F. Goethals

Dietary oligofructose lowers triglycerides, phospholipids and cholesterol in serum and very low density lipoproteins of rats

The present study was aimed at answering the question why feeding rats an oligofructose (OFS) supplemented diet could cause a significant reduction in plasma lipid levels. Daily administration of a 10% (w/w) OFS-containing diet to normolipidemic male rats resulted in a decrease in plasma triglycerides, phospholipids and cholesterol. The triglyceride-lowering effect was observed after one week and lasted for at least 16 wk and was associated with a reduction in plasma very low density lipoproteins, indicating that the hypolipidemic effect of OFS may be due to changes in liver lipid metabolism. We therefore tested whether OFS feeding modified the capacity of the liver to synthesize triglycerides from free fatty acids. Hepatocytes isolated from livers of control and OFS-fed rats were incubated in the presence of [1-14C]palmitate, and both intracellular and extracellular [14C]triglyceride formation were quantified. We found that chronic feeding of an OFS-supplemented diet to rats significantly reduced the capacity of isolated hepatocytes to synthesize triglycerides from palmitate. The results suggest that, like other soluble dietary fibers, OFS significantly alters liver lipid metabolism, resulting over time in a significant reduction in plasma triglyceride, phospholipid and cholesterol levels.

Interference of chemicals with glycogen metabolism in isolated hepatocytes.

Freshly isolated hepatocytes in suspension were used to evaluate the possible effects of certain chemicals. Conditions including the choice of the incubation medium have been defined for maintaining the cells competent for a sufficient length of time. Using paracetamol alone or in combination with diethylmaleate, we have been able to show that these chemicals markedly alter the metabolic state of the cells, as indicated by an inhibition of glycogen synthesis and even by an enhancement of glycogen degradation, without modifying membrane integrity. These effects are dose-dependent and probably mediated through modification of glycogen phosphorylase activity.

Critical biochemical functions of isolated hepatocytes as sensitive indicators of chemical toxicity

Isolated hepatocytes from adult male Wistar rats are a suitable experimental model to study the cytotoxicity of chemicals. Indeed, the isolated cells incubated in suspension in a Waymouth medium supplemented with 10% newborn calf serum maintain critical biochemical functions such as cytochrome P-450-dependent monooxygenase activity, glycogen, and protein synthesis capacities. This cellular model is used to detect the early biochemical effects of various xenobiotics, i.e., chlorpromazine, promethazine, bromobenzene, paracetamol, and isoniazid. Both cellular lysis (measured by the LDH leakage) and metabolic competence of the hepatocytes (glycogen deposits and protein synthesis) are modified as a function of both the duration of exposure to, and the concentration of, the chemicals. These results point out that the evaluation of metabolic functions of isolated cells surviving in suspension might be a sensitive test to predict early cell injury. Indeed, changes in the cellular behavior may occur before or without cell death. Furthermore, since both the cytochrome P-450 content and its dependent monooxygenase activity together with critical biochemical functions of the isolated cells remain stable, this model is of significant interest in ascertaining the mechanisms of toxicity.

Investigation into the mechanism of tetracycline-induced steatosis: study in isolated hepatocytes.

Tetracycline is known to cause hepatic dysfunction in humans by inducing steatosis. Accumulation of fat in the liver could result from biochemical effects at various levels in the sequence from protein and triglyceride synthesis to lipoprotein secretion. The effects of tetracycline on the synthesis and secretion of triglycerides and proteins were studied in isolated rat hepatocytes surviving in suspension for up to 2.5 hr. Interpretation of the results obtained for tetracycline was made by comparison with results obtained, under the same experimental conditions, for the well-known steatogenic compounds, cycloheximide and colchicine. The data indicate that tetracycline produces a concentration-dependent inhibition of 14C-triglyceride secretion without affecting triglyceride synthesis. This inhibition explains the intracellular triglyceride accumulation. However, tetracycline does not affect protein secretion. Furthermore, it was demonstrated that the effect of tetracycline on protein synthesis was not related to inhibition of triglyceride release. In conclusion, it is proposed that the effect of tetracycline could be at the level of the association between triglycerides and apoproteins to form lipoproteins.

Toxicity of the antitumoral drug datelliptium in hepatic cells: Use of models in vitro for the prediction of toxicity in vivo

Datelliptium is a DNA-intercalating agent derived from ellipticine. The drug has potent antitumoral activity in vitro and in vivo. The first clinical use of the drug revealed unexpected hepatotoxic effects in humans that had not been observed in animals. Using different hepatic models in vitro (rat hepatocytes in suspension and in culture, cultured human hepatocytes and rat and human hepatoma cell lines), the possibility of prediction in vitro of the hepatic toxicity of a drug has been investigated. Cytotoxic effects were evaluated by measuring the leakage of intracellular lactate dehydrogenase and the ability of cells to reduce MTT after exposure to concentrations of datelliptium ranging from 0.1 to 1000 mum. According to these endpoint parameters, the concentrations of the drug that produced 50% of maximal inhibitory effect (IC(50)) were in the range 100 to 195 mum in rat hepatocyte suspension and hepatocyte cultures after 2 hr of treatment, 7-9 mum in cultured rat and human hepatocytes after 23 hr of treatment, and about 200-320 mum in HepG2 and FaO cells after 23 hr of treatment. Metabolic parameters were generally more sensitive than cytotoxic endpoints for detecting toxic effects of datelliptium on hepatocytes in the two experimental models used. Metabolic effects on rat hepatocyte suspension and culture were evaluated respectively after 2 and 23 hr of exposure to the drug. Triglyceride secretion was the most sensitive parameter and the IC(10) values (concentration causing 10% of maximal inhibitory effect) were 0.03 and 0.9 mum in hepatocyte suspension and culture, respectively. Glycogen, albumin and cellular protein synthesis were similarly altered in both cellular systems and the IC(10) values were in the range 0.5-3.5 mum. Ureogenesis and gluconeogenesis were relatively insensitive parameters in cell suspensions (IC(10) values 16.4 and 10.3 mum, respectively) compared with those in hepatocyte culture (IC(10) values 3.6 and 3.1 mum, respectively). The concentrations of datelliptium reported in blood, and particularly in liver, are higher than the concentrations that produce impairment of cell metabolism in vitro. This may be an indicator of the toxicological risk of datelliptium and anticipates the hepatotoxicity observed in vivo.

Synergism between aflatoxin B1 and oxytetracycline on fatty acid esterification in isolated rat hepatocytes.

The individual and combined effects of aflatoxin B1 (AFB1) and oxytetracycline (OXT) on the synthesis and secretion of triacylglycerols in isolated rat hepatocytes maintained in suspension during 2.5 h were studied. Secretion of triacylglycerols was inhibited by both drugs when administered separately. This inhibition was accompanied by a concomitant elevation of intracellular triacylglycerols only at the highest AFB1 dose tested. Total synthesis of triacylglycerols was not inhibited by AFB1 or by OXT. When the two drugs were simultaneously added to the incubation medium, the AFB1-induced accumulation of intracellular triacylglycerols was no longer observed; the inhibition of secretion was nevertheless identical to that observed with AFB1 alone. Finally, total esterification of palmitate was inhibited by 20% compared to the AFB1-treated cells. These data suggest that OXT inhibits lipid accumulation induced by AFB1 but that this effect is due to an inhibition of total synthesis of triacylglycerols. The mechanism of AFB1s effect and of the interaction between both molecules is discussed.

Biochemical Effects of Methotrexate in Isolated Hepatocytes in Relation To its Steatogenic Activity

Methotrexate (MTX) is known to induce steatosis in humans. The effect of MTX on the synthesis and secretion of triglycerides and proteins as well as on RNA synthesis were evaluated using isolated rat hepatocytes in suspension as an experimental model. MTX significantly inhibited protein synthesis (48% inhibition after 180 min) and RNA synthesis (72% inhibition after 180 min) at 10(-3)m but not at a concentration of 10(-4)m. Protein secretion was not affected by MTX at any of the concentrations tested. At 10(-3)m, but not at 10(-4)m, MTX inhibited triglyceride secretion (44% inhibition after 180 min). At both 10(-3) and 10(-4)m, MTX significantly depleted the intracellular triglyceride, and significantly inhibited total triglyceride esterification (18 and 28% inhibition at 10(-4) and 10(-3)m-MTX, respectively). These data support the hypothesis that MTX affects protein and fatty-acid metabolism.

Alternatives in Testing for Hepatotoxicity

Toxicology studies the properties of chemicals and their effects on living organisms with the aim to assess their potential adverse effects so as to help preserve and protect human health.