Toxic consequence of the abrupt depletion of glutathione in cultured rat hepatocytes

Cultured hepatocytes were exposed to two chemicals, dinitrofluorobenzene (DNFB) and diethyl maleate (DEM), that abruptly deplete cellular stores of glutathione. Upon the loss of GSH, lipid peroxidation was evidenced by an accumulation of malondialdehyde in the cultures followed by the death of the hepatocytes. Pretreatment of the hepatocytes with a ferric iron chelator, deferoxamine, or the addition of an antioxidant, N,N'-diphenyl-p-phenylenediamine (DPPD), to the culture medium prevented both the lipid peroxidation and the cell death produced by either DNFB or DEM. However, neither deferoxamine nor DPPD prevented the depletion of GSH caused by either agent. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or inhibition of catalase by aminotriazole sensitized the hepatocytes to the cytotoxicity of DNFB. In a similar manner, pretreatment with BCNU potentiated the cell killing by DEM. DPPD and deferoxamine protected hepatocytes pretreated with BCNU and then exposed to DNFB or DEM. These data indicate that an abrupt depletion of GSH leads to lipid peroxidation and cell death in cultured hepatocytes. It is proposed that GSH depletion sensitizes the hepatocyte to its constitutive flux of partially reduced oxygen species. Such an oxidative stress is normally detoxified by GSH-dependent mechanisms. However, with GSH depletion these activated oxygen species are toxic as a result of the iron-dependent formation of a potent oxidizing species.     

The effect of dexamethasone on transferrin secretion by cultured fetal rat hepatocytes

Cultured fetal rat hepatocytes derived from 12, 15 and 19-day gestation rats are capable of secreting transferrin. When dexamethasone is added to the medium an increased secretion rate is observed. The changes in secretion rates in control as well as dexamethasone-treated cells during culture have been shown to correlate with the level of mRNA coding for transferrin. Immunocytochemical experiments show that initially all hepatocytes contain transferrin which is localized in the lumina of the perinuclear space, rough endoplasmic reticulum and in the saccules and vesicles of the Golgi apparatus. During culture, particularly in control cells, the intensity of labelling varies from cell to cell. In addition, adjacent cells are observed to label more intensely in different intracellular organelles.     

The effect of chylomicron remnants on bile acid synthesis in cultured rat hepatocytes

The effect of chylomicron remnants on bile acid synthesis in isolated rat hepatocytes in monolayer cultures was investigated. Production of bile acids by the cells in the presence of chylomicron remnants at a cholesterol concentration of 7.8-9 nmol/ml was increased by approx. 75% after 17 h and 25% after 24 h incubation. Similar concentrations of cholesterol added to the cells in the form of chylomicrons had no significant effect on bile acid synthesis. These results suggest that cholesterol taken up in chylomicron remnants may be an important source of substrate for bile acid synthesis.     

Species-specific toxicity of diclofenac and troglitazone in primary human and rat hepatocytes

Troglitazone was withdrawn from the market shortly after approval for diabetes type II therapy because of strong hepatotoxic effects in man that could not be predicted from regulatory animal or in vitro studies. Another pharmaceutical that is regularly associated with adverse effects on the liver, sometimes leading to acute liver failure, is the widely used non-steroidal anti-inflammatory drug (NSAID) diclofenac. Since the underlying molecular mechanisms are not yet fully known, we treated primary rat and human hepatocyte monolayer cultures for 24 h with different doses of troglitazone and diclofenac to analyze species differences related to toxicity in vitro. Metformin an antidiabetic drug which does not cause severe adverse reactions served as negative control. Human hepatocytes showed a higher sensitivity to troglitazone than rat hepatocytes, while diclofenac-induced cytotoxicity at fairly similar concentrations. By co-treatment with specific inhibitors for cytochrome P450 (CYP) 2C and CYP3A - the major phase I enzymes involved in liver xenobiotic metabolism - we could confirm the prominent role of CYP3A in the bioactivation of troglitazone as well as the role of CYP3A and CYP2C in the activation of diclofenac. Inhibition of these enzymes increased the viability of treated cells in both species. Furthermore, we were able to demonstrate marked species differences in gene expression patterns of troglitazone treated rat and human hepatocytes. In contrast to rat hepatocytes, human cells showed distinct upregulation of various CYPs, regulators of xenobiotic metabolism and marker genes for oxidative stress. In contrast, gene expression alterations in rat and human hepatocytes treated with Diclofenac were rather similar. Altogether our study showed that species-specific effects as well as indications for the mode of action of compounds can be addressed by the use of primary hepatocyte cultures from various species in combination with gene expression profiling.     

Toxic injury from mercuric chloride in rat hepatocytes

The relationship between cytosolic free Ca2+, mitochondrial membrane potential, ATP depletion, pyridine nucleotide fluorescence, cell surface blebbing, and cell death was evaluated in rat hepatocytes exposed to HgCl2. In cell suspensions, 50 microM HgCl2 oxidized pyridine nucleotides between 1/2 and 2 min, caused ATP depletion between 2 and 5 min, and produced an 89% loss of cell viability after 20 min. Rates of cell killing were identical in high (1.2 mM) and low (2.6 microM) Ca2+ buffers. Cytosolic free Ca2+ was determined in 1-day cultured hepatocytes by ratio imaging of Fura-2 employing multiparameter digitized video microscopy. In high Ca2+ medium, HgCl2 caused a 3-4-fold increase of free Ca2+ beginning after 6-7 min, but free Ca2+ did not change in low Ca2+ medium. Bleb formation occurred after about 4-5 min in both buffers prior to any increase of free Ca2+. Subsequently, in high Ca2+ medium, blebs became hot spots of free Ca2+ (greater than 600 nM). After about 2 min of exposure to HgCl2, rhodamine 123 fluorescence redistributed from mitochondrial to cytosolic compartments signifying collapse of the mitochondrial membrane potential. The results taken together demonstrate that bleb formation, ATP depletion, and the onset of cell death are not dependent on an increase of cytosolic free Ca2+. HgCl2 toxicity appears to be a consequence of inhibition of oxidative phosphorylation leading to ATP depletion and cell death.     

Sialyltransferase activities in cultured rat hepatocytes

Previous studies on the age and sex dependency of the ganglioside patterns in rat liver in vivo and the concomitant determination of the activities of some enzymes involved in these pathways revealed the prominent role of the sialylation of GM3 to GD3 in determining the flow to the mono (a)- and polysialo (b)-series, respectively. Here, the influence of hormones on the activities of GM3 and GD3 synthases in isolated hepatocytes was studied. The combination of several factors (insulin, glucagon, epidermal growth factor, glucocorticoids) was found to be necessary for maintaining in vivo activity levels of GD3- but not of GM3-synthase.     

Effects of troglitazone and insulin on glucose production in cultured hepatocytes isolated from rats on high fat diet

Increased dietary fat intake in general, and saturated fat specifically, will lead to the impairment of insulin action. The aim of this study was to find out the changes in hepatic glucose output in dependence of fat diet and a possible direct action of insulin and trogitazone in hepatocytes. Hepatocytes were isolated by a collagenase perfusion technique and cultured for 24 h in M 199 serum-free medium. The glucose production in hepatocytes isolated from rats on high fat diet (unsaturated fat) was 79% higher compared to control and even 139% higher than in rats on high-fat diet (saturated fat). Troglitazone significantly decreased the glucose production in hepatocytes obtained from rats on unsaturated fat diet. The troglitazone in presence of insulin totally normalized glucose production but also only in hepatocytes obtained from rats on unsaturated-fat diet. The troglitazone showed an insulinomimetic as well as insulin-sensitizing effect but only in rats on unsaturated-fat diet.     

Secretion of alpha-tocopherol from cultured rat hepatocytes

Primary cultures of rat hepatocytes and rat liver perfusions were used to study hepatic secretion of alpha-tocopherol. The secretion of alpha-tocopherol from hepatocytes in culture was linear with time for 4 h. Ultracentrifugation of the medium revealed that 89.4 +/- 2.1% of alpha-tocopherol secreted during 4 h incubation was associated with the very-low density lipoprotein fraction (VLDL, d less than 1.006 g/ml). Oleic acid had no significant effect on the secretory rate of alpha-tocopherol, whereas eicosapentaenoic acid reduced the amount of alpha-tocopherol secreted to 48.4 +/- 12.7% of the control value after 20 h incubation (P less than 0.01). Monensin, a known inhibitor of VLDL secretion, reduced the secretion of alpha-tocopherol to 14.1 +/- 4.3% of the control value (P less than 0.02). Colchicine and chloroquine inhibited the secretion of alpha-tocopherol in the same order of magnitude as monensin. Hepatic perfusion after intravenous injection of in vivo labeled alpha-[3H]tocopherol lymph, showed that about 75% of the secreted radioactivity was in the VLDL fraction. From these results we conclude that most alpha-tocopherol is secreted from the liver associated with nascent VLDL in rats.     

Lipid synthesis in permeabilized cultured rat hepatocytes

Hepatic lipid synthesis was verified and studied in lysolecithin-permeabilized cultured rat hepatocytes and compared to that of intact liver cells. Triacylglycerol synthesis in permeabilized cells incubated in the presence of glycerol 3-phosphate and long chain fatty acids approached that of intact hepatocytes. Similarly, phosphatidylcholine synthesis in permeable cells incubated in the presence of exogenous CDP-choline was similar to that of intact hepatocytes and at the expense of microsomal neutral lipid synthesis. Phosphatidic acid accumulation in lysolecithin-permeabilized liver cells was remarkably increased as compared to that of intact cells, and its synthesis was mostly accounted for by the activity of mitochondrial glycerol-3-phosphate acyltransferase. Mitochondrial-generated phosphatidate was found to migrate to the endoplasmic reticulum, thus establishing a novel lipid esterification pathway which begins in mitochondrial glycerol 3-phosphate acylation and results in microsomal triacylglycerol and phospholipid synthesis. The free access of permeabilized liver cells to substrates and modulators of lipid synthesis, while maintaining an overall synthetic pattern similar to that of intact hepatocytes, makes them a system of choice for studying hepatic lipid synthesis in general and the microsomal/mitochondrial distribution of fluxes in particular.     

Oxygen uptake rates in cultured rat hepatocytes

One potential treatment of acute liver failure involves the use of an extracorporeal device composed of functional hepatocytes. A major issue in the design of such a large-scale device is providing the hepatocytes with a sufficient supply of oxygen and other nutrients. In this study, we have designed and characterized a simple perfusion system hepatocytes using this system. The OUR of hepatocytes was determined during the first day after seeding on a single collagen gel and during the long-term stable culture after the addition of a top layer of collagen. The OUR increased to 20.7 +/- 0.57 pmol/sec/mug DNA during the first 13 hours of culture on a single collagen gel, while during the next 11 hours, the OUR declined to 10.6 +/- 1.5 pmol/sec/mug DNA. In parallel with the increase in OUR during the first 10 hours, we observed significant cell spreading, suggesting that the oxygen supply to the cells may be critical for the spreading and adaptation of the anchorage-dependent hepatocytes following isolation. Addition of a top layer of collagen to hepatocyte cultures for 24 hours of culture on a single collagen layer resulted in a stable OUR for 15 days. These results indicate that OUR of hepatocytes in culture may vary depending on the phase of culture (i.e., early vs. late) and on the extracellular environment. (c) 1992 John Wiley & Sons, Inc.     

Effect of epidermal growth factor on cultured adult rat hepatocytes

When adult rat hepatocytes were cultured in plastic Petri dishes in a medium containing insulin and glucagon, supplementation with epidermal growth factor (EGF) had a pronounced effect on their viability, morphology, and biochemical integrity. Transmission and scanning electron microscopic studies showed that after 1 week cells denied EGF accumulated numerous non-electron-dense bodies and filamentous whorls, had irregular nuclei, and exhibited atypical cell surfaces. In contrast, cells grown for 2-3 weeks in the presence of EGF had well-preserved cellular organelles and remained as an epithelial-like monolayer. After 3 weeks EGF-exposed cultures were still inducible for liver-specific tyrosine aminotransferase, and both rat albumin and rat transferrin were recoverable from the culture medium. Virtually no viable cells were present at 3 weeks in EGF-deprived cultures.     

Influence of rat strain on P-glycoprotein expression in cultured hepatocytes

The amount and activity of the multi-drug transport P-glycoprotein (Pgp) have been measured in cultured hepatocytes derived from different rat strains. A marked increase in Pgp, as revealed by Western blotting, occurred 48 h after seeding in hepatocytes from Sprague-Dawley, Wistar and Fischer 344 rats, the last showing the highest value. The addition of dexamethasone (DEX) to culture medium delayed Pgp overexpression in all the strains, proportionally to the protein amount in the absence of hormone. The R-123 functional test for Pgp showed that Fischer 344 hepatocytes had the lowest ability to extrude the fluorescent dye as compared with Sprague-Dawley or Wistar rats. These results suggest that the Fischer 344 rat is more prone than other strains to culture stressing conditions, leading to an overexpression of Pgp that is not necessarily functional.Abbreviations DEX dexamethasone - FCS fetal calf serum - HRP horseradish peroxide - Pgp P-glycoprotein     

Absence of bicarbonate abolishes the glycogenic effect of cortisol in cultured fetal rat hepatocytes

The glycogenic action of cortisol in cultured fetal rat hepatocytes was completely abolished by the absence of NaHCO3 from the medium, while its presence stimulated the action in relation to its concentration. The absence of NaHCO3 slightly reduced glycogen storage by insulin but did not affect glucose-dependent glycogen deposition in the basal state. Also, the cortisol-induced increase in glycogen synthase a activity was reduced but that in total synthase activity was not affected. The absence of NaHCO3 did not reduce the cortisol-induced increase in tyrosine aminotransferase activity and the incorporation of [3H]dexamethasone into the nuclei. These results show that the absence of NaHCO3 specifically inhibits the glycogenic action of glucocorticoids in cultured fetal rat hepatocytes and indicate the need for further investigation into the role of HCO3- in universally used bicarbonate-buffered media.     

Modulation of functional activities in cultured rat hepatocytes

Rat hepatocytes isolated by enzymatic dissociation of the liver must attach in order to survive for more than a few hours. In conventional culture conditions, they rapidly lose their highly differentiated functions, e.g. adult isozymic forms, enzyme response to specific hormones and cytochrome P-450-dependent monooxygenase activities. Incompletely differentiated cells such as perinatal and regenerating hepatocytes, can transiently exhibit a more differentiated state. Therefore, regulation of hepatic functions, particularly enzyme activities cannot be studied for more than a few days. Hepatocyte survival rate and maintenance of specific functions are dependent on nutrient composition of the medium as well as the substrate. Complex matrices, particularly that derived from the connective liver biomatrix, appear to have an important favorable effect. However, regardless of culture conditions specific functions cannot be quantitatively maintained for more than several days. Recent observations strongly suggest that such a problem may be overcome by mimicking in vivo specific cell-cell interactions. Thus when co-cultured with a liver epithelial cell line, probably derived from biliary ductular cells, adult hepatocytes remain able to synthesize high levels of albumin and to conjugate drugs. In these conditions, the cells secrete an abundant heterogeneous extracellular material. The co-cultures can be maintained in a serum-free medium and specific liver functions can be altered experimentally. Such a model could be appropriate for studying long-term induction and modulation of liver enzyme activities under defined experimental conditions.     

Identification of glutathione conjugates of troglitazone in human hepatocytes

Troglitazone (TGZ) is an orally active antihyperglycemic agent used in the treatment of noninsulin-dependent diabetes mellitus. Several cases of liver failure following TGZ administration led to its withdrawal from the market. The mechanism of toxicity is still not understood. The formation of toxic metabolites is believed to play an important role. Herein, we report the biotransformation of TGZ in human hepatocytes. TGZ at 50 microM concentration was incubated with cryopreserved human hepatocytes. Four metabolites were found-glucuronide, sulfate, and two glutathione (GSH) conjugates of TGZ. The two GSH metabolites could be conjugation at the 6-hydroxychromane nucleus and the thiazolidinedione ring. Alternatively, the conjugation could be one of the two rings, with the two GSH metabolites are diastereomers. The sulfate conjugate was the major metabolite found. The cytochrome P450 (CYP) inhibitors furafylline (CYP1A1/2), omeprazole (CYP2C19), ketoconazole (CYP3A4), and sulfaphenazole (CYP2C9) had no inhibitory effect on the TGZ metabolism suggesting that several P450s may play a role in the TGZ metabolic pathway. Previous studies in our laboratory have shown a large interindividual variation between different donors in cytotoxicity after dosing with TGZ. Based on EC(50) values, donors were classified as sensitive or resistant. The sensitive human donors were found to form significantly less troglitazone GSH conjugates and glucuronides than the resistant donors.     

Effects of insulin on inositol phosphate production in cultured rat hepatocytes

Addition of vasopressin (100 nM) to rat hepatocytes prelabelled with [3H]inositol stimulated the production of inositol phosphates in the presence of 20 mM Li+. Preincubation of hepatocytes with insulin (50 nM) or glucagon (10 nM) had no significant effect alone but enhanced the effects of vasopressin after a lag period of at least 1 min. The effects of insulin and glucagon appeared additive in this respect. Insulin also enhanced the norepinephrine-mediated stimulation of inositol phosphate accumulation. The enhancement by insulin of the effects of vasopressin required at least 0.5-5 nM insulin and did not involve changes in [3H]inositol lipid labelling or IP3 phosphatase activity. The effect of insulin appeared insensitive to prior treatment of hepatocytes with pertussis toxin (200 ng/ml for 18-24 h) or cholera toxin (100 ng/ml for 3-4 h). The glucagon enhancement of the effects of vasopressin was not affected by pertussis toxin but was mimicked by cholera toxin. The response of hepatocytes to vasopressin in the absence of Li+ was smaller and more transient. Under these conditions a 5 min prior incubation with insulin inhibited the stimulation by vasopressin of inositol phosphate accumulation. A similar inhibitory effect of prior insulin exposure on the transient activation by vasopressin of exogenous phosphatidylinositol 4,5-bisphosphate breakdown by hepatocyte homogenates was also seen. These data indicate that insulin, although having no effect on basal inositol phosphate accumulation, can either enhance or antagonise the effects of vasopressin in primary rat liver hepatocyte cultures depending on the experimental conditions.     

Modulation of alkaline phosphodiesterase I in cultured rat hepatocytes

Alkaline phosphodiesterase I activity was measured in adult and foetal rat hepatocytes maintained in primary culture under various conditions. This enzyme was found to be expressed in both cell populations and could be resolved into two bands having apparent molecular weights of 130,000 and 250,000, respectively. Alkaline phosphodiesterase I activity was already at high levels in 15 day foetal liver and, as early as the 19th day of gestation, it reached adult levels. Alkaline phosphodiesterase I levels were well maintained during culture. In the absence of serum, its level continued to increase with time in foetal cells. It dramatically increased by days 4 and 5, in adult cells maintained on fibronectin and plastic, respectively. Dexamethasone stimulated alkaline phosphodiesterase I activity after a lag phase of 8 h, with a maximum reached after 40 h. As this induction was prevented by addition of actinomycin D or cycloheximide, it could be concluded that it required RNA and protein synthesis. Only the major Mr 250,000 form responded to dexamethasone and was sensitive to serum.     

Excretion of glutathione conjugates by primary cultured rat hepatocytes

Conjugation of xenobiotics with glutathione occurs commonly within the liver, and these glutathione conjugates are then preferentially excreted into bile. We have characterized this excretory process using primary cultured hepatocytes (24 h). 1-Chloro-2,4-dinitrobenzene rapidly entered the cells and formed a glutathione conjugate, S-(dinitrophenyl)glutathione, irrespective of the temperature of incubation. In contrast, the efflux of the glutathione conjugate was essentially absent in the cold but recovered rapidly upon rewarming of the cells. Therefore, initial rates of efflux of the conjugate at 37 degrees C were measured from cells preloaded biosynthetically at 10 degrees C. Efflux was a saturable process with respect to intracellular S-(dinitrophenyl)glutathione with an apparent Km of 0.58 +/- 0.12 mM and Vmax of 0.15 +/- 0.05 nmol/min/mg of protein. The excretion of S-(dinitrophenyl)glutathione had an energy of activation of 15.3 kcal/mol. The glutathione conjugate of p-nitrobenzylchloride when formed within the hepatocytes acted as a competitive inhibitor of S-(dinitrophenyl)glutathione efflux. Cultured hepatocytes, therefore, appeared to have a specific transport process for the excretion of glutathione conjugates. The addition of S-(dinitrophenyl)glutathione, but not GSH, GSSG, or methionine, to the medium caused a decrease in the rate of efflux of radiolabeled S-(dinitrophenyl)glutathione. The hepatocytes were able, however, to excrete the glutathione conjugate against an excess of extracellular S-(dinitrophenyl)glutathione. This observation suggested that extracellular S-(dinitrophenyl)glutathione, although capable of binding to the carrier, entered the hepatocytes quite slowly relative to rates of efflux. This carrier may function in a manner that would minimize the reuptake by hepatocytes of conjugates that have been excreted into the bile.     

Adenylate energy charge of rat and human cultured hepatocytes

Summary A simple and rapid method for the assay of adenine nucleotides (ATP, ADP, and AMP) was established to evaluate the adenylate energy charge (ATP+ADP/2)/(ATP+ADP+AMP) of cultured hepatocytes. The effects of inhibitors of glycolysis, fatty acid oxidation, or oxidative phosphorylation on the energy charge were examined. The energy charges of cultured hepatocytes in rats and human were almost identical and were maintained at a high level between 6 and 24 h after changing the media (rat: 0.908±0.008n=9, human: 0.918±0.014n=6, mean ± SD). Inhibition of glycolysis with sodium fluoride or oxidative phosphorylation with antimycin A irreversibly reduced both the adenine nucleotide contents and the energy charge. However, the inhibition of fatty acid oxidation with 2-tetradecylglycidic acid did not affect the nucleotide contents, and the energy charge only decreased transiently to recover within 8 h. When the inhibitor of oxidative phosphorylation was removed, the recovery in the energy charge preceded the recovery in the adenine nucleotide contents. These findings suggest that the adenylate energy charge is a more sensitive measure of the changes in energy metabolism than the adenine nucleotide contents. Furthermore, energy charge regulates adenine nucleotide contents in cultured hepatocytes. It is important to confirm that the high energy charge of the cultured hepatocytes is maintained when these cells are used for metabolic studies.