The use of VPA in the treatment of epilepsy has, on rare occasions, been associated with irreversible hepatic failure. Several possible biochemical mechanisms responsible for the hepatotoxicity have been proposed, but the actual causes have not been conclusively determined. It is believed that the hepatotoxicity resultes from an interaction of different factors. The aim of this study was to examine the impact of additional stress factors on an in vitro-model of freshly isolated hepatocytes of rats. The present experiments studied the effects of VPA in combination with starvation and with oxidative stress on hepatotoxicity and the metabolism. Prooxidative agents such as the hydroperoxides CuOOH and t-BuOOH and the GSH-depletor BrH were used. It was especially important to investigate the role of GSH, lipid peroxidation, alterations of VPA metabolism and the ultrastructure of the liver cells. Hepatocytes were isolated from adult male Wistar rats. The rats received either tap water and food ad libitum or the food was withdrawn 20 hours before the beginning of the experiment. In the first series of experiments, the influence of starvation as well as VPA and prooxidative agents alone on viability (Trypan blue test, LDH release, intracellular potassium and sodium concentration) and antioxidant capacity (intracellulare GSH concentration, MDA formation) was studied in liver cells. With combinations of VPA and hydroperoxides, the influence of hydroperoxides, VPA and nutritional status on these parameters was examined. In a second series of experiments, the combinations of BrH and VPA were used to investigate the influence of the GSH depletor BrH, VPA and the nutritional status on these parameters. Several concentrations of VPA and the prooxidative compounds were applied to test for dose dependency in both series. For selected groups, VPA and its metabolites were determined with GC/MS and the ultrastructure of the hepatocytes was studied with the electron microscope. Starvation resulted in a reduction of body and liver weight. There was no influence on viability. The initial GSH content was decreased by 40-50 %. GSH synthesis or regeneration occurred. MDA formation was not increased. Independent of the nutritional status, there were no alterations of the viability of liver cells caused by prooxidative agents alone. Only with starvation and the highest dose of VPA the treatment with VPA alone resulted in a decline of viability measured by the sensitive parameters potassium and sodium concentration and by the Trypan blue test. Increased MDA formation was never observed. With and without starvation, the hydroperoxides caused no changes in the GSH content of the liver cells. The pretreatment with BrH resulted in a GSH depletion of the liver cells by 60-80 % throughout the incubation period. Only with starvation were the losses of GSH maintained on this level. There was a GSH synthesis or regeneration. VPA led to a dose- and time-dependent reduction of GSH with and without starvation. Higher VPA doses lowered the capability of GSH synthesis or regeneration. During the whole incubation period starvation resulted in significantly diminished GSH values. Nether prooxidative agents nor VPA alone caused an major alteration of the hepatocellular ultrastructure. Only a minor bleb formation could be found. With the highest VPA concentration, lipid droplets were occasionally observed. The structure of the mitochondria was unaltered. Independent of the VPA dose, the same metabolite pattern was found. Generally, the percentage of ?-metabolites was higher than the percentage of ?-metabolites. With a high VPA dose the percentage of all metabolites was lower than with a low VPA dose. Starvation did not change the metabolite pattern in principle. But with 1 mM VPA the formation of metabolites was slightly inhibited and only with 10 mM VPA was there a significant inhibition of the formation of ?- and ?1-metabolites and of the ?-metabolite 2-en-VPA. The combination of VPA with hydroperoxides or BrH only in conjunction with starvation caused a strong dose- and time-dependent lipid peroxidation with a delayed or simultaneous dose- and time-dependent deterioration of the viability of the liver cells. The GSH content was decreased through VPA and hydroperoxides in a dose- and time-dependent manner independent of the nutritional status. The influence of VPA was more important. Naturally, with the combination of BrH and VPA, the effect of BrH on GSH was stronger. Starvation led to significantly decreased GSH values in all combinations. The antioxidative capability of the liver cells was sufficient in all non- starvation tested combination groups. The damages in the liver cells were not serious and could be repared by protective cellular systems like GSH or antioxidative agents. The results of the group V10T400 provide a first indication of the important role of GSH and lipid peroxidation in the mechanism of cytotoxicity of VPA. The only slightly increased lipid peroxidation and the deterioration of viability in the groups with starvation and low concentrations of the combined agents could be the result of the compensation for the threatening cellular damage through the above mentioned protective systems. It is assumed that the strong lipid peroxidation in groups with starvation and high concentrations of the various substances used in the experiments is the reason for cellular damage. It is impossible to decide whether VPA, hydroperoxides or the combined action of these factors was the cause of lipid peroxidation in experiments with combinations of VPA, hydroperoxides and starvation. It must be supposed that all factors are involved. However, the starvation induced decrease of the GSH level and the additional dose- and time-dependent reduction of the GSH content by VPA and hydroperoxides play the key role in the mechanism of cytotoxicity. It is worth stating that the results of the experiments with the GSH depletor BrH indicate that VPA may induce lipid peroxidation if an unfortunate set of circumstances like starvation, GSH depletion and a high VPA dose occurs. Over and above that, these experiments underline the importance of starvation. In all groups with strong lipid peroxidation, the determined GSH values at the end of the incubation period were between 15-30 % of the initial values of groups without starvation. This implies a critical decrease of the GSH level and a resulting impairment of the cell´s defense against toxic actions leading to cell injury and death. The mitochondrial GSH pool is assumed to play a critical role. The electron microscopic studies showed that the combination of the highest concentrations of VPA and CuOOH without starvation only caused an unimportant bleb formation in a reversible stage. In a few cases there were less microvilli. The mitochondria remained unchanged. On the other hand, with combined treatments and starvation and heavily increased MDA formation, a loss of microvilli and more frequent appearance of large blebs occurred. Severely damaged mitochondria characterized by swelling, matrix alterations, cristolysis and loss of cristae were observed. Lipid vacuoles and dilated endoplasmatic reticulum were also found. In all the combined groups with and without starvation there were no fundamental alterations of metabolite patterns. In the experiments with varying amounts of VPA with or without the combination of prooxidative agents, only the highest VPA dose in conjunction with starvation caused an inhibition of the formation of VPA metabolites. In contrast to this, with and without starvation, the percentage of all metabolites were reduced. In treatments with 1 mM VPA, prooxidative agents and starvation caused, dependent on the kind of substances, a significant reduction of metabolism of VPA. Especially in the groups V1T400 and B500V1 with starvation and increased MDA formation the ?/?1-metabolism and the ?-metabolism in certain stages were inhibited. In these combinations, a stronger lipid peroxidation was accompanied by an inhibition of ?-oxidation at an earlier stage. In addition, the hepatotoxic metabolite 4-en-VPA was detected. There were no clear differences in VPA metabolism between 10 mM VPA combination groups and 10 mM VPA treatment alone, although strong lipid peroxidation was determined only in combined groups. As well as in the group V10, the ?-oxidations and the ?-oxidation to 2-en-VPA were inhibited with starvation. With 10 mM VPA and starvation the hepatotoxic metabolite 4-en-VPA and the subsequent products could not be detected. Various hypotheses claiming the involvement of an altered metabolism in the hepatotoxicity of VPA were discussed in connection with the chosen conditions and the results of the investigation of the parameters and morphology. It is believed that free radical metabolites or free radicals generated during VPA metabolism contribute to this process, because the lack of GSH prevents their sufficient detoxification. In conclusion it can be stated that only the simultaneous interaction of a combination of detrimental factors leads to VPA mediated damage of liver cells. This dissertation supports the hypothesis that hepatotoxicity is caused by a failure of the free radical scavenger system. A simple statement as to whether changes in VPA metabolism are the cause or result of cytotoxicity is impossible. These alterations of VPA metabolism seem to play a role but have only an effect after a diminution of the antioxidant capacity through negative circumstances. From the results of this in vitro model, it follows that the combination of starvation, oxidative stress and high VPA doses is necessary for fatal liver injury to occur.
