G. Krack

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.

Subcellular fractionation of isolated rat hepatocytes. A comparison with liver homogenate.

An improved method for the homogenization and the subsequent subcellular fractionation of hepatocytes isolated from adult rat liver is described. The homogenization procedure developed in the present study allows the preservation of the integrity of subcellular structures, as demonstrated by measurement of the activities of representative enzymes as well as by determination of their latency. The activities of representative marker enzymes, as calculated on subcellular fractions obtained by differential centrifugation of the homogenate, are identical whether the homogenate arises from isolated hepatocytes or from the whole liver. Moreover, there is a close similitude between the kinetic parameters (Km and V) of two microsomal cytochrome P450-dependent mixed-function oxidases, namely aniline hydroxylase and aminopyrine demethylase determined on microsomal preparations obtained either from isolated cells or from the whole liver.

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.