Effect of dietary copper and iron restriction on aconitase activity and antioxidant capacity of liver, kidney and heart from growing rats.

Dietary copper- and iron restriction was achieved by application of the whole milk diet to growing rats in the course of 50 days. Three distinct responses of cytosolic and mitochondrial aconitases as well as of antioxidant defense system (CuZnSOD, MnSOD, catalase and GSH) to the dietary copper- and iron deficiency were established in liver, kidney and heart from experimental rats. The results were discussed with a view to the participation of ROS-generating processes in copper- and iron-deficient state. Differences in oxidative stability of cytosolic and mitochondrial aconitase activity of both control and experimental rats were also found. The in vitro increased aconitase activity of cytosol and the unchanged one of mitochondria from liver upon exposure of preparations to air were proved in vivo upon dietary copper- and iron restriction. This finding was interpreted to suggest the existence of putative aconitase activity.

Effects of cimetidine and its metal complexes on nitroblue tetrazolium and ferricytochrome c reduction by superoxide radicals.

The effects of cimetidine, a potent histamine-H2-receptor antagonist and a good -OH scavenger, on the kinetics of ferricytochrome c and NBT reduction by superoxide anions were studied. The drug dose-dependently inhibited ferricytochrome c and NBT reduction by O2- radicals, generated either in xanthine oxidase system or photochemically or directly by KO2. The inhibitory effect of cimetidine remained unchanged in the presence of catalase or mannitol. Cimetidine and its complexes with Cu(II) and Fe(III) ions inhibited ferricytochrome c and NBT reduction even when metal chelators were added to the reaction medium. The results suggest the reaction of cimetidine with O2-radicals.

Studies on paraquat-induced oxidative stress in rat liver.

Oxidative injury of liver was studied 20 hr after a single oral administration of 150 mg/kg paraquat (PQ) to rats. PQ exerted no effect on cytosolic superoxide dismutase (SOD) activity but increased mitochondrial SOD activity by 14%. The level of GSH was decreased by 30%, and GSH/GSSG ratio was diminished almost twice. The correlation between the enhancement of mitochondrial SOD activity and the diminution of GSH level by PQ implicates O2- in the liver toxicity of the drug. Mitochondrial aconitase activity was slightly decreased (by 9%) while cytosolic aconitase activity was not affected. The results cast additional light on the responses of both aconitases to oxidative stress.

In vitro effects of alloxan-vanadium combination on lipid peroxidation and on antioxidant enzyme activity.

1. The in vitro effects of alloxan, dialuric acid and vanadium ions, alone or in combination, on lipid peroxidation and on antioxidant enzyme activity in rat liver and kidney were studied. 2. Unlike alloxan, alloxan-glutathione (GSH) and dialuric acid increased lipid peroxidation, which could be explained by the decreased activity of catalase and GSH peroxidase during incubation. 3. Vanadium(IV) ions increased the amount of thiobarbituric acid-reacting substances, but neither vanadium(IV) nor vanadium(V) changed the enzyme activity. 4. The combination of vanadium ions and alloxan-GSH or dialuric acid had no additive effect on lipid peroxidation. Vanadium ions decreased the dialuric acid-induced inhibition of catalase activity. 5. The present results suggest the therapeutic value of vanadium as an antidiabetic agent.

Hydroxyl radicals production in the vanadium ions/dialuric acid systems.

1. The effects of vanadium ions on the .OH radical production in the presence of and in the absence of dialuric acid were studied. 2. Dialuric acid enhanced deoxyribose degradation. 3. Vanadium ions and vanadium/EDTA complexes decreased the degradation of deoxyribose in the presence and in the absence of dialuric acid. 4. The question as to whether or not free .OH radicals are formed via reaction of vanadium ions with H2O2 in the presence of dialuric acid is discussed. 5. The results are interpreted with a view to the vanadium ability to decrease the toxic effects of dialuric acid.

Lipid peroxidation induced by ultrasonication in Ehrlich ascitic tumor cells.

The mechanism of sonodynamic action in tumor cells is poorly investigated. It is known that ultrasound generates free radicals in phosphatidylcholine liposomes used as a membrane model. The participation of lipid peroxidation products in the mechanisms of physiological suppression of cell multiplication has been investigated for some tumor cells. In the present work ultrasound-induced lipid peroxidation in Ehrlich ascitic tumor cells was studied. Ultrasonication increased the level of lipid peroxidation quantified by the TBARS method in homogenates from Ehrlich ascitic tumor cells. Changes in the fatty acid composition of lipids from Ehrlich ascitic tumor cells irradiated by sonication were observed. TBARS production obtained by ultrasound was compared to TBARS production obtained by widely used chemical inductors. The free-radical processes evoked by ultrasound are of interest in antitumor therapy.

Aconitase activity in rat liver.

This paper presents data about the subcellular distribution of aconitases in rat liver and some properties of the aconitase activity in cytosol, mitochondria and soluble mitochondrial protein (SMP). The cytosolic and mitochondrial aconitase activity was 64.8% or 61.0% and 20.1% or 19.4% of the total rat liver aconitase activity when cis-aconitate or isocitrate was used as substrate. Aconitase activity of stored SMP and mitochondria with phosphate buffer (pH 7.4) and 0.25 M sucrose (pH 7.4) as isolation medium respectively, was reduced to an equal extent upon exposure to air. Fresh SMP preparations immediately and three hr after isolation had the same aconitase activity. It is concluded that phosphate has no role in the oxidative degradation of mitochondrial aconitase and does not inhibit it. Complete restoration of the decreased mitochondrial aconitase activity to the initial level was achieved with thiomalate and Fe2+ under anaerobic conditions or 60-70% was restored during the long period (60 min) of incubation with exogenous substrate. The aconitase activity of cytosol and mitochondria increased upon exposure to air for 7 1/2 hr. This finding is interpreted to suggest the existence of putative aconitase activity.

Lipid peroxidation and antioxidant enzyme activity in aspirin-treated rats.

1. Malondialdehyde formation and antioxidant enzyme activity after oral or intraperitoneal treatment of rats with various doses of aspirin was studied. 2. Aspirin, orally, had no effect on spontaneous, Fe(II)- or Fe(II)/ascorbate-induced malondialdehyde formation in liver homogenates; orally, ascorbate-induced malondialdehyde production was inhibited but only after 5-day treatment with 500 mg/kg aspirin; after intraperitoneal injection, the drug inhibited ascorbate- and Fe(II)/ascorbate-induced production of malondialdehyde. 3. Aspirin had no effect on malondialdehyde formation in erythrocytes, irrespective of the dose and route of drug administration. 4. Aspirin increased glutathione peroxidase activity in liver after 5-day treatment with an oral dose of 500 mg/kg and decreased enzyme activity in both liver and erythrocytes, 24 hr after a single injection of the same dose. 5. Aspirin, in vivo slightly affected lipid peroxidation and antioxidant enzyme activity.

In vitro effects of aspirin on malondialdehyde formation and on activity of antioxidant and some metal-containing enzymes.

The in vitro effects of aspirin in different concentrations on malondialdehyde formation and on the activity of antioxidant and some metal-containing enzymes in rat liver homogenate and erythrocytes were studied. Aspirin showed a biphasic dependence on concentration: low concentrations (to 1.0 mM) stimulated the spontaneously formed malondialdehyde in the liver homogenate and the high concentration (5.0 mM) inhibited it; all aspirin concentrations tested had no effect on the liposomes; 5.0 mM aspirin inhibited the Fe(2+)-induced lipid peroxidation in the liver homogenate but had an opposite effect on the liposomes. Aspirin dose-dependently inhibited nitro-blue tetrazolium reduction and decreased deoxyribose degradation. The higher aspirin concentrations inhibited the activity of antioxidant and some metal-containing enzymes. It is suggested that these aspirin effects are determined by the aspirin-metal complexes formed rather than by aspirin itself.

Do indomethacin and cimetidine or Cu(cimetadine)2 affect the nature of superoxide dismutase activity in the liver of copper-deficient rats?

1. Control and copper-deficient rats were treated with: (i) indomethacin; (ii) indomethacin in the presence of cimetidine; and (iii) indomethacin in the presence of Cu(cimetidine)2. The levels of copper, zinc and manganese as well as the nature of superoxide dismutase activity in the liver were studied. 2. Copper deficiency caused a decrease of enzyme SOD activity, EDTA-insensitive (by 84%) and the appearance of nonenzyme SOD-like activity, EDTA-sensitive. The levels of copper and zinc decreased by 67% and 40% and the manganese level increased by 53%. 3. The above-mentioned treatments (i, ii, iii) of copper-deficient rats induced a progressive increase of enzyme SOD activity (by 19, 90 and 176%, respectively) without, however, changing nonenzyme SOD-like activity. It was only indomethacin treatment in the presence of Cu(cimetidine)2 that increased the copper level in control (by 82%) and copper-deficient (by 182%) rats. 4. The liver contained 4 CuZnSOD- and 1 MnSOD-isoenzymes, whose number and position on the gel were affected neither by copper deficiency nor by indomethacin treatment in the presence of Cu(cimetidine)2. Copper deficiency significantly increased the MnSOD-band and reduced the CuZnSOD-bands, particularly that with pI approximately 5.7. Indomethacin in the presence of Cu(cimetidine)2 changed neither the MnSOD-band nor the reduced CuZnSOD-band with pI approximately 5.7, but restored to normal all the other CuZnSOD-bands.

Lipid peroxidation and antioxidant enzymes in vanadate-treated rats.

Male Wistar rats received an aqueous solution of ammonium metavanadate (AMV) of 0.15 mg/V/ml concentration instead of water for 14 days. The erythrocyte count and haemoglobin level in blood were not changed; the haematocrit index was slightly increased. The spontaneous lipid peroxidation in kidney and liver homogenates was increased. The Fe(II)- or ascorbate-induced lipid peroxidation was more pronounced in the kidney than in the liver. No changes in lipid peroxidation were observed in erythrocytes after AMV treatment. The AMV treatment resulted in a decrease in the activity of the antioxidant enzymes, catalase and glutathione peroxidase in the kidney and liver; the cytosolic Cu,Zn-SOD and mitochondrial Mn-SOD were unchanged. The activity of the enzymes in blood was not changed. The results are discussed with a view to the participation of lipid peroxidation in vanadium toxicity.

Liver superoxide dismutases after copper deficiency and/or indomethacin treatment of rats.

Copper deficiency in rats resulted in a decrease of liver cytosolic and lysosomal CuZnSOD activity (by 71% and by 55%, respectively) and in an increase of mitochondrial MnSOD (by 185%). The content of copper and zinc decreased by 64% and 38%, respectively, and that of manganese increased by 47%. Cytosolic CuZnSOD activity, both in control and copper-deficient rats, increased (by 71.5 units/mg protein and by 83.0 units/mg protein, respectively) after indomethacin treatment. Rat liver contained four CuZnSOD (cytosolic and lysosomal) and one MnSOD isoenzyme. Neither copper deficiency nor indomethacin treatment changed the position and the number of bands. The CuZnSOD-band with pI approximately 5.7 was greatly decreased by copper deficiency and was not restored to normal after indomethacin treatment.

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.

Do the copper complexes of histamine, histidine and of two H2-antagonists react with O2-?

The effects of cimetidine, ranitidine, histamine and histidine, as well as of their copper complexes, have been examined in an enzymic and chemical O2- generated systems. Copper complexes like CuZnSOD inhibited both the reduction of cytochrome c and NBT2+ in xanthine-xanthine oxidase systems, but their inhibitory action was due to a certain extent to the copper-induced inhibition of xanthine oxidase. EDTA abolished the inhibitory effect of all copper complexes studied. Luminol chemiluminescence in NADH2-PMS system was inhibited by CuZnSOD while it was enhanced by copper complexes. The copper-accelerating effect gradually increased up to about 1 microM Cu and decreased, reaching the control values up to 10 microM Cu. In the presence of low copper concentrations chemiluminescence was inhibited by CuZnSOD only, while in the presence of high copper concentrations it was inhibited by catalase and mannitol, but not by CuZnSOD. The ligands however, have been ineffective in the two O2- generated systems.

Effect of pregnancy and fetal development on sheep liver superoxide dismutase activity.

Liver cytosolic CuZn SOD activity of four-week and 12-week pregnant sheep was twice as high as that of their fetuses and almost equal to that of control, barren ewes. By the 20th week of pregnancy activity had decreased by about 70 per cent in the maternal liver and increased to a value similar to that of the controls in the fetuses. The lysosomal CuZn SOD activity remained almost unchanged during pregnancy both in the maternal and fetal livers. Cytosolic and lysosomal CuZn SOD activities of 20-week pregnant sheep and their fetuses showed a similar electrophoretic pattern and low electrophoretic mobility. Hepatic Mn SOD activity increased sharply during fetal development but remained lower than that in both the control and maternal livers. It is proposed that the changes in CuZn SOD and Mn SOD activities are associated with changes in copper metabolism and oxygen utilisation, respectively. The low electrophoretic mobility of CuZn SOD is assumed to be a species specificity.

Vanadyl- and vanadate-induced lipid peroxidation in mitochondria and in phosphatidylcholine suspensions.

Vanadyl (V(IV] was found to induce rapidly developing lipid peroxidation in intact and sonicated mitochondria as well as in phosphatidylcholine suspension. The ability of vanadate (V(V] to induce lipid peroxidation was much less pronounced compared to that of vanadyl. The peroxidative action of vanadate on phosphatidylcholine much increased in the presence of NADH and ascorbate. Preincubation of vanadate with glucose had the same effect. Vanadyl-induced lipid peroxidation was not essentially influenced by SOD, catalase and ethanol but was completely inhibited by butylated hydroxytoluene. All these effects of vanadyl and vanadate are thought to participate in the insulin-like and other biological actions of vanadium.

Studies on the nature of superoxide dismutase activity in sheep liver subcellular fractions.

In the absence or in the presence of 10(-4)M EDTA cytosolic and lysosomal CuZnSOD and MnSOD activities were differentiated as enzymatic and nonenzymatic (SOD-like). Both activities were about three times lower in mitochondrial matrix compared to those in cytosol and lysosomes. The cytosolic and lysosomal CuZnSOD showed an equal electrophoretic pattern: three peaks of SOD activity with Rf values of 1 = 0.10 and 0.12; 2 = 0.16 and 0.18; 3 = 0.21 and 0.22, respectively. The low anodic mobility was explained by the high isoelectric points (pI) of the three CuZnSOD isoenzymes (approximately 5.7, approximately 6.5 and approximately 7.5) contained both in cytosol and in lysosomes.

Phosphatidylcholine peroxidation induced by Cu(II) and some Cu(II) complexes.

We established that Cu(II) and its complexes with aspirin, indomethacin and cimetidine produced lipid peroxidation in phosphatidylcholine suspension. Ionic copper was found to have the most pronounced effect, while the effect of the complexes depends on the ligand. Neither of the ligands exerted a prooxidative effect. NADH sharply increased the effects of Cu(II) and its complexes. In contrast, in the presence of ascorbate the prooxidative effect of Cu(II) and its complexes decreased. Cu(II) and its complexes strongly inhibited ascorbate-induced lipid peroxidation. Using antioxidant substances, enzymes and copper-chelating agents, we attempted to explain the observed effects of Cu(II) and its complexes.