P. Caraceni

Source of oxygen free radicals produced by rat hepatocytes during postanoxic reoxygenation

The aim of this study was to determine the cellular source of oxygen free radicals generated by isolated hepatocytes during post-anoxic reoxygenation. Superoxide anions (O2.-) were detected by lucigenin chemiluminescence. Cell damage was assessed by LDH release. During anoxia, the chemiluminescence decreased to background levels while LDH release increased 8-fold. During reoxygenation, O2.- formation increased 15-fold within 15 min then declined towards control levels. LDH release increased from 161 to 285 mU/min in the first 30 min of reoxygenation, then declined toward the control rate. Allopurinol, an inhibitor of the xanthine-xanthine oxidase system, did not inhibit O2.- formation nor LDH release. Antimycin, a mitochondrial complex III inhibitor that does not block O2.- formation, increased both O2.- generation and LDH release 82 and 133% respectively. Diphenyleneiodonium (DPI), a mitochondrial and microsomal NADPH oxidase inhibitor, reduced O2.- and LDH release 60-70%. SOD, which catalyzes the dismutation of O2.- to H2O2, was without effect on O2.- and LDH release, but TEMPO, a stable nitroxide which mimics SOD and easily penetrates the cell membrane, decreased O2.-86% without affecting LDH. These results suggest that mitochondria or microsomes are the principal sites of O2.- production during reoxygenation of isolated hepatocytes, whereas the cytosolic xanthine/xanthine oxidase system is apparently not involved.

Proteins but not Nucleic Acids are Molecular Targets for the Free Radical Attack During Reoxygenation of Rat Hepatocytes

Isolated rat hepatocytes generate large amounts of reactive oxygen species and suffer a significant cell injury during postanoxic reoxygenation. The aim of this study was to determine whether oxidation of proteins and nucleic acids occurs during reoxygenation and whether their damage is related to the development of hepatocyte injury. Isolated perfused rat hepatocytes were exposed sequentially to 1 h of aerobic control, 2.5 h of anoxia, and 2 h of reoxygenation. Protein oxidation was determined by measuring the hepatocyte protein carbonyl content. DNA and RNA oxidation was assessed by measuring the 8-hydroxydeoxyguanosine and 8-hydroxyguanosine adducts, respectively. The control preanoxic carbonyl content was 6.48 +/- 1.03 nmol/mg protein. The preanoxic 8-8 hydroxydeoxyguanosine and 8-hydroxyguanosine levels were 4.76 +/- 1.22 pmol/ml and 14.19 +/- 2.17 pmol/ml, respectively. During anoxia, protein and nucleic acid oxidation did not change significantly. With reoxygenation, the protein carbonyl content increased significantly within 30 min, reaching a value of 10.25 +/- 1.58 nmol/mg. The nucleic acid oxidation level remained stable. Perfusion with 100 muM of during reoxygenation abolished protein oxidation. These results indicate that in rat hepatocytes during the early phase of reoxygenation: (1) the protein oxidation level increased significantly above the preanoxic aerobic values; (2) DNA and RNA oxidation does not appear to occur; and (3) free metal-mediated free radical reactions are involved in the oxidative protein damage.

Adaptation of subcellular glutathione detoxification system to stress conditions in choline-deficient diet induced rat fatty liver

The response of fatty liver to stress conditions (t-butyl hydroperoxide [t-BH] or 36 h of fasting) was investigated by assessing intracellular glutathione (GSH) compartmentation and redox status, GSH peroxidase (GSH-Px) and reductase (GSSG-Rx) activities, lipid peroxidation (TBARs) and serum ALT levels in rats on a choline-deficient diet. Baseline cytosolic GSH was similar between fatty and normal livers, while the mitochondrial GSH content was significantly lower in fatty livers. With the except of cytosolic GSH-Px activity, steatosis was associated with significantly higher GSH-related enzymes activities. Liver TBARs and serum ALT levels were also higher. Administration of t-BH significantly decreased the concentration of cytosolic GSH, increased GSSG levels in all the compartments, and increased TBARs levels in cytosol and mitochondria and serum ALT; all these alterations were more marked in rats with fatty liver. Fasting decreased the concentration of GSH in all the compartments both in normal and fatty livers, increased GSSG, TBARs and ALT levels, and decreased by 50% the activities of GSH-related enzymes. Administration of diethylmaleimide (DEM) resulted in cytosolic and microsomal GSH pool depletion. Administration of t-BH to DEM-treated rats further affected cytosolic GSH and enhanced ALT levels, whereas the application of fasting to GSH depleted rats mainly altered the mitochondrial GSH system, especially in fatty livers. This study shows that fatty livers have a weak compensation of hepatic GSH regulation, which fails under stress conditions, thus increasing the fatty livers susceptibility to oxidative damage. Differences emerge among subcellular compartments which point to differential adaptation of these organelles to fatty degeneration.

Effect of vitamin E on reoxygenation injury experienced by isolated rat hepatocytes

The pathogenic role of lipid peroxidation in the reperfusion injury of the liver is still controversial. This study was performed to determine whether the damage caused by oxygen free radicals during reoxygenation in perfused rat hepatocytes is related to lipid peroxidation. Superoxide anion was detected by lucigenin-enhanced chemiluminescence. Lipid peroxidation and cell injury were assessed by the release of malondialdehyde and lactic dehydrogenase. Upon reoxygenation following 2.5 h of anoxia, isolated hepatocytes generated considerable amount of O2-. Following O2- formation, a significant increase in malondialdehyde release was measured. Cell injury was temporally delayed relative to O2- generation, but preceded the occurrence of a significant lipid peroxidation. Treatment with Vitamin E abolished lipid peroxidation but had no effect upon superoxide anion formation and cell injury. These results suggest that in perfused rat hepatocytes non-peroxidative mechanisms are more important than peroxidative mechanisms in the pathogenesis of the early phases of reoxygenation injury.