-induced lipid peroxidation without a protective effect on GFR. These data indicate that this pathway is not be important in cyclosporine-induced nephrotoxicity. Administration of both drugs (CsA + sili) increased the specific content of cytochrome P-450 in liver microsomes. This suggests that the effect of silibinin on cyclosporine biotransformation in the liver is via cytochrome P-450.

Amelioriation of chemically-induced hepatocyte injury by cyclosporine A.

Cyclosporine A, beside its current applications, possesses potential hepatoprotective effects. This study was directed to investigate the effect of Cyclosporine A pretreatment on hepatic injury due to carbon tetrachloride (CCl4) and D-galactosamine. Rats were injected by two successive doses of Cyclosporine A (5 mg kg-1 day-1). Six hours after the second dose, 1 ml kg-1 of CCl4 was administered i.p. Effects associated with Cyclosporine A pretreatment were examined by using isolated hepatocytes and hepatocytes that were immobilized and continuously perfused. D-Galactosamine (5 mM) was added directly to the perfusion medium. After isolation, hepatocytes were examined histologically by light and electron microscopy, immobilized and perfused for further metabolic functional activity evaluation. Cyclosporine A pretreatment in vivo produced hepatoameliorative effects of various degrees which were statistically significant as manifested by: (1) an increased trypan blue exclusion after CCl4; (2) an improved ureagenesis after CCl4; (3) a reduction in the lipid droplets accumulation in the cytoplasm produced by CCl4 administration; (4) well preserved cytoplasmic organelles as mitochondria, endoplasmic reticulum ER, nuclear chromatin structures that were altered by CCl4; and (5) an increased hepatocytes survival in the agarose gel matrix, reduction of LD leakage and improvement of ureagenesis after D-galactosamine addition to the perfusion medium. The beneficial effect of Cyclosporine A pretreatment in modifying hepatotoxicity of chemical insults merits further studies.

Response of immobilized hepatocytes in a perfusion system to anoxia/reoxygenation: effect of cyclosporine A pretreatment.

The present study was designed to investigate the ameliorative effect of cyclosporine A (CsA) pretreatment on an anoxia/reoxygenation injury model by using immobilized perfused hepatocytes. Rats received an i.p. injection of two successive doses of CsA (5 mg/kg/day). Twenty-four hours later hepatocytes were isolated from CsA-treated and control rats. After hepatocyte isolation, immobilization, perfusion, induction of anoxia/reoxygenation, the structural and functional integrity of the hepatocytes was followed in a perfusion medium by measuring the leakage of lactate dehydrogenase (LD) and the time course of urea biosynthesis. CsA pretreatment reduced the initial rate of urea synthesis during normoxia but reduced the drop in the relative percentage rate of urea synthesis during the period of anoxia. LD leakage was increased threefold by anoxia and sevenfold by reoxygenation in cells of untreated animals. After CsA pretreatment in vivo, hepatocytes showed no increase in LD leakage into the medium. These findings demonstrate that the perfused immobilized hepatocytes can be used as a cellular model to assess the effects of liver insults such as anoxia/reoxygenation injury and that CsA modulates the injury. The mechanisms of CsA beneficial effects at the experimental level remain to be elucidated.

The in vitro effect of sodium mercaptoundecahydrododecaborate (borocaptate) on sulfhydryl groups of different rat tissues.

The effect of borocaptate on structural protein sulfhydryl (SH) groups of different organs and kidney subcellular fractions was studied in vitro. It was shown that the binding capacity of borocaptate is not the same for different tissues and subcellular fractions. The highest binding capacity was in the liver, while kidney and brain values were significantly lower. Therefore, it appears that the same concentration of borocaptate may have different effects in various organs.

The concept of application of immobilized and perfused mammalian cells (a bioreactor model) in biomedical research.

An overview of the concept of cellular immobilization and perfusion as a small laboratory bioreactor model is presented. The cellular systems currently used may be described as static. This is due to conditions of hypoxia and waste product build-up that affect cell physiology. Cellular immobilization and perfusion is, therefore, expected to maintain the cells for very long periods of time under approximately physiological conditions. A number of applications of immobilized perfused hepatocytes and other cellular systems such as adipocytes and Sertoli cells are described in addition to various other cell lines. Moreover, it is suggested that the bioreactor may have potential use as a bioartificial organ.

Preparation of functionally active immobilized and perfused mammalian cells: an example of the hepatocyte bioreactor.

In the present study, a method has been employed for hepatocyte immobilization in agarose threads which allows for cell perfusion. The rat hepatocytes are isolated from the liver. A 1.8% low-gelling agarose solution is prepared in warm Krebs-Henseleit solution. The agarose solution is mixed 1:1 with the hepatocytes and the cells are immobilized in agarose threads by extruding the agarose-cell mixture through cooled Chemfluor teflon (TFE) tubing. Light and electron microscopy studies indicated the integrity of the hepatocytes in the gel matrix. This system allows for liver cell perfusion and viability studies to be carried out non-invasively on the cells and provides data that are comparable to those obtained with a perfused isolated liver. Immobilized hepatocytes are an in vitro system worthy of further evaluation which may be useful in the studies of liver cell metabolism and the response of the liver to foreign chemicals.

Application of the hepatocytes bioreactor to xenobiotic biotransformation.

This study deals with the application of the previously developed immobilized and perfused isolated hepatocytes as a cellular system for the study of representative phase I and phase II of biotransformation reactions. To illustrate phase I reactions, aminopyrine (0.17-4.25 mmol/l) and hexobarbital (0.2 mmol/l) were selected. For phase II reactions, glutathione transferase activity was evaluated by using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate (0.125-2.0 mmol/l). Formaldehyde, that was formed from aminopyrine, increased steadily in the perfusion medium with time. The perfused hepatocytes eliminated hexobarbital at a much higher rate than the hepatocytes in suspension. At several time points the amount of CDNB-glutathione conjugate formed per one million hepatocytes in the bioreactor was almost twice the amount formed by the hepatocytes in suspension. The present data illustrate the successful application of the hepatocyte bioreactor in phase I and phase II of xenobiotic metabolism and indicate that the cells were metabolically more active than the cells in suspension.

A preliminary evaluation of drug biotransformation in hepatocytes of genetically defined rat strains.

This study was directed to use the genetically developed isoprenaline-sensitive (S), isoprenaline-resistant (R) and spontaneous hypertensive rats (SHR) as standard diseased animal models for in vitro liver function evaluation of drug biotransformation. Hepatic hexobarbital hydroxylase and glutathione transferase (GST) were evaluated by using hexobarbital and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates, at concentrations of 0.21 mmol/l and 1 mmol/l, respectively. The assay was conducted by using isolated hepatocytes in suspension and hepatocytes in a bioreactor configuration. The data demonstrate that there are certain cellular pharmacokinetic differences in hexobarbital hydroxylase and GST activities in hepatocytes obtained from Wistar, SHR, R and S strains which can be better demonstrated, when using the model of perfused and immobilized hepatocytes.

Muramyl dipeptide and carbon tetrachloride hepatotoxicity in rats: involvement of plasma membrane and calcium homeostasis in protective effect.

The present study was carried out to investigate the effect of single and multiple doses of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) on CCl4-induced hepatotoxicity, and hence some of the mechanisms involved. MDP (8.26 mumol/kg i.v.) was administered to rats according to different protocols followed by a single dose of CCl4 (5.2 mmol/kg i.p.), and either the hepatocytes were subsequently isolated and tested for viability and lipid peroxides formation or the level of serum aminotransferases and lactate dehydrogenase (LD) was measured. The results clearly indicate that MDP pretreatment in a single dose reduced CCl4 hepatotoxicity as judged from viability tests as well as reduction of elevated lipid peroxides induced by CCl4 administration. The level of lactate dehydrogenase was also brought to normal value by single MDP administration. MDP also decreased significantly the CCl4-elevated Ca2+ content of isolated hepatocytes and postmicrosomal supernatant Ca2+. 14C-palmitic acid incorporation was increased significantly for neutral lipids and/or phospholipids in hepatocytes and certain subcellular fractions under MDP treatment in vivo. A different effect was seen after multiple MDP administration which further increased CCl4-induced elevation of aminotransferases. Even the repeated administration of MDP without CCl4 increased the level of the latter enzymes. It may be concluded that a single administration of MDP can protect liver cells from CCl4 injury by a mechanism affecting the plasma membrane.

Study of relationship between allogeneic transplantability of tumors and their effects on drug-metabolizing system in rats.

Effects of three different fibrosarcomas on the hepatic mixed-function oxidase system were studied in males of the Lewis inbred strain of rats. No association between graded potentiality of these tumors to grow across histocompatibility barriers and their suppressive effects upon the microsomal drug-metabolizing system was found.