Factors affecting the function of the mitochondrial membrane permeability transition pore and their role in evaluation of calcium retention capacity values.

Values of the calcium retention capacity (CRC) of rat liver mitochondria are highly dependent on the experimental conditions used. When increasing amounts of added calcium chloride are used (1.25-10 nmol), the values of the CRC increase 3-fold. When calcium is added in 75 s intervals, the CRC values increase by 30 % compared with 150 s interval additions. CRC values are not dependent on the calcium/protein ratio in the measured sample in our experimental design. We also show that a more detailed evaluation of the fluorescence curves can provide new information about mitochondrial permeability transition pore opening after calcium is added.

The effect of oleic and palmitic acid on induction of steatosis and cytotoxicity on rat hepatocytes in primary culture.

In vitro models serve as a tool for studies of steatosis. Palmitic and oleic acids can induce steatosis in cultured hepatocytes. The aim of our study was to verify steatogenic and cytotoxic effects of palmitic acid (PA), oleic acid (OA) and their combinations as well as their impact on functional capacity of rat primary hepatocytes. Hepatocytes were exposed to OA or PA (0.125-2 mmol/l) or their combination at ratios of 3:1, 2:1 or 1:1 at the final concentrations of 0.5-1 mmol/l. Both OA and PA caused a dose-dependent increase in triacylglycerol content in hepatocytes. PA was more steatogenic at 0.25 and 0.5 mmol/l while OA at 0.75 and 1 mmol/l. PA exhibited a dose-dependent cytotoxic effect associated with ROS production, present markers of apoptosis and necrosis and a decrease in albumin production. OA induced a damage of the cytoplasmic membrane from 1 mM concentration. Mixture of OA and PA induced lower cytotoxicity with less weakened functional capacity than did PA alone. Extent of steatosis was comparable to that after exposure to OA alone. In conclusion, OA or combination of OA with PA is more suitable for simulation of simple steatosis than PA alone.

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.

Transcriptome temporal and functional analysis of liver regeneration termination.

Decades of liver regeneration studies still left the termination phase least elucidated. However regeneration ending mechanisms are clinicaly relevant. We aimed to analyse the timing and transcriptional control of the latest phase of liver regeneration, both controversial. Male Wistar rats were subjected to 2/3 partial hepatectomy with recovery lasting from 1 to 14 days. Time-series microarray data were assessed by innovative combination of hierarchical clustering and principal component analysis and validated by real-time RT-PCR. Hierarchical clustering and principal component analysis in agreement distinguished three temporal phases of liver regeneration. We found 359 genes specifically altered during late phase regeneration. Gene enrichment analysis and manual review of microarray data suggested five pathways worth further study: PPAR signalling pathway; lipid metabolism; complement, coagulation and fibrinolytic cascades; ECM remodelling and xenobiotic biotransformation. Microarray findings pertinent for termination phase were substantiated by real-time RT-PCR. In conclusion, transcriptional profiling mapped late phase of liver regeneration beyond 5(th) day of recovery and revealed 5 pathways specifically acting at this time. Inclusion of longer post-surgery intervals brought improved coverage of regeneration time dynamics and is advisable for further works. Investigation into the workings of suggested pathways might prove helpful in preventing and managing liver tumours.

Steatotic rat hepatocytes in primary culture are more susceptible to the acute toxic effect of acetaminophen.

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in humans. Non-alcoholic fatty liver disease is the most frequent chronic liver disease in developed countries. The aim of our work was to compare the effect of APAP on intact rat hepatocytes and hepatocytes isolated from steatotic liver in primary cultures. Male Wistar rats were fed with standard diet (10 % energy from fat) and high-fat diet (71 % energy from fat) for 6 weeks and then hepatocytes were isolated. After cell attachment, APAP (1; 2.5; 3.75 and 5 mM) was added to culture media (William's E medium) and hepatocytes were cultured for up to 24 hours. APAP caused more severe dose-dependent damage of steatotic hepatocytes as documented by increased release of lactate dehydrogenase (LDH) and LDH leakage, decreased activity of cellular dehydrogenases (WST-1 test) and reduced albumin production. Intact steatotic hepatocytes contained lower amount of reduced glutathione (GSH). Treatment with APAP (1 and 2.5 mmol/l) caused more pronounced decrease in GSH in steatotic hepatocytes. ROS (reactive oxygen species) formation after 24-hour incubation was significantly higher in fatty hepatocytes using APAP at concentration of 3.75 and 5 mmol/l. Interleukin 6 (IL-6) production was elevated in 2.5 mM APAP-treated nonsteatotic and steatotic hepatocyte cultures at 8 hours, compared to appropriate controls. In conclusions, our results indicate that steatotic hepatocytes exert higher sensitivity to the toxic action of APAP. This sensitivity may be caused by lower content of GSH in intact steatotic hepatocytes and by more pronounced APAP-induced decrease in intracellular concentration of GSH.

Developmental changes of the sensitivity of cardiac and liver mitochondrial permeability transition pore to calcium load and oxidative stress.

Opening of the mitochondrial membrane permeability transition pore (MPTP) is an important factor in the activation of apoptotic and necrotic processes in mammalian cells. In a previous paper we have shown that cardiac mitochondria from neonatal rats are more resistant to calcium load than mitochondria from adult animals. In this study we have analyzed the ontogenetic development of this parameter both in heart and in liver mitochondria. We found that the high resistance of heart mitochondria decreases from day 14 to adulthood. On the other hand, we did not observe a similar age-dependent sensitivity in liver mitochondria, particularly in the neonatal period. Some significant but relatively smaller increase could be observed only after day 30. When compared with liver mitochondria cardiac mitochondria were more resistant also to the peroxide activating effect on calcium-induced mitochondrial swelling. These data thus indicate that the MPTP of heart mitochondria is better protected against damaging effects of the calcium load and oxidative stress. We can only speculate that the lower sensitivity to calcium-induced swelling may be related to the higher ischemic tolerance of the neonatal heart.