Comparative study of the hepatoprotective effect of silymarin and silybin on isolated rat hepatocytes.

The flavonoid silymarin and its main active component silybin have been used in the treatment of toxic liver diseases. In order to evaluate the hepatoprotective potency of both these compounds, their effects on the viability, lipid peroxidation and reduced glutathione (GSH) depletion induced by allyl alcohol (AA) and tert-butyl hydroperoxide (t-BuOOH) in suspensions of isolated hepatocytes were investigated. Cells were preincubated for 30 min with silymarin and silybin before the addition of AA or t-BuOOH.

The effects of silymarin on the attachment and viability of primary cultures of rat hepatocytes.

The effects of the hepatoprotective compound silymarin on hepatocytes in primary culture were studied. Exposure of cells in primary culture (both conventional cells and perivenous or periportal cells isolated by the digitonin-collagenase perfusion technique) to high concentrations of silymarin surprisingly demonstrated that silymarin per se was cytotoxic. Incubation of cells for 18 hr with silymarin at concentrations exceeding 25 mum abruptly increased cell damage, whereas viability decreased in a more linear fashion with increasing concentrations of its major constituent, silybin.

Hepatoprotective mechanism of silymarin: no evidence for involvement of cytochrome P450 2E1.

The involvement of the alcohol-inducible cytochrome P450 2E1 in the hepatoprotective mechanism of the plant flavonoid extract silymarin, and its main active component silybin, was investigated in isolated hepatocytes. Allyl alcohol toxicity, associated lipid peroxidation and GSH depletion was efficiently counteracted by silymarin (0.01-0.

Zonation of acetaminophen metabolism and cytochrome P450 2E1-mediated toxicity studied in isolated periportal and perivenous hepatocytes.

To study the mechanism of centrilobular damage developing in the centrilobular region after high doses of acetaminophen (APAP), its metabolism and toxicity were compared in periportal and perivenous hepatocytes isolated by digitonin/collagenase perfusion. Contrary to earlier reports, based on perfusions, no evidence for a periportal dominance of APAP sulfation could be observed. Glucuronidation, the dominant pathway of conjugation at high (5 mM) APAP concentration, was faster in perivenous cells.

Evidence for cytochrome P450 2E1-mediated toxicity of N-nitrosodimethylamine in cultured perivenous hepatocytes from ethanol treated rats.

The involvement of cytochrome P450 in the liver toxicity of the potent carcinogen, N-nitrosodimethylamine (NDMA) was investigated in hepatocytes isolated from the periportal or perivenous region by digitonin-collagenase perfusion. Exposure of hepatocytes in culture to NDMA (0.5 or 5 mM) for up to 18 hrs caused little damage, but after 42 hr loss of cell viability became evident, and the extent of cell death was higher in perivenous cells than in periportal cells.

Futile cycling of a sulfate conjugate by isolated hepatocytes.

The sulfate conjugate of the model compound 4-methylumbelliferone was taken up and hydrolyzed considerably more rapidly by isolated hepatocytes than was the glucuronide conjugate. Using intact hepatocytes or homogenates of hepatocytes, compounds were identified that either inhibited 4-methylumbelliferyl sulfate hydrolysis via arylsulfatase or impaired its uptake into cells. For example, sodium sulfate inhibited hydrolysis of 4-methylumbelliferyl sulfate by intact hepatocytes (half-maximal inhibition, 0.

Differences in glycolytic capacity and hypoxia tolerance between hepatoma cells and hepatocytes.

Viability, glycolytic capacity and energy metabolism under anaerobic conditions were studied in the hepatoma cell lines HTC, FU5 and HepG2 and in rat and human hepatocytes using glucose and fructose as glycolytic precursors. During 6 hr of anaerobic incubation without additional substrate, viability decreased rapidly in FU5 and HTC cells, whereas viability of HepG2 cells was not significantly affected. In all tumor cells, 10 mmol/L glucose prevented hypoxic cell injury almost completely.

Hypoxic liver cell death: critical Po2 and dependence of viability on glycolysis.

Viability of isolated hepatocytes was not significantly dependent on oxygen at oxygen partial pressures (Po2) from 70 to 0.3 mmHg. The critical Po2 for induction of hypoxic cell death was close to 0.

Adenine nucleotides and carbohydrates in subpopulations of hepatic nodules with normal and compromised microcirculation.

Fluorescein-isothiocyanate dextran (FITC-dextran), a dye confined to the vascular space, was infused via the hepatic artery and portal vein into perfused livers from fed rats treated with diethylnitrosamine for 4 to 5 months. Fluorescence due to FITC-dextran was detected with fiberoptic microlight guides placed on surface nodules of about 5 mm in diameter. Nodules were categorized into groups with normal and compromised microcirculation based on their fluorescence following infusion of FITC-dextran.

Hypoxia-reoxygenation injury and the generation of reactive oxygen in isolated hepatocytes.

Reoxygenation following hypoxia enhanced loss of viability of isolated hepatocytes compared to cells maintained under hypoxic conditions. Cell damage due to reoxygenation was not dependent on the conversion of xanthine dehydrogenase to xanthine oxidase which occurred at a time when almost all the hepatocytes had lost their viability. The effect of reoxygenation was critically linked to the duration of the hypoxic period.

Fructose prevents hypoxic cell death in liver.

Perfusion of livers from fasted rats with nitrogen-saturated buffer caused hepatocellular damage within 30 min. Lactate dehydrogenase (LDH) was released at maximal rates of approximately 300 U . g-1 .

Gluconeogenesis from fructose predominates in periportal regions of the liver lobule.

Gluconeogenesis from fructose was studied in periportal and pericentral regions of the liver lobule in perfused livers from fasted, phenobarbital-treated rats. When fructose was infused in increasing concentrations from 0.25 to 4 mM, corresponding stepwise increases in glucose formation by the perfused liver were observed as expected.

Hydrolysis of 4-methylumbelliferyl sulfate in periportal and pericentral areas of the liver lobule.

4-Methylumbelliferyl sulfate was used to characterize sulfatase activity in periportal and pericentral regions of the liver lobule in the perfused rat liver. Following infusion of 1.5 mM of this organic sulfatester, free 4-methylumbelliferone and 4-methylumbelliferyl glucuronide were formed at rates of 13 and 9 mumoles/g/h, respectively, in livers from fasted, phenobarbital-treated rats.

Total protection from hypoxic liver damage by fructose.

The purpose of these experiments was to evaluate the effect of carbohydrates on hypoxic cell death in the perfused rat liver. Lactate dehydrogenase (LDH) was released maximally at rates up to 300 U/g/h after 40 to 60 min of hypoxia in control livers from fasted rats or in livers perfused with glucose (20 mM). Fructose (20 mM) prevented the release of LDH completely.

Hydrolysis of organic sulfates in periportal and pericentral regions of the liver lobule: studies with 4-methylumbelliferyl sulfate in the perfused rat liver.

The hydrolysis of 4-methylumbelliferyl sulfate by liver sulfatases to free fluorescent 4-methylumbelliferone and the subsequent formation of the glucuronide conjugate were studied in the isolated perfused rat liver. In livers from fed, phenobarbital-treated rats, 4-methylumbelliferyl sulfate (0.25-1.

Effects of selenite on O2 consumption, glutathione oxidation and NADPH levels in isolated hepatocytes and the role of redox changes in selenite toxicity.

Isolated hepatocytes incubated with selenite (30-100 microM) exhibited changes in the glutathione redox system as shown by an increase in O2 consumption, oxidation of glutathione and loss of NADPH. Selenite (50 microM) raised O2 consumption within the 1 h and induced an partial depletion of thiols with a concomitant increase in oxidized glutathione, as well as a decrease in NADPH levels within 2 h. With 100 microM selenite more pronounced effects were obtained such as a total depletion of thiols.

Selenite biotransformation to volatile metabolites in an isolated hepatocyte model system.

The biotransformation of selenite to dimethylselenide was studied in an oxygenated hepatocyte model system. The concentrations of selenite used were 20-100 microM. A lag period of one hour or more, during which no net formation of selenide could be detected characterized the system.

Absorption of selenite in the rat small intestine: interactions with glutathione.

Incubation of isolated rat intestinal epithelial cells with selenite resulted in a rapid decrease of intracellular glutathione (GSH) and about 5 nmol of Se (added as 75Se labelled selenite, 50 microM) was accumulated per 10(6) cells during 20 min. Addition of exogenous GSH enhanced cellular accumulation of Se, while the gamma-glutamyl transferase inhibitor, serine-borate, decreased uptake. The added exogenous GSH was rapidly consumed (oxidized) by selenite.

The effectiveness of N-acetylcysteine in isolated hepatocytes, against the toxicity of paracetamol, acrolein, and paraquat.

The protective effect of N-acetylcysteine against the toxicity of paracetamol, acrolein, and paraquat was investigated using isolated hepatocytes as the experimental system. N-acetylcysteine protects against paracetamol toxicity by acting as a precursor for intracellular glutathione. N-acetylcysteine protects against acrolein toxicity by providing a source of sulfhydryl groups, and is effective without prior conversion.

Toxic effects of free and DNA-linked daunorubicin on isolated rat cardiac myocytes.

Isolated cardiac myocytes from adult rats were used as a model to study the cardiotoxicity of free and DNA-linked daunorubicin. The toxic effects on the myocytes were evaluated by studying morphological changes, trypan blue exclusion and cell membrane permeability to NADH, as determined by LDH-activity. At a concentration of 100 microM daunorubicin caused an increased plasma membrane permeability within 30 min.

Involvement of glutathione reductase in selenite metabolism and toxicity, studied in isolated rat hepatocytes.

Cellular lysis in freshly isolated hepatocytes incubated with varying concentrations of selenite could be related to the reductive metabolism of selenite. A decrease in intracellular GSH levels was observed concomitant with an increased rate of accumulation of oxidized glutathione in the incubation medium. Pretreatment of hepatocytes with an inhibitor of GSSG-reductase (1,3-bis(2-chloroethyl)-1-nitrosourea), prior to the addition of 50 microM selenite, resulted in substantially lower GSH-levels.

Metabolism of toxic substances in isolated hepatocytes.

Chloroform and selenite toxicity have been studied in isolated hepatocytes. Two different toxic mechanisms, which lead to cellular lysis after distinct lag periods, are compared. Chloroform toxicity can be divided into 2 phases, a first phase characterized by chloroform metabolism and a second phase characterized by lipid peroxidation.

Glutathione depletion in isolated hepatocytes: its relation to lipid peroxidation and cell damage.

The effects of glutathione depletion in isolated hepatocytes have been studied. A list of compounds which depleted glutathione and induced lipid peroxidation and cell lysis is given. The effects of halogenated acetamides were studied in more detail and results of studies on the interaction of iodoacetamide with cellular constituents are presented.