In vivo effects of indomethacin on the activity of metal-containing enzymes.

1. Indomethacin injected subcutaneously at a single "ulcerogenic" dose decreased aminooxidase and leucine aminopeptidase activity and did not change alcohol dehydrogenase and ceruloplasmin activity. 2. Indomethacin administered at an oral "therapeutic" dose inhibited aminooxidase activity in small intestinal mucosa but not in liver and did not change leucine aminopeptidase activity in blood, liver and intestinal mucosa; it, however, increased alcohol dehydrogenase and ceruloplasmin activity. 3. The decreased activity of ceruloplasmin and alcohol dehydrogenase by metal deficiency increased after oral indomethacin treatment, reaching the control values when indomethacin was chelated with copper. 4. The results suggest the participation of endogenous metals in the indomethacin effect.

In vivo effects of indomethacin--I. Activity of antioxidant enzymes and lipid peroxidation.

1. The in vivo effects of indomethacin on the activity of antioxidant enzymes and on lipid peroxidation in erythrocytes, liver and small intestines of rats were examined. 2. The activity of the enzymes studied increased or remained unchanged depending on the preparation and model used: treatment with "therapeutic" or "ulcerogenic" dose of indomethacin. 3. Indomethacin inhibited lipid peroxidation in the liver but not in the erythrocytes. 4. The results suggest that the stimulation of antioxidant enzymes, probably through in vivo formed metal complexes, is an alternative mechanism of the antiinflammatory action of indomethacin.

In vivo effects of indomethacin--II. Antioxidant enzymes in metal-deficient rats.

1. The in vivo effects of indomethacin on the activity of antioxidant enzymes in erythrocytes, liver and small intestinal mucosa of rats fed a metal-deficient diet were studied. 2. Metal deficiency led to a significant decrease in the activity of the enzymes studied. 3. Neither with the "ulcerogenic" nor with the "therapeutic" dose of indomethacin significant alterations in the enzyme activity were observed. 4. The oral treatment of metal-deficient rats with a copper complex of indomethacin caused a significant increase in the activity of the enzymes studied. 5. The results suggest the participation of indomethacin in the regulation and redistribution of metals in the organism, which is probably effected through in vivo chelation of endogenous metals.

Anti-oxidant capacity of spinal cord and erythrocytes of guinea pigs in case of experimental allergic encephalomyelitis and after disease suppression.

In the lumbar spinal cord of EAE guinea pigs a significant increase in SOD activity, lipid hydroperoxides content (more than 60%) and Fe2(+)-ascorbate-induced lipid peroxidation was observed. Multiple injections of cytochrome C-vitamin B2-vitamin PP (CV-combination) during the disease latent period resulted in suppression of EAE development. Supplementation with vitamin C, vitamin B12 or ATP eliminated this suppressive effect. Upon treatment with CV-combination beginning on the day of the first EAE clinical signs a half of the sick animals recovered. In their erythrocytes the ratio between SOD and catalase activities was normalized, though on a higher level. In the lumbar spinal cord the concentration of lipid hydroperoxides was decreased to the control one. Oxidative damage of the central nervous system is one of the mechanisms underlying the pathogenesis of lethal EAE.

Inhibition of phospholipid hydrolysis by soluble phospholipase A2 in biological membranes of different origin after lipid peroxidation.

In peroxidized rat liver microsomal membranes phospholipid hydrolysis catalyzed by porcine pancreas phospholipase A2 was found to be inhibited. The extent of inhibition depended on the amount of lipid peroxidation products (MDA) accumulated in the membrane. This effect was not due to the direct action of lipid peroxidation products on the enzyme but to membrane modification. The same inhibitory effect was also found with other membranes--rabbit skeletal muscle sarcoplasmic reticulum, bovine retina rod outer segments and rat brain synaptosomes--differing in phospholipid and fatty acid composition. The inhibition of phospholipase reaction by lipid peroxidation depended at least on three factors: decrease in the amount of phosphatidylethanolamine; decrease in the level of phospholipids, containing polyunsaturated fatty acid residues and occurrence of membrane structural rearrangements resulting in unavailability of phospholipid substrates for phospholipase A2 attack. Membrane destruction with anionic detergent--sodium cholate--led to a sharp increase of phospholipase hydrolysis rate.

Effect of chronic copper loading on the activity of rat liver antioxidative enzymes.

The activity of antioxidative enzymes SOD, catalase, glutathione peroxidase and the related glutathione reductase, glucose-6-phosphate dehydrogenase and NADPH-isocitrate dehydrogenase was examined in liver cytosol and large granule fraction (mitochondria) from control and copper-loaded rats. An increase of SOD activity (more than 100%) and a decrease of both catalase (by 60%) and glutathione peroxidase activity (by 30%) in large granule fraction were observed after copper loading. The cytosolic glutathione peroxidase activity was also markedly decreased: glutathione peroxidase I (EC 1.11.1.9)--by 35% and glutathione peroxidase II (EC 2.5.1.18)--by 75%. Cytosolic catalase activity and the glutathione reductase, glucose-6-phosphate dehydrogenase and NADPH-isocitrate dehydrogenase activities in cytosol and in mitochondria of copper-loaded rats were unchanged. It is concluded that under chronic copper loading the primary mechanisms of copper toxicity are accompanied by disturbances of the antioxidative enzyme function.

Enzymes of oxygen metabolism and lipid peroxidation on erythrocytes from copper-deficient rats.

1. Copper deficiency in the rat results in a high decrease of erythrocyte superoxide dismutase activity (by 70%), an increase of glutathione peroxidase activity (by 17%) and glucose 6-phosphate dehydrogenase activity (by 40%) and no change in catalase activity. 2. Ascorbate (30 nM) and copper (10 and 50 nmol/mg protein) enhance about 2-fold the lipid peroxidation of erythrocyte membranes from copper-deficient rats. 3. The osmotic stability of copper-deficient rat erythrocytes is higher compared with that of the controls.

Decorporation of copper from liver subcellular fractions after alimentary loading of rats.

Subcellular copper distribution was studied after single alimentary loading of rats with copper sulfate and after 2- and 4-day spontaneous decorporation of the metal. Copper content in mitochondria and in the soluble fraction was found to rise as early as the 1st day. Then it decreased reaching the normal values after 2-day and 4-day autodecorporation for cytosol and mitochondria, respectively. The activities of rotenone-sensitive NADH-cytochrome c reductase, succinate-cytochrome c reductase and cytochrome oxidase were inhibited by copper treatment but after a 4-day decorporation they became normal. On the contrary, rotenone-insensitive NADH-cytochrome c reductase was activated. Single alimentary copper treatment induced changes in electron transport and oxidative phosphorylation with succinate and glutamate as substrates. The changes established were in accordance with the decreased enzyme activities. After chronic copper loading (7-8 weeks) the interruption of copper-enriched diet for 5 days led to restoration of the copper content in the subcellular fractions. Treatment with unitiol did not change the spontaneous decorporation.

Effect of chronic copper loading on the functions of rat liver mitochondria.

Male albino rats were kept on copper-enriched diet for 2, 4, 6 and 8 weeks. Experiments were made to study the electron transported, oxidative phosphorylation and the activity of some respiratory enzymes (rotenone-insensitive NAD. H-cytochrome c-reductase, NAD. H-DCPIP-reductase, succinate-cytochrome c(DCPIP)-reductase and succinate dehydrogenase) depending on the duration of copper sulphate treatment and hepatic copper level. Copper content is found to rise as early as the 2nd week, after which it remains relatively constant. Oxygen consumption in State 3 decreases strongly during the 2nd week and remains low throughout the period studied. Oxygen consumption in State 4 also decreases in the 2nd week, after which it rises and reaches the values of the control animals. The enzyme activities studied are also strongly inhibited (32-57%) after a 14-day treatment, later they are recovered gradually, reaching 50-79% of the control values. The probable compensatory mechanism of copper metabolism in the liver and the participation of thiol groups in it are discussed.