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.

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.

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.

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.

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.

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.

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.

A study on the ability of copper complexes to act as active oxygen species scavengers.

The ability of two low-molecular-weight copper complexes to influence the hemolysis of human erythrocytes caused by active oxygen species-generating systems was studied. Cu(II) (glycine)2 and Cu(II) (tyrosine)2 did not inhibit hemolysis due to O-2 and H2O2 generated by xanthine oxidase plus acetaldehyde but rather has a prooxidant effect. The same copper complexes as well as Cu(II) strongly inhibited the hemolysis caused by the 1O2-generating system (Rose Bengal + light). It was found that except for 1O2 the other active oxygen species (O-2, H2O2 and OH.) did not participate in the Rose Bengal + light-induced hemolysis. Thus we examined whether the inhibitory effect of copper complexes was due to 1O2 quenching. Cu(II) (glycine)2 inhibited the Rose Bengal + light-induced oxidation of compounds known to react chemically with 1O2 and its effects were analogous to the effects of physical 1O2 quenchers, e. g. NaN3 and NiCl2. The oxygen consumption upon NADH-photooxidation in the presence of Rose Bengal was inhibited competitively by Cu(II) (glycine)2 but when concentration of Rose Bengal or light intensity were varied the extent of Cu(II) (glycine)2-caused inhibition was not changed. It is concluded that the effects of Cu(II) (glycine)2 and possibly of Cu(II) (tyrosine)2 are due to quenching of 1O2 but quenching of the excited state of the dye could not be excluded.

Reactions of oxyphenbutazone with active oxygen species.

The ability of Oxyphenbutazone (a non-steroidal antiinflammatory drug) to react with singlet oxygen and superoxide anions, possible mediators of the damage to the lipids of the cell membranes during inflammation was studied. Oxyphenbutazone inhibited the reduction of nitroblue tetrazolium in aerobic riboflavin-photosensitized oxidation of methionine, but did not influence the cytochrome C-reduction by superoxide-generating system xanthine-xanthine oxidase. Oxyphenbutazone was photooxidized in the presence of Rose Bengal, the latter being a photosensitizer. The increase of the reaction rate of Oxyphenbutazone-oxidation in D2O as compared to H2O, as well as the inhibition of oxidation by singlet oxygen-quencher sodium azide confirmed the participation of singlet oxygen in this process. It was found that Oxyphenbutazone reacted with singlet oxygen, but did not react with superoxide anions. This was supported by the observed protection of erythrocyte membranes from the hemolytic action of the singlet oxygen-generating system Rose Bengal + light.

The effects of aspirin, indomethacin and their copper complexes on phospholipase activity and on lipid peroxidation in rat liver microsomes.

Aspirin and indomethacin decreased the hydrolysis of microsomal phospholipids by exogenous soluble phospholipase A2 and increased lipid peroxidation in rat liver microsomes. Copper chelates of the non-steroidal anti-inflammatory drugs tested more strongly decreased the activity of soluble phospholipase A2 as compared to the ligands. In contrast to the stimulant effects of aspirin and indomethacin these chelates exerted dose-dependent inhibitory effects on enzymatic and non-enzymatic lipid peroxidation. The effects of CuSO4 were similar to those of Cu(II)-aspirin and Cu(II)-indomethacin. Bathocuproine sulfonate, a specific chelator for Cu+, completely prevented the inhibitory effects of copper complexes and of CuSO4 both on lipid peroxidation and on microsomal NADPH-oxidation. Therefore, cupric ions, free or chelated, as well as their reduction to Cu+ by microsomes did not affect the activity of NADPH-dependent cytochrome P-450 reductase. These data are explained by drug-induced changes in the membrane structure as well as by the redox cycling of cupric ions in the copper complexes of aspirin and indomethacin and the subsequent and/or simultaneous interaction of Cu2+/Cu+ with some component(s) of the reaction medium.

Heterogeneity of copper-induced superoxide dismutase in rat liver lysosomes.

Preparations of liver large granule fraction from control and copper-injected rats were treated with digitonin (0.01-0.16 mg/mg protein) and supernatants rich in lysosomal matrix and in mitochondrial intermembrane space were obtained. After copper injection the superoxide dismutase activity in all supernatants was significantly increased. The granular CuZn-superoxide dismutase in the two animal groups was localized in lysosomes only. The cytosolic and lysosomal CuZn-superoxide dismutase in control preparations showed an equal electrophoretic pattern (two peaks with Rf = 0.42 and 0.47). After copper injection three new electrophoretic peaks of the lysosomal CuZn-superoxide dismutase activity (with Rf = 0.37, 0.52 and 0.62 respectively) appeared. The increased heterogeneity of the granular CuZn-superoxide dismutase activity after copper injection is explained by the oxidative degradation of the CuZn-superoxide dismutase in lysosomes.

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.

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.

Differences in the in vivo effects of cuprizone on superoxide dismutase activity in rat liver cytosol and mitochondrial intermembrane space.

Through aerobic NBT2+ photoreduction in the absence or in the presence of 10(-4) M EDTA and through o-dianisidine photooxidation superoxide dismutase (SOD) activity was differentiated as enzymatic and nonenzymatic. SOD activity in cytosol and mitochondrial matrix was enzymatic, while 50% of the SOD activity in the intermembrane space was nonenzymatic. Cuprizone inhibited by 40% the cytosolic SOD activity and by more than 50% the nonenzymatic SOD activity in intermembrane space without, however, changing the enzymatic SOD activity in the latter fraction. Single copper injection increased enzymatic SOD activity: the cuprizone-inhibited cytosolic dismutase activity restored to normal and the unaltered dismutase activity in the intermembrane space exceeded that in the controls.

Copper deficiency-induced changes in the fatty acid composition of mitochondrial and microsomal membranes of rat liver.

The effects of copper deficiency on the fatty acid composition of mitochondrial and microsomal phospholipids in rat liver were studied. Copper deficiency was induced by a milk powder diet. To evaluate the effect of the milk diet on the fatty acid pattern of mitochondrial and microsomal phospholipids, one group of rats was fed Cusupplemented powdered milk. A decrease in the relative proportion of linoleic acid and an increase in the level of oleic and docosahexaenoic acids in membrane phospholipids were found in this group. However, no changes in the fatty acid pattern characteristic of essential fatty acid deficiency were observed. Dietary copper deficiency produced a significant decrease in the relative amounts of linoleic and arachidonic acids, as well as an increase in the docosahexaenoic acid content in both mitochondrial and microsomal membranes compared to the nondeficient controls. The disproportionate quantities of polyunsaturated fatty acids are discussed with a view to the disturbances of membrane function in copper deficiency.

Copper increases superoxide dismutase activity in rat liver.

Superoxide dismutase (SOD) activity in rat liver cytosol and submitochondrial fractions was characterized as enzymatic and nonenzymatic (due to the SOD-like activity of copper) by four approaches: (i) aerobic NBT2+ (nitroblue tetrazolium) photoreduction in the absence of EDTA; (ii) aerobic NBT2+ photoreduction in the presence of 10(-4)M EDTA; (iii) anaerobic NBT2+ photoreduction; and (iv) o-dianisidine photooxidation. Under normal conditions nonenzymatic SOD activity has been observed only in the intermembrane space. The single subcutaneous injection of rats with CuSO4 solution (5 mg Cu/kg body wt) led to (i) an elevation of the copper level in all submitochondrial fractions; (ii) an increase in enzymatic SOD activity in only cytosol and intermembrane spaces; (iii) the appearance of a new electrophoretic SOD activity band in the intermembrane space preparations; and (iv) the appearance of nonenzymatic SOD-like activity in the outer and inner mitochondrial membranes, and a twofold increase in lipid hydroperoxides. This suggests that the increased nonenzymatic copper in vivo has a prooxidant effect, and does not catalyze the dismutation of O2- as it has been shown in in vitro experiments [E. M. Russanov, S. G. Ljutakova, and S. I. Leutcher (1982) Arch. Biochem. Biophys. 215, 220-229]. The peculiarities of the SOD activity in the intermembrane space are explained by the lysosomal localization of the granular CuZnSOD.

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.