Effects of Inducers of Drug Metabolism on Cytosolic Glutathione S-transferase Activity in Rat Hepatoma-derived Fa32 Cells
Paul J. Dierickx
Established Fa32 cells, derived from a rat hepatoma, were investigated for their glutathione S-transferase (GST) induction capacity, which is an important characteristic of the detoxification capacity in normal liver. The cells were exposed to inducers of drug metabolism for 3 days in complete medium (containing 10% fetal calf serum). Neither dimethyl sulphoxide nor dimethyl formamide could be used as a vehicle to transport the inducers into the cells, because they also interacted with GST. Phenobarbital, butylated hydroxyanisole, allyl isothiocyanate and dimethyl fumarate (but not fumaric acid) all effectively increased the total specific GST activity. None of the test chemicals produced a very pronounced induction of specific GST subunits, but subunit 2 and subunit 8 were increased more than the others. The effects of inducers of drug metabolism on the GST activity in Fa32 cells are comparable with those in rat liver. These cells can therefore be used as a valuable alternative model for GST-dependent metabolic interactions in rat liver.
Cytotoxicity of the MEIC Reference Chemicals in Antioxidant-enriched Rat Hepatoma-derived Fa32 cells
Paul J. Dierickx, Claudia Smit and Ellen M. Scheers
Since vitamin E increases the antioxidant status of cells, its influence on cytotoxicity was investigated. The neutral red uptake (NRU) inhibition effects of 39 MEIC reference chemicals were measured after treatment of rat hepatoma-derived Fa32 cells in the presence of vitamin E for 30 minutes. The results were quantified in terms of the NI50, the concentration of test compound required to reduce the NRU by 50%. Sodium chloride was the only chemical that was more toxic in the presence of vitamin E. This effect was related to the concentration of vitamin E in the cell culture medium. A vitamin E dose-related response was also observed for the decreased toxicity of paracetamol and caffeine. Glutathione levels were slightly increased in the presence of vitamin E, which could contribute to the protective effect of vitamin E. Of the remaining chemicals, 50% were less toxic in the presence of vitamin E, but the correlation with the acute human toxicity data of the MEIC study was not improved. The results imply that reactive oxygen species interfere with the toxicity of a high proportion of toxic chemicals. The assay described provides a quick and easy method for checking whether reactive oxygen species contribute to the toxicity of a chemical.
Cytotoxicity of the Dicarboximide Fungicides, Vinclozolin and Iprodione, in Rat Hepatoma-derived Fa32 cells
Paul J. Dierickx
Dicarboximide fungicides are widely used to control various fungal species. Their primary action is not known, due to a lack of knowledge concerning the mechanism of action of the dicarboximide group. The cytotoxicities of vinclozolin and iprodione in rat hepatoma-derived Fa32 cells were investigated. Cytotoxicity was measured by neutral red uptake inhibition after treatment for 24 hours. Iprodione was more toxic than vinclozolin. Vinclozolin was less toxic in glutathione-depleted cells than in control cells. This was also true for iprodione at lower concentrations, but iprodione became more toxic at higher concentrations. Both the fungicides increased the endogenous glutathione content by 20% after 1 hour. After 24 hours, the glutathione content was doubled by vinclozolin, but was not affected by iprodione. No effect on glutathione S-transferase activity or reactive oxygen species formation could be observed. Cytochrome P450-dependent ethoxyresorufin-O-deethylase and pentoxyresorufin-O-depentylase activities were moderately activated by iprodione and strongly activated by vinclozolin. A glutathione-related cytochrome P450-dependent metabolic attack of vinclozolin and iprodione could be responsible for their cytotoxicity in Fa32 cells. Further research is needed to fully elucidate these (or other) mechanisms.