Isolated Rat Hepatocytes In Suspension Research Articles

Functional and toxicological consequences of metabolic bioactivation of methapyrilene via thiophene S-oxidation: Induction of cell defence, apoptosis and hepatic necrosis

Methapyrilene, [ N, N-dimethyl- N′-pyridyl- N′(2-thienylmethyl)-1,2-ethanediamine] (MP) was withdrawn from, clinical use due to reported periportal hepatic necrosis and hepatocarcinogenicity in the rat, via S-oxidation of the thiophene group. In this study MP is used as a model hepatotoxin to further characterise the functional consequences of S-oxidation of the thiophene group in vivo, in rat models and in vitro, in freshly isolated rat hepatocyte suspensions. In vivo histological studies revealed the early depletion of glutathione (GSH), which was confined to the damaged periportal area, in contrast to an increase in GSH levels in the centrilobular region. Additionally, the induction of cell defence was demonstrated by an increase in the protein levels of heme-oxygenase 1 (HO-1) and glutamate cysteine ligase, catalytic subunit (GCLC) in vivo. Histological examination demonstrated that cytotoxicity progresses initially via apoptosis before an increase in necrosis over the 3-day administration. An apoptotic-like mechanism was observed in vitro via the measurement of cytochrome c release and caspase activation. Conclusion: This study provides evidence for a complex pathway of MP-induced hepatotoxicity which progresses through early adaptation, apoptosis, necrosis and inflammation, all underpinned by the zonal induction and depletion of GSH within the liver.

Beta-carotene prevents bile acid-induced cytotoxicity in the rat hepatocyte: Evidence for an antioxidant and anti-apoptotic role of beta-carotene in vitro.

Hydrophobic bile acids are implicated in the pathogenesis of cholestatic liver disorders through mechanisms involving oxidative stress and mitochondrial dysfunction. Antioxidants ameliorate bile acid-induced cytotoxicity in rat hepatocyte suspensions. The purpose of the current study was to evaluate the potential protective role of beta-carotene (betaC), a putative fat-soluble antioxidant that is reduced in patients with cholestasis, against bile acid-induced hepatotoxicity. In freshly isolated rat hepatocyte suspensions that were exposed to the toxic hydrophobic bile acid glycochenodeoxycholic acid (100 or 500 microM), betaC (100 microM) decreased generation of reactive oxygen species by >50%, similar to the inhibition afforded by alpha-tocopherol. Commensurate with this antioxidant effect, 100 microM betaC also protected hepatocytes against both glycochenodeoxycholic acid-induced cellular necrosis and apoptosis, which was associated with reduction in caspase 3 activation, inhibition of mitochondrial cytochrome c release in rat hepatocytes, and prevention of the mitochondrial permeability transition in both liver mitochondria and rat hepatocytes. A lower concentration of betaC (50 microM) produced similar antioxidant and anti-apoptotic protection but with less inhibition against cell necrosis, suggesting that the higher concentration of betaC may have conferred additional cytoprotection not directly related to its antioxidant function. These results demonstrate that the antioxidant effects of betaC may provide hepatoprotection against cholestatic liver injury by preventing bile acid-induced oxidative stress and mitochondrial perturbations.

Hepatotoxicity of 3,4-methylenedioxyamphetamine and alpha-methyldopamine in isolated rat hepatocytes: formation of glutathione conjugates.

The amphetamine designer drugs 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy") and its N-demethylated analogue 3,4-methylenedioxyamphetamine (MDA or "love") have been extensively used as recreational drugs of abuse. MDA itself is a main MDMA metabolite. MDMA abuse in humans has been associated with numerous reports of hepatocellular damage. Although MDMA undergoes extensive hepatic metabolism, the role of metabolites in MDMA-induced hepatotoxicity remains unclear. Thus, the aim of the present study was to evaluate the effects of MDA and alpha-methyldopamine (alpha-MeDA), a major metabolite of MDA, in freshly isolated rat hepatocyte suspensions. The cells were incubated with MDA or alpha-MeDA at final concentrations of 0.1, 0.2, 0.4, 0.8, or 1.6 mM for 3 h. The toxic effects induced following incubation of hepatocyte suspensions with these metabolites were evaluated by measuring cell viability, the extent of lipid peroxidation, levels of glutathione (GSH) and glutathione disulfide (GSSG), the formation of GSH conjugates, and the activities of GSSG reductase (GR), GSH peroxidase (GPX), and GSH S-transferase (GST). MDA induced a concentration- and time-dependent GSH depletion, but had a negligible effect on lipid peroxidation, cell viability, or on the activities of GR, GPX, and GST. In contrast, alpha-MeDA (1.6 mM, 3 h) induced a marked depletion of GSH accompanied by a loss on cell viability, and decreases in GR, GPX and GST activities, although no significant effect on lipid peroxidation was found. For both metabolites, GSH depletion was not accompanied by increases in GSSG levels; rather, 2-(glutathion- S-yl)-alpha-MeDA and 5-(glutathion- S-yl)-alpha-MeDA were identified by HPLC-DAD/EC within cells incubated with MDA or alpha-MeDA. The results provide evidence that one of the early consequences of MDMA metabolism is a disruption of thiol homeostasis, which may result in loss of protein function and the initiation of a cascade of events leading to cellular damage.

Beta-alanine protection against hypoxic liver injury in the rat

Liver hypoxia still represents an important cause of liver injury during shock and liver transplantation. We have investigated the protective effects of β-alanine against hypoxic injury using isolated perfused rat livers and isolated rat hepatocyte suspensions. Perfusion with hypoxic Krebs–Henseleit buffer increased liver weight and caused a progressive release of lactate dehydrogenase (LDH) in the effluent perfusate. The addition of 5 mmol/l β-alanine to the perfusion buffer completely prevented both weight increase and LDH leakage. These findings were confirmed by histological examinations showing that β-alanine blocked the staining by trypan blue of either liver parenchymal and sinusoidal cells. Studies performed in isolated hepatocytes revealed that β-alanine exerted its protective effects by interfering with Na + accumulation induced by hypoxia. The addition of γ-amino-butyric acid, which interfered with β-alanine uptake by the hepatocytes or of Na +/H + ionophore monensin, reverted β-alanine protection in either hepatocyte suspensions or isolated perfused livers. We also observed that liver receiving β-alanine were also protected against LDH leakage and weight increase caused by the perfusion with an hyposmotic (205 mosm) hypoxic buffer obtained by decreasing NaCl content from 118 to 60 mmol/l. This latter effect was not reverted by blocking K + efflux from hepatocyte with BaCl 2 (1mmol/l). Altogether these results indicated that β-alanine protected against hypoxic liver injury by preventing Na + overload and by increasing liver resistance to osmotic stress consequent to the impairment of ion homeostasis during hypoxia.

Caffeine Potentiation of Allyl Alcohol-Induced Hepatotoxicity. II. In Vitro Study*

We examined the effects of caffeine (C) on allyl alcohol (AA)- and acrolein (A)-induced hepatotoxicity on freshly-isolated, rat hepatocytes obtained from livers of adult, male, Sprague-Dawley rats. Isolated rat hepatocytes in suspension were incubated in each test with one of the following: 0, 1.0, or 2.5 mM of AA alone; or with 0, 2.5, or 5 mM of C alone; or a combination of AA and C at the same range of concentrations as used alone, for 15, 30, 60, 90, and 120 minutes at 37 degrees C. A dose- and time-dependent potentiation of cytotoxicity as measured by cellular viability (using trypan blue exclusion) were observed. The AA (2.5 mM)-induced lactate dehydrogenase (LDH) leakage observed after 60 minutes incubation was completely prevented when pretreated for 15 minutes with 4-methylpyrazole (MP) (0.5 mM). Such pretreatment, even with a double dose of 4-MP, only partially, and not significantly, prevented LDH leakage when the hepatocytes were incubated with a mixture of 2.5 mM AA and 5 mM C. The depletion of hepatocyte nonprotein sulfhydryl (NPSH) content caused by AA was further enhanced in the presence of C, as early as 15 minutes after their exposure. The AA-induced increase in lipid peroxidation was also potentiated by C; however, potentiation started later, and only after sufficient depletion of NPSH (mostly glutathione) occurred resulting from the presence of AA plus C. A significant loss of protein sulfhydryls in rat hepatocytes could be noted following a 60-minute incubation period with either AA (1 mM) or AA (1 mM) plus C (5 mM). Similarly, C produced a dose-and time-dependent potentiation of A-induced liver cytotoxicity, which was preceded by severe loss of NPSH content within 15 minutes of exposure, whereas the potentiation of lipid peroxidation (LPO) resulting from A plus C was found to be a relatively late event, as with AA plus C. Furthermore, combined treatment with AA and C produced a significantly higher cytotoxicity (as measured by cellular viability) than that due to the combined treatment with A plus C based on equimolar concentration. These results suggest that two increased bioactivation pathways of AA involving the P-450 mixed-function oxidase system resulting from C may be involved in the potentiation of AA hepatotoxicity.

Glutathione-dependent regulation of nitric oxide production in isolated rat hepatocyte suspensions.

Freshly isolated suspensions of rat parenchymal liver cells (hepatocytes) spontaneously produce large amounts of nitrite following collagenase isolation. Our previous studies indicate that nitrite production is associated with the expression of inducible nitric oxide synthase (iNOS) and reflects NO production. Depletion of glutathione (GSH) with diethylmaleate (DEM) inhibited nitrite production, and this inhibition was time-dependent. DEM was more effective in blocking nitrite production if it was added within the first 1 hr of the start of the incubation. The reducing agent dithiothreitol (DTT) and the alkylating agent ethyl methanesulfonate (EMS) also inhibited hepatocyte nitrite production, and this inhibition was also greatest if they were added within 1 hr of initiating the incubation. However, EMS added at 3 hr still reduced 6-hr nitrite production by about 70%. This reduction in nitrite production by EMS added at 3 hr may be due to the direct modification of thiol groups on the iNOS protein because we have determined that iNOS activity is inhibited by the sulfhydryl modifying reagent N-ethylmaleimide (NEM). Western blots also indicate that the iNOS protein is expressed when EMS is added at 3 hr. The addition of DEM, DTT, or EMS at 0 time greatly reduced the levels of cellular iNOS mRNA relative to controls as determined by quantitative RT-PCR. Based on our results with mRNA levels, both DTT and depletion of cellular GSH appear to inhibit the early signaling events leading to iNOS expression and suggest that the control of iNOS induction in hepatocytes is sensitive to the thiol redox status of the cell.

Effect of hydrazine upon vitamin B 12-dependent methionine synthase activity and the sulphur amino acid pathway in isolated rat hepatocytes

The effect of the industrial chemical, hydrazine (4–12 mM), on methionine synthase (EC 2.1.1.13) activity and levels of the sulphur amino acids homocysteine, cysteine, and taurine as well as GSH were investigated in vitro in isolated rat hepatocyte suspensions and monolayers in order to explain some of the adverse in vivo effects of hydrazine. None of the concentrations of hydrazine were overtly cytotoxic in hepatocyte suspensions (measured as lactate dehydrogenase [LDH] leakage) after 3 hr. However, after 24 hr in culture cells treated with 12 mM, hydrazine showed a significant increase in LDH leakage. Methionine synthase activity was reduced by hydrazine (8 and 12 mM) in suspensions (by 45 and 55%, after 3 hr) and monolayers (12 mM; 65–80% after 24 hr). This was not due to nitric oxide production and the inhibitor of nitric oxide synthase, N ω-nitro- l-arginine, failed to protect against the hydrazine-induced loss of ATP and GSH and the reduction in urea synthesis at 24 hr. Homocysteine export was increased by 6 mM hydrazine, and total taurine content of treated cells was increased by 12 mM hydrazine. Thus, hydrazine was found to have several important and possibly deleterious effects on some parts of the sulphur amino acid pathway.

Propofol preserves the viability of isolated rat hepatocyte suspensions under an oxidant stress.

The purpose of this study was to investigate whether propofol protects rat hepatocyte suspensions against an oxidant attack by a free radical generator 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH). Rat hepatocyte suspensions (2 x 10(6) cells/mL) were prepared using Seglen's collagenase perfusion technique. Suspensions were treated with AAPH (50 mM) alone, propofol (28 microM) plus AAPH, or, in a separate experiment, with either AAPH alone or 10% intralipid (0.5 microL/mL) plus AAPH. Each experiment had untreated control suspensions. Cell viability was measured at 1, 2, and 3 h using the trypan blue exclusion test and expressed as a percentage of the initial number of viable cells. Cells taken from control at time 0 h and each experimental group at 1 h from five separate hepatocyte preparations were examined by electron microscopy. Control cell viability decreased with time. The addition of AAPH significantly reduced viability compared with control (P < 0.0001); pretreatment with propofol significantly attenuated this effect at 1 h (P = 0.0008), but 10% intralipid had no effect. Electron microscopy revealed structural changes in cell membranes that could have accounted for the inability to exclude trypan blue. In conclusion, a 28-microM concentration of propofol protects rat hepatocytes from an oxidant stress sufficient to cause cell death at 1 h. Oxidants contribute to tissue injury in a variety of situations. We have shown that the anesthetic propofol improves survival of liver cells exposed to oxidant injury at blood concentrations achieved in anesthetized patients. These effects may be relevant during transplantation and critical illness.

Propofol Preserves the Viability of Isolated Rat Hepatocyte Suspensions Under an Oxidant Stress

The purpose of this study was to investigate whether propofol protects rat hepatocyte suspensions against an oxidant attack by a free radical generator 2,2[prime]-azobis (2-amidinopropane) dihydrochloride (AAPH). Rat hepatocyte suspensions (2 x 106 cells/mL) were prepared using Seglen's collagenase perfusion technique. Suspensions were treated with AAPH (50 mM) alone, propofol (28 [micro sign]M) plus AAPH, or, in a separate experiment, with either AAPH alone or 10% intralipid (0.5 [micro sign]L/mL) plus AAPH. Each experiment had untreated control suspensions. Cell viability was measured at 1, 2, and 3 h using the trypan blue exclusion test and expressed as a percentage of the initial number of viable cells. Cells taken from control at time 0 h and each experimental group at 1 h from five separate hepatocyte preparations were examined by electron microscopy. Control cell viability decreased with time. The addition of AAPH significantly reduced viability compared with control (P < 0.0001); pretreatment with propofol significantly attenuated this effect at 1 h (P = 0.0008), but 10% intralipid had no effect. Electron microscopy revealed structural changes in cell membranes that could have accounted for the inability to exclude trypan blue. In conclusion, a 28-[micro sign]M concentration of propofol protects rat hepatocytes from an oxidant stress sufficient to cause cell death at 1 h. Implications: Oxidants contribute to tissue injury in a variety of situations. We have shown that the anesthetic propofol improves survival of liver cells exposed to oxidant injury at blood concentrations achieved in anesthetized patients. These effects may be relevant during transplantation and critical illness. (Anesth Analg 1998;87:1152-7)

The effect of idebenone, a coenzyme Q analogue, on hydrophobic bile acid toxicity to isolated rat hepatocytes and hepatic mitochondria

Oxidant stress induced by hydrophobic bile acids has been implicated in the pathogenesis of liver injury in cholestatic liver disorders. We evaluated the effect of idebenone, a coenzyme Q analogue, on taurochenodeoxycholic acid (TCDC)-induced cell injury and oxidant stress in isolated rat hepatocytes and on glycochenodeoxycholic acid (GCDC)-induced generation of hydroperoxides in fresh hepatic mitochondria. Isolated rat hepatocytes in suspension under 9% oxygen atmosphere were preincubated with 0, 50, and 100 μmol/l idebenone for 30 min and then exposed to 1000 μmol/l TCDC for 4 h. LDH release (cell injury) and thiobarbituric acid reactive substances (measure of lipid peroxidation) increased after TCDC exposure but were markedly suppressed by idebenone pretreatment. In a second set of experiments, the addition of 100 μmol/l idebenone up to 3 h after hepatocytes were exposed to 1000 μmol/l TCDC resulted in abrogation of subsequent cell injury and markedly reduced oxidant damage to hepatocytes. Chenodeoxycholic acid concentrations increased to 5.15 nmol/10 6 cells after 2 h and to 7.05 after 4 h of incubation of hepatocytes with 1000 μmol/l TCDC, and did not differ in the presence of idebenone. In freshly isolated rat hepatic mitochondria, when respiration was stimulated by succinate, 10 μmol/l idebenone abrogated the generation of hydroperoxides during a 90-minute exposure to 400 μmol/l GCDC. These data demonstrate that idebenone functions as a potent protective hepatocyte antioxidant during hydrophobic bile acid toxicity, perhaps by reducing generation of oxygen free radicals in mitochondria.

Metabolism of exogenous S-adenosylmethionine in isolated rat hepatocyte suspensions: methylation of plasma-membrane phospholipids without intracellular uptake.

Administration of S-adenosylmethionine (AdoMet), the main biological methyl donor, has been shown to exert favourable effects on liver disorders in man and animal models. The mechanism of action of AdoMet has, however, remained elusive, mainly owing to controversies with respect to its capacity to enter intact liver cells. Incubation of isolated rat hepatocytes with 2 or 50 microM -methyl-14C-AdoMet showed that it was utilized predominantly to methylate cellular phospholipids, forming mainly phosphatidylcholine, although less than 0.2% of labelled AdoMet was found inside the cells. The concentration of neither AdoMet nor S-adenosylhomocysteine (AdoHcy), its demethylation product, was significantly elevated inside the cells. A slight elevation of intracellular AdoMet was only recorded on incubation with concentrations of AdoMet above 200 microM. AdoHcy, which does not penetrate cells, inhibited phospholipid methylation from [methyl-14C]AdoMet but not from [methyl-14C]Met. Elevation of intracellular AdoHcy by adenosine dialdehyde, an inhibitor of AdoHcy hydrolase, inhibited phospholipid methylation from [methyl-14C]Met, but virtually not at all from [methyl-14C]AdoMet. Taken together, these data indicate that exogenous AdoMet does not penetrate hepatocytes significantly but is utilized for phospholipid methylation on the outer surface of the plasma membrane.

Assessment of isolated rat hepatocyte suspensions as a model system to examine the effect of P-glycoprotein inhibitors on daunorubicin and etoposide hepatobiliary disposition: [formula omitted] Division of Pharmaceutics, School of Pharmacy, University of North Carolina, Chapel Hill, and ∗Department of Drug Metabolism, Glaxo Wellcome, Research Triangle Park, NC

Assessment of isolated rat hepatocyte suspensions as a model system to examine the effect of P-glycoprotein inhibitors on daunorubicin and etoposide hepatobiliary disposition: [formula omitted] Division of Pharmaceutics, School of Pharmacy, University of North Carolina, Chapel Hill, and ∗Department of Drug Metabolism, Glaxo Wellcome, Research Triangle Park, NC

Regulation of liver cell volume and proteolysis by glucagon and insulin

The effects of insulin and glucagon on liver cell volume and proteolysis were studied in isolated perfused rat liver. The rate of proteolysis was assessed as [3H]leucine release from single-pass-perfused livers from rats which had been prelabelled in vivo by intraperitoneal injection of [3H]leucine. The intracellular water space was determined from the wash-out profiles of simultaneously added [3H]inulin and [14C]urea. In normo-osmotic (305 mosM) control perfusions the intracellular water space was 548 +/- 10 microliters/g wet mass (n = 44) and was increased by 16.5 +/- 2.6% (n = 6), i.e. by 85 +/- 14 microliters/g, after hypoosmotic exposure (225 mosM). Glucagon (0.1 microM) decreased the intracellular water space by 17 +/- 4% (n = 4), whereas insulin (35 nM) increased the intracellular water space by 9.3 +/- 1.4% (n = 15). Also, in isolated rat hepatocyte suspensions insulin (100 nM) caused cell swelling by 10.7 +/- 1.8% (n = 16), which was fully reversed by glucagon. In perfused liver, insulin-induced cell swelling was accompanied by a hepatic net K+ uptake (4.5 +/- 0.2 mumol/g) and an inhibition of proteolysis by 21 +/- 2% (n = 12); further addition of glucagon led to a net K+ release of 3.8 +/- 0.2 mumol/g (n = 7) and fully reversed the insulin effects on both cell volume and proteolysis. Similarly, insulin-induced cell swelling and inhibition of proteolysis were completely antagonized by hyperosmotic (385 mosM) cell shrinkage. Furthermore, cell swelling and inhibition of proteolysis after hypo-osmotic exposure or amino acid addition were reversed by glucagon-induced cell shrinkage. There was a close relationship between the extent of cell swelling and the inhibition of proteolysis, regardless of whether cell volume was modified by insulin, glucagon or aniso-osmotic exposure. The data show that glucagon and insulin are potent modulators of liver cell volume, at least in part by alterations of cellular K+ balance, and that their opposing effects on hepatic proteolysis can largely be explained by opposing effects on cell volume. It is hypothesized that hormone-induced alterations of cell volume may represent an important, not yet recognized, mechanism mediating hormonal effects on metabolism.

Inhibition of long-chain ACYL-CoA synthetase by the peroxisome proliferator perfluorodecanoic acid in rat hepatocytes

Perfluorodecanoic acid (PFDA) is a potent peroxisome proliferator and is known to affect hepatic lipid metabolism in rats. The effects of PFDA on fatty acid utilization were examined in isolated rat hepatocyte suspensions and in rat liver mitochondria and microsomes. PFDA inhibited the oxidation of palmitic acid but not octanoic or pyruvic acids when hepatocytes were incubated with 1 mM PFDA. At this PFDA concentration the esterification of palmitic acid into triacylglycerols was also reduced. The activity of long-chain acyl-CoA synthetase (ACS), an enzyme essential for both oxidation and esterification of fatty acids, was reduced in hepatocytes incubated with 1 mM PFDA. Carnitine palmitoyltransferase (CPT), an important enzyme for the oxidation of long-chain fatty acids, was not altered in hepatocytes incubated with this PFDA concentration. In rat liver mitochondria, palmitate oxidation and ACS activity were reduced significantly (P<0.01) at a PFDA concentration that had no effect on CPT activity. The inhibition of ACS by PFDA was similar in liver mitochondria and microsome preparations. In mitochondria incubated with PFDA, the inhibition of ACS appears to be noncompetitive for the substrates palmitic acid and CoA. However, the ACS inhibition by PFDA appeared to be competitive for the ATP binding site of the enzyme. Several chain length perfluorinated fatty acids were examined for their ability to inhibit mitochondrial ACS. Short-chain perfluorinated fatty acids (perfluoroproprionic and -butyric acid) did not inhibit ACS activity. However, medium-chain perfluorinated acids (perfluorooctanoic, -ananoic and -decanoic acid) were found to be potent inhibitors of ACS in isolated mitochondria. Whether ACS inhibition is causally related to PFDA-induced peroxisome proliferation and altered lipid metabolism seen in vivo is yet to be determined.

Ultrastructural Study of Isolated Rat Hepatocyte Suspensions Incubated in Incomplete or Complete Culture Medium

Ultrastructural changes in isolated rat hepatocytes (IHC) in suspension were examined after 1, 3, 5 and 10 hr incubation, and the influence of Krebs-Henseleit (K-H) buffer as an incomplete medium and GIT culture medium (Nihon Pharmaceutical Co.) as a complete culture medium were investigated. The viability was approximately 60-75% after 1, 3 and 5 hr incubation, and was slightly higher in GIT medium than K-H buffer. Immediately after isolation, IHC showed a cuboidal shape, and after 3 and 5 hr incubation, a round shape with numerous surface microvilli. Cytoplasmic organelles appeared almost normal. After 10 hr incubation, cells reaggregated and the number of autophagic vacuoles increased. Cellular changes, such as slight dilatation of the endosplasmic reticulum and Golgi apparatus, and bleb formation in the cell surface, were more often observed in IHC incubated in K-H buffer than in GIT medium. IHC incubated in GIT medium contained glycogen particles in the cytoplasm, and lipoprotein-like particles were observed in the cisternae of Golgi apparatus and smooth endoplasmic reticulum. The lipoprotein-like particles were found in the intercellular spaces after 10 hr incubation. These results suggest that GIT medium is superior to K-H buffer in its ability to preserve normal structure and function of IHC.

Propranolol metabolism by isolated hepatocytes from normal and cirrhotic rat livers: the effect of albumin

Several recent reports have shown that the hepatic uptake and subsequent elimination of some substrates is faster in the presence of albumin than in its absence, as if some of the substrate bound to albumin was also available for uptake. In the present study, we examined the effect of albumin on the clearance of propranolol by isolated rat hepatocyte suspensions. The clearance of total drug decreased progressively as albumin concentration increased. There was also a progressive decrease in the free fraction of propranolol and the net result was an increase in the clearance of unbound drug (+50% at 40 g/L albumin). This increase was not due to an oncotic pressure effect of albumin, nor to the presence of fatty acids bound to albumin. The clearance of propranolol by isolated hepatocytes from cirrhotic rats was decreased compared with controls (-50%), and albumin also increased propranolol free clearance, albeit to a lesser extent than in control animals. Our results indicate that albumin facilitates the elimination of propranolol by hepatocytes, possibly because of surface-mediated catalysis of the albumin-propranolol complexes.

Effect of membrane potential and cellular ATP on glutathione efflux from isolated rat hepatocytes.

total glutathione (GSH) efflux was studied in isolated rat hepatocyte suspensions at repleted GSH content (45-55 nmol/10(6) cells). The increase in concentrations of medium K+ in place of Na+ caused a parallel fall in membrane potential and total GSH efflux. Ouabain (1 mM) and replacement of Na+ with choline caused a gradual fall in membrane potential and GSH efflux. Hyperpolarization of hepatocytes with lipophilic anions, thiocyanate, and nitrate was associated with significantly increased efflux. Total GSH efflux was inhibited by increasing concentrations of fructose, antimycin A, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and there was a direct relationship between the rate of efflux and cellular ATP. Changes in total GSH efflux were paralleled by changes in GSH determined by high-performance liquid chromatography. Vanadate markedly inhibited efflux but caused only a modest decrease in cellular ATP. Fructose, antimycin A, and vanadate did not affect membrane potential or cell volume under the conditions at which efflux was inhibited. These results suggest independent requirements for both membrane potential and ATP in the transport of GSH.

Role of glutathione reductase during menadione-induced nadph oxidation in isolated rat hepatocytes

Metabolism of menadione (2-methyl-1,4-naphthoquinone) results in the rapid oxidation of NADPH within isolated rat hepatocytes. The glutathione redox cycle is thought to play a major role in the consumption of NADPH during menadione metabolism, chiefly through glutathione reductase (GSSG-reductase). This enzyme reduces oxidized glutathione (GSSG), formed via the glutathione-peroxidase reaction, with the concomitant oxidation of NADPH. To explore the relationship between GSSG-reductase and the consumption of NADPH during menadione metabolism, isolated rat hepatocyte suspensions were exposed to non-lethal and lethal menadione concentrations (100 and 300 μM respectively) following the inhibition of GSSG-reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Menadione produced a concentration-related depletion of GSH (measured as non-protein sulfhydryl content) which was potentiated markedly by BCNU. Menadione toxicity was potentiated at either concentration by BCNU based on lactate dehydrogenase leakage at 2hr. In addition, the NADPH content of isolated hepatocytes rapidly declined following exposure to either concentration of menadione. However, at the lower menadione concentration (100 μM), the NADPH content returned to control values or above by 60 min, whereas the NADPH content of cells exposed to 300 μM menadione with or without BCNU remained depressed for the duration of the incubation. These data suggest that, although NADPH is required by GSSG-reductase for the reduction of GSSG to GSH during quinone-induced oxidative stress, this pathway does not appear to be the major route by which NADPH is consumed during the metabolism of menadione in isolated hepatocytes.

Metabolism-related spectral characterization and subcellular distribution of polychlorinated biphenyl congeners in isolated rat hepatocytes

The disposition and biotransformation of 4,4'-dichlorobiphenyl (4-DCB), 2,2',3,3',6,6'-hexachlorobiphenyl (236-HCB), and 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB) were studied in isolated rat hepatocyte suspensions. The polychlorinated biphenyls (PCBs) were taken up rapidly by the cells but incompletely metabolized. Metabolism followed first-order Michaelis-Menten kinetics for 20 min and plateaued by 60 min, at which point only 32% of 4-DCB (0.005 to 100 μM) and 60% of 236-HCB (0.001 to 100 μM) were metabolized, while metabolism of 245-HCB was not detected (0.1 to 200 μM). Kinetic studies revealed that both 4-DCB and 236-HCB were metabolized by two Michaelis-Menten processes, displaying high- and low-affinity binding. Readdition of congener once metabolism plateaued resulted in a reinitiation of metabolism with the same proportion of metabolites produced. The termination of metabolism was not due to destruction of the mixed-function oxidases or to depletion of cofactors. The metabolism of PCB congeners is influenced by the affinity of the congener for cytochrome P-450 and partitioning of the congener within the hepatocyte. Analysis of absorbance differences (Δ absorbance 390-240 nm) of equimolar concentrations of congener (100 μM) revealed that 236-HCB displayed the greatest affinity of binding to cytochrome P-450 followed by 4-DCB, while 245-HCB showed virtually no binding. Microsomal preparations demonstrated equivalent but greater absorbance values. Subcellular distribution of 14C-labeled congener and its metabolites showed that the majority of radioactivity appeared in the cytosolic fraction, representing 70% of the dose added for each congener. Cytosolic binding of congener and metabolites may influence both the availability of congener to cytochrome P-450 and the excretion rate of metabolites from the cell.

A characterization of haem uptake and intracellular distribution by isolated hepatocytes.

The uptake and intracellular distribution of haem by isolated rat hepatocyte suspensions was studied. An increase in cell haem content occurred after a challenge with 5, 10 or 20 microM haem, supplied as methaemalbumin. The rate of haem uptake was temperature dependent; no non-specific binding occurred. Intracellular haem distribution data are consistent with a rapid association of haem with the endoplasmic reticulum fraction prior to its accumulation in the cytosol and at the mitochondrion.