The effect of alpha-tocopheryl succinate on succinate respiration in rat liver mitochondria.

We compared the effect of alpha-tocopheryl succinate (TOS) on succinate-dependent respiration in rat liver mitochondria, homogenate and permeabilized hepatocytes in both a coupled and uncoupled state. In isolated mitochondria, a significant inhibitory effect was observed at a concentration of 5 microM, in liver homogenate at 25 microM and in permeabilized hepatocytes at 50 microM. The inhibitory effect of TOS on succinate respiration in an uncoupled state was less pronounced than in a coupled state in all the experimental models tested. When the concentration dependence of the TOS inhibitory effect was tested, the most sensitive in both states were isolated mitochondria; the most resistant were permeabilized hepatocytes.

The effect of D-galactosamine on lean and steatotic rat hepatocytes in primary culture.

The aim of our work was to compare the effect of D-galactosamine (GalN) on primary cultures of lean and steatotic rat hepatocytes isolated from intact and fatty liver, respectively. GalN caused more severe injury to steatotic hepatocytes than to lean cells as documented by lactate dehydrogenase leakage. Necrotic mode of cell death strongly prevails over apoptosis since we did not observe any significant increase in activities of caspase 3, 8 and 9 in any group of hepatocytes treated with GalN. Reactive oxygen species (ROS) formation and lipid peroxidation were elevated in a dose-dependent manner by GalN and were significantly more pronounced in fatty hepatocytes. A decrease in the percentage of hepatocytes with energized mitochondria was observed from 30 mM and 10 mM GalN in lean and steatotic hepatocytes, respectively. Our results undoubtedly indicate that steatotic hepatocytes exert higher sensitivity to the toxic effect of GalN. This sensitivity may be caused by more intensive GalN-induced ROS production and lipid peroxidation and by higher susceptibility of mitochondria to loss of mitochondrial membrane potential in steatotic hepatocytes. In our experimental arrangement, apoptosis does not seem to participate considerably on hepatotoxic action of GalN in either group of hepatocytes.

Comparison of acetaminophen toxicity in primary hepatocytes isolated from transgenic mice with different appolipoprotein E alleles.

The nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor, important for combating electrophilic and oxidative stress in the liver and other organs. This encompasses detoxification of hepatotoxic drugs, including acetaminophen (APAP). Recently, an association between apolipoprotein E (ApoE) genotype and Nrf2 expression was described. We compared the toxicity of APAP on primary culture hepatocytes isolated from transgenic mice carrying two different human ApoE alleles and wild-type controls. The cells were exposed to APAP in concentrations from 0.5 to 4 mM for up to 24 hours. APAP led to a dose-dependent hepatotoxicity from 1 mM after 16 h exposure in all mice tested. The toxicity was higher in hepatocytes isolated from both transgenic strains than in wild-type controls and most pronounced in ApoE3 mice. Concurrently, there was a decline in mitochondrial membrane potential, especially in ApoE3 hepatocytes. The formation of reactive oxygen species was increased after 24 hours with 2.5 mM APAP in hepatocytes of all strains tested, with the highest increase being in the ApoE3 genotype. The activity of caspases 3 and 7 did not differ among groups and was minimal after 24 hour incubation with 4 mM APAP. We observed higher lipid accumulation in hepatocytes isolated from both transgenic strains than in wild-type controls. The expression of Nrf2-dependent genes was higher in ApoE3 than in ApoE4 hepatocytes and some of these genes were induced by APAP treatment. In conclusion, transgenic mice with ApoE4 and ApoE3 alleles displayed higher susceptibility to acute APAP toxicity in vitro than wild-type mice. Of the two transgenic genotypes tested, ApoE3 allele carriers were more prone to injury.