Zn-SOD Activity Research Articles

Deferiprone protects the isolated atria from cardiotoxicity induced by doxorubicin

To investigate the effects of deferiprone on doxorubicin-induced cardiotoxicity and determine its protection on cardiac contractility in vivo at tissue level. Spontaneously-beating isolated atria from rats were pretreated with deferiprone for 10 min at 1.2 mmol/L or 0.3 mmol/L, respectively before co-incubation with doxorubicin (DOX) at 0.03 mmol/L for 60 min. Contractility (dF/dt) was assessed every 10 min during the incubation. After that, the tissues around the sinuatrial nodes were fixed for ultrastructural study; succinate dehydrogenase (SDH) and Cu, Zn superoxide dismutase (Cu, Zn-SOD) activity, as well as malondialdehyde (MDA) level of the atria were assayed. Treatment with DOX alone resulted in a 49.34% reduction of the contractility, mitochondria swelling, disruption of mitochondrial crista and decreased electron density of the matrices. Conversely, with the presence of deferiprone, the negative inotropic effect and lesions in the cardiac mitochondria structure induced by DOX were attenuated. Cu, Zn-SOD activity increased by 12.97%-12.11%, the MDA level decreased by 29.12%-39.82% and succinate dehydrogenase (SDH) activity was ameliorated by 25.15%-34.76%. Deferiprone can efficiently preserve cardiac contractility. Moreover, the results of this study indicate that deferiprone is able to protect mitochondrial function and structure form damage induced by DOX. This cardiac protective potential of deferiprone could be due to its defense capability against oxidative damage.

Oxidative damage induced in hearts, kidneys and spleens of mice by landfill leachate

The effects of the Xingou landfill leachate on levels of thiobarbituric acid reactive substances (TBARS) and the activities of Cu, Zn-superoxide dismutase (Cu, Zn-SOD), Se-dependent glutathione peroxidase (Se-dependent GPx) and catalase (CAT) were investigated in hearts, kidneys and spleens of Kunming albino mice of both sexes. Exposure to leachate caused significant increases of TBARS levels in the organs tested from mice of both sexes. For hearts, Cu, Zn-SOD, Se-dependent GPx and CAT activities were significantly increased at high concentrations for male mice, but the activities of these antioxidant enzymes were significantly increased at low concentration and decreased at high concentrations for female mice. For kidneys, Cu, Zn-SOD and Se-dependent GPx activities were significantly increased at high concentrations for male mice, but the activities were significantly increased at low concentrations and the ratio of increase was reduced with the increasing of concentration for female mice; CAT activities remained unchanged for male mice and were significantly increased at all concentrations tested for female mice. For spleens, Cu, Zn-SOD and Se-dependent GPx activities were significantly increased at high concentrations for male mice, but the activities were significantly increased at low concentrations and decreased at high concentrations for female mice; CAT activities remained unchanged for male mice and were significantly increased at high concentrations for female mice. The results suggest that leachate exposure can cause oxidative damage on hearts, kidneys and spleens of mice, and there were sex difference and organ difference in the response of antioxidant status.

Superoxide targets calcineurin signaling in vascular endothelium

Superoxide emerges as key regulatory molecule in many aspects of vascular physiology and disease, but identification of superoxide targets in the vasculature remains elusive. In this work, we investigated the possibility of inhibition of protein phosphatase calcineurin by superoxide in endothelial cells. We employed a redox cycler 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) to generate superoxide inside the cells. DMNQ caused inhibition of cellular calcineurin phosphatase activity, which was reversible upon DMNQ removal. Inhibition was suppressed by pre-incubating the cells with copper/zinc superoxide dismutase (Cu,ZnSOD). In addition, reducing cellular Cu,ZnSOD activity by diethylthiocarbamic acid treatment resulted in calcineurin inhibition and enhanced sensitivity to DMNQ. Further, we could show that DMNQ inhibits calcineurin-dependent nuclear translocation and transcriptional activation of NFAT transcription factor, and Cu,ZnSOD or superoxide scavenger Tiron reduced the inhibition. Thus, superoxide generation in endothelial cells results in inhibition of calcineurin signaling, which could have important pathophysiological implications in the vasculature.

Copper–zinc superoxide dismutase activity in healthy and inflamed human dental pulp

To examine copper-zinc superoxide dismutase (Cu, Zn-SOD) activity in clinically healthy and symptomatic human dental pulps. Twenty-five systemically healthy patients, 14 females and 11 males (age: 13.1-34.6 years; mean: 21.7 +/- 6.3), were the source of the pulp tissue. The condition of the pulps was assessed using clinical and radiographic evaluation. The pulp tissue was collected by longitudinally grooving and splitting the teeth (if extracted) or during endodontic treatment, and were age- and sex-matched between the healthy and the irreversible symptomatic pulpitis tissue groups. Cu, Zn-SOD activity was determined through spectrophotometric methods and a Mann-Whitney test assessed the significance of differences between the groups. The enzyme activities were 144.8 +/- 42.2 and 68.1 +/- 25.0 U mg(-1) in the healthy and irreversible symptomatic pulp tissue, respectively. The difference between the groups was statistically significant (P < 0.001). These results demonstrate a potential role for Cu, Zn-SOD during dental pulp inflammation in humans.

Purification and partial characterization of Cu, Zn containing superoxide dismutase from entomogenous fungal species Cordyceps militaris

Cordyceps militaris mycelium produced mainly Cu, Zn containing superoxide dismutase (Cu, Zn-SOD). Cu, Zn-SOD activity was detectable in the culture filtrates, and intracellular Cu, Zn-SOD activity as a proportion protein was highest in early log phase culture. The effects of Cu 2+, Zn 2+, Mn 2+ and Fe 2+ on enzyme biosynthesis were studied. The Cu, Zn-SOD was isolated and purified to homogeneity from C. militaris mycelium and partially characterized. The purification was performed through four steps: (NH 4) 2SO 4 precipitation, DEAE-sepharose™ fast flow anion-exchange chromatography, CM-650 cation-exchange chromatography, and Sephadex G-100 gel filtration chromatography. The purified enzyme had a molecular weight of 35070 ± 400 Da and consisted of two equal-sized subunits each having a Cu and Zn element. Isoelectric point value of 7.0 was obtained for the purified enzyme. The N-terminal amino acid sequence of the purified enzyme was determined for 12 amino acid residues and the sequences was compared with other Cu, Zn-SODs. The optimum pH of the purified enzyme was obtained to be 8.2–8.8. The purified enzyme remained stable at pH 5.8–9.8, 25 °C and up to 50 °C at pH 7.8 for 1.5 h incubation. The purified enzyme was sensitive to H 2O 2, KCN. 2.5 mM NaN 3, PMSF, Triton X-100, β-mercaptoethanol and DTT showed no significant inhibition effect on the purified enzyme within 5 h incubation period.

Neuoscience: Molecular Pathology and Epilepsy

Abstract Yoshiya L. Murashima*, Jiro Suzuki*, and Mitsunobu Yoshii* *Department of Neural Plasticity, Tokyo Institute of Psychiatry, Tokyo, Japan . Purpose: We recently observed inducible nitric oxide synthase (iNOS) expression and decreased Cu, Zn-superoxide dismutase (Cu,Zn-SOD) activities in the hippocampus of epileptic mutant EL mice at age 30 weeks. In addition, DNA fragmentation was detected preferentially in region CA1 of the hippocampus and in the parietal cortex of the EL mouse brain. The results suggest that abnormalities in the region-specific iNOS isoenzyme activities and decreased Cu, Zn-SOD activities might produce spatially specific free radicals, leading to DNA fragmentation. However, neuronal cell loss is not found in the hippocampus. Furthermore, the immediate early gene (IEG), c-fos, also is expressed in the interictal period at age 30 weeks. These findings suggest that some protein cascades occur during epileptogenesis. In the present study, we used epileptic mutant EL mice to examine whether the altered equilibrium of the proapoptotic Bax and anti-apoptotic Bcl-2/Bcl-XL induces DNA fragmentation without cell loss. We also examined whether neurotrophic factors modulate the developmental process of epileptogenesis in EL mouse brain. Methods: Developmental changes of NOS isoenzymes were determined by using immunoblotting. Formalin-fixed mouse brains were stained by hematoxylin–eosin to examine cell loss in the tissue. IEG expression in the interictal period was analyzed with in situ hybridization by using the 35S x-ray emulsion method. Developmental changes of Bcl-2, Bcl-XL, and Bax were examined by immunoblotting in the hippocampus of EL mice compared with the control DDY mice. Alterations of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and fibroblast growth factor-2 (FGF-2) during development were investigated by immunoblotting. Results: Unexpectedly, the eNOS content in EL mouse brain was very low although eNOS appears to be responsible for NO that mediates an increase in local cerebral blood flow during focal seizures. Neuronal NOS, iNOS, and relatively lower concentration eNOS are essential to establish either ictogenesis or epileptogenesis. No cell loss was found in the hippocampus. Even in the interictal period, EL mice expressed the IEG, c-fos, continuously, preferentially in the parietal cortex and hippocampal CA1 when the seizure thresholds were very low. In the control DDY mice, very low steady-state levels of Bcl-2 and Bax were observed throughout development. In EL mice, the Bcl-2 and Bax levels increased predominantly after frequent seizures. The BDNF level in EL mice showed a significant increase during development, in the period of ictogenesis and epileptogenesis. Conclusions: The results indicate that in EL mice, the susceptibility of hippocampal neurons to DNA fragmentation increases after experiencing repetitive seizures during development. This effect probably is due to a change in the balance between the protective mechanism and inactivation of the proapoptotic pathway. BDNF may play a role in the ictogenesis and epileptogenesis by promoting abnormal synaptic plasticity.

Glutathione peroxidase, glutathione-S-transferase, catalase, xanthine oxidase, Cu–Zn superoxide dismutase activities, total glutathione, nitric oxide, and malondialdehyde levels in erythrocytes of patients with small cell and non-small cell lung cancer

Lung cancer is a common pathology with high mortality due to late diagnosis. Glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST), catalase (CAT), xanthine oxidase (XO), Cu–Zn superoxide dismutase (Cu–Zn SOD) activities, total glutathione (TGSH), nitric oxide (NO ), and malondialdehyde (MDA) levels were investigated in erythrocytes of patients with non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), and healthy control group. We aimed to investigate serum GSH, GSH-dependent enzymes activities (GSH-Px and GST), XO, CAT, Cu–Zn SOD activity, and NO , and MDA levels in patients with NSCLC and with SCLC and correlate with the cancer stage. Erythrocyte MDA, NO , TGSH levels and erythrocyte SOD, CAT and XO activities were significantly higher in patients with NSCLC and SCLC than in controls. Slightly increased erythrocyte GSH-Px and GST activities were not significantly different from the controls. Erythrocyte MDA level positively correlated with erythrocyte NO levels in patients with early stage (I+II) in NSCLC groups. Erythrocyte MDA level positively correlated with erythrocyte XO activity in patients with advanced stage (III+IV) in NSCLC groups. However, no other correlation could be found among the parameters in healthy controls and patients with NSCLC and with SCLC. Results obtained in this study indicate significant changes in antioxidant defence system in NSCLC and SCLC patients, which may lead to enhanced action of oxygen radical, resulting in lipid peroxidation.

A possible protective role of coenzyme Q10 on antioxidant defense system in the heart of rats treated with cadmium

The effect of cadmium (Cd), coenzyme Q10 (CoQ10) and Cd+CoQ10 on the activities of superoxide dismutases (total SOD), manganese containing superoxide dismutase (Mn SOD) and copper-zinc containing superoxide dismutase (Cu,Zn SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione-Stransferase (GST), glutathione reductase (GR) and concentrations of ascorbic acid (AsA) and vitamin E (Vit E) in the heart of male Wistar albino rats were studied in comparison to the controls and cadmium treated animals. Cd induces a significant increase of total SOD, Cu, Zn SOD and GSH-Px activities, as well as AsA and Vit E concentrations, but leads to a significant decrease of CAT and GR activities. CoQ10 induces a significant increase of total SOD, Mn SOD, Cu, Zn SOD and GSH-Px activities, as well as AsA and Vit E concentrations. In the same group of animals the activities of CAT, GST and GR were significantly decreased. By concomitant treatment of rats with Cd+CoQ10 the activities of total SOD, Mn SOD and GSH-Px, as well as concentrations of AsA and Vit E were markedly increased. In the same group of animals the activities of Cu, Zn SOD, CAT and GR were significantly decreased. In respect to the Cd treated rats in Cd+CoQ10 partially are reversed changes (Cu,Zn SOD) of antioxidant defense system in the heart.

Age-related changes of antioxidant enzyme activities, glutathione status and lipid peroxidation in rat erythrocytes after heat stress

The aim of this study was to determine whether exposure to heat stress would lead to oxidative stress and whether this effect varied with different exposure periods. We kept 1-, 6- and 12-month-old male Wistar rats at an ambient temperature of either 22 °C or 40 °C for 3 and 7 days and measured glucose-6-phosphate dehydrogenase (G-6-PD), Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase (CAT), selenium-dependent glutathione peroxidase (Se-GSH-Px) and glutathione-S-transferase (GST) activities and levels of thiobarbituric acid-reactive substances (TBARS), reduced glutathione (GSH) and oxidized glutathione (GSSG) in erythrocytes and determined GSH/GSSG ratio, total glutathione and the redox index. G-6-PD and CAT activities were found to be significantly increased in 1- and 6-month-old rats after 3 and 7 days of heat stress, but G-6-PD activities decreased in 12-month-old rats. Cu, Zn-SOD activity decreased in 1-month-old rats after heat stress, whereas it increased in 6- and 12-month-old rats. GST activity increased in all groups. GSH and total GSH levels and GSH/GSSG ratios decreased in 1- and 6-month-old rats but they increased in 12-month-old rats after heat stress. GSSG levels increased in 1- and 6-month-old rats but decreased in 12-month-old rats after heat stress. TBARS levels increased in all groups. Seven days of stress is more effective in altering enzyme activities and levels of GSH, GSSG and TBARS. When the effects of both heat stress and aging were examined together, it was interesting to note that they mostly influenced G-6-PD activity.

Differences in antioxidant enzyme activity in adverse drug reactions

Rationale In adverse drug reactions with an immunological basis (ADRIB) innate systems can also be activated, amplifying the specific response and making it more severe and long-lasting. Reactive oxygen species (ROS) are involved in inflammatory processes including allergic diseases. We studied differences in the in vivo antioxidant enzyme activities in patients with immediate, accelerated and delayed reactions to drugs. Methods Determinations of enzymatic activity of catalase, copper-zinc superoxide dismutase (Cu, Zn-SOD) and cellular glutathione peroxidase (cGPX), and oxidative damage (TBARS and carbonyl content) were undertaken in peripheral blood mononuclear cells using spectrophotometry. Results We evaluated 38 patients with ADRIB: 12 immediate, 8 accelerated and 18 delayed. Catalase and Cu, Zn-SOD activity were increased in patients with immediate and delayed reactions versus controls (p=0.011 and p=0.004 for catalase and p=0.002 and p=0.001 for Cu, Zn-SOD). However cGPX showed a significant decrease compared to controls in immediate, accelerated and delayed reactions (p=0.035, p=0.002 and p=0.002). The oxidative damage was significantly increased in TBARS and in carbonyl in all types of reaction. Conclusions An oxidative damage in ADRIB exists. Although there is a significant increase in catalase and superoxide dismutase in immediate and delayed reactions, the important decrease in all three groups of glutathione peroxidase seems to be related with the oxidative damage observed. Whether this is a primary drug effect in a subset of individuals or a consequence of the reaction is at present unknown.

Effects of octreotide on acute necrotizing pancreatitis in rabbits.

To assess the role of oxygen-derived free radicals and cytokines in the pathogenesis of taurocholic acid-induced acute pancreatitis, and to evaluate the preventive effects of octreotide towards the development of acute pancreatitis. Acute pancreatitis was induced in male New Zealand white rabbits by retrograde injection of 0.8 mL/kg.b.m. of 50 g/L sodium taurocholate (NaTC) in the pancreatic duct. Sham-operated animals served as control. Octreotide 1 mg/kg.b.m. was administered subcutaneously before the induction of pancreatitis. Blood was taken from the jugular vein before and at 1, 3, 6, 12 and 24 h after pancreatitis induction. Serum activities of amylase, IL-6 and TNF-alpha and levels of malonyl dialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GPx), catalase and superoxide dismutase (Mn-, Cu-, and Zn-SOD) in pancreatic tissue were measured. Serum TNF-alpha and IL-6 levels increased significantly 3 h after the onset of pancreatitis, and then returned to control level. The tissue concentration of MDA was significantly elevated at 24 h, while the GSH level and GP-x, catalase, Mn-SOD, Cu-, Zn-SOD activities were all significantly decreased in animals with pancreatitis as compared to the control. Octreotide pretreatment significantly reversed the changes in cytokines and reactive oxygen metabolites. Octreotide treatment did not alter the serum amylase activity and did not have any beneficial effects on the development of histopathological changes. Oxygen-derived free radicals and proinflammatory cytokines are generated at an early stage of NaTc-induced acute pancreatitis in rabbits. Prophylactic octreotide treatment can prevent release of cytokines and generation of reactive oxygen metabolites, but does not have any beneficial effects on the development of necrotizing pancreatitis.

Glutathione peroxidase, glutathione reductase, Cu–Zn superoxide dismutase activities, glutathione, nitric oxide, and malondialdehyde concentrations in serum of patients with chronic lymphocytic leukemia

Background: Chronic lymphocytic leukemia (CLL) is a rare neoplasm that comprises a substantial proportion of all leukemias in middle-aged persons and is the most common type among elderly persons. The major causes are not known nor is there a detailed understanding about how the elusive origin(s) may relate to clinical expression, basic biological mechanisms, or pathogenesis. Methods: Glutathione peroxidase (GSH-Px), glutathione reductase (GRD), Cu–Zn superoxide dismutase (Cu–Zn SOD) activities, glutathione (GSH), nitric oxide (NO ), and malondialdehyde (MDA) concentrations were measured in serum of patients with CLL and a healthy control group. Results: Serum GSH-Px, Cu–Zn SOD activities, GSH concentration were lower in patients with CLL while serum NO and MDA concentrations were higher in these patients compared with the control group. Serum GRD activity was not statistically significant in patients with CLL compared with the control. However, there was no statistically significant difference in the parameters on the basis of stages in these patients. Serum GSH concentration negatively correlated with serum MDA ( r=30.63, p<0.05) and NO concentrations ( r=0.72, p<0.05) in patients with advanced stage (III+IV). However, no other correlation could be found among the parameters in healthy controls and patients with CLL Conclusions: There is significant changes in antioxidant defense system in CLL cases, which may lead to enhanced action of oxygen radical, resulting in lipid peroxidation.

Replacement of a cytosolic copper/zinc superoxide dismutase by a novel cytosolic manganese superoxide dismutase in crustaceans that use copper (haemocyanin) for oxygen transport.

The blue crab, Callinectes sapidus, which uses the copper-dependent protein haemocyanin for oxygen transport, lacks the ubiquitous cytosolic copper-dependent enzyme copper/zinc superoxide dismutase (Cu,ZnSOD) as evidenced by undetectable levels of Cu,ZnSOD activity, protein and mRNA in the hepatopancreas (the site of haemocyanin synthesis) and gills. Instead, the crab has an unusual cytosolic manganese SOD (cytMnSOD), which is retained in the cytosol, because it lacks a mitochondrial transit peptide. A second familiar MnSOD is present in the mitochondria (mtMnSOD). This unique phenomenon occurs in all Crustacea that use haemocyanin for oxygen transport. Molecular phylogeny analysis suggests the MnSOD gene duplication is as old as the origin of the arthropod phylum. cytMnSOD activity in the hepatopancreas changes during the moulting cycle of the crab. Activity is high in intermoult crabs and non-detectable in postmoult papershell crabs. mtMnSOD is present in all stages of the moulting cycle. Despite the lack of cytCu,ZnSOD, crabs have an extracellular Cu,ZnSOD (ecCu,ZnSOD) that is produced by haemocytes, and is part of a large, approx. 160 kDa, covalently-linked protein complex. ecCu,ZnSOD is absent from the hepatopancreas of intermoult crabs, but appears in this tissue at premoult. However, no ecCu,ZnSOD mRNA can be detected, suggesting that the protein is recruited from the haemolymph. Screening of different taxa of the arthropod phylum for Cu,ZnSOD activity shows that those crustaceans that use haemoglobin for oxygen transport have retained cytCu,ZnSOD. It appears, therefore, that the replacement of cytCu,ZnSOD with cytMnSOD is part of an adaptive response to the dynamic, haemocyanin-linked, fluctuations in copper metabolism that occur during the moulting cycle of the crab.

Contribution of Xanthine Oxidase-Derived Oxygen Free Radicals to the Development of Carbon Tetrachloride-Induced Acute Liver Injury in Rats

We examined how oxygen free radicals derived from xanthine oxidase (XOD) contribute to the development of carbon tetrachloride (CCl4)-induced acute liver injury in rats. In rats treated with CCl4 (1ml/kg), liver injury appeared 3h after the treatment and developed by 24h, judged from the serum levels of transaminases. Hepatic and serum XOD activities and serum uric acid concentration did not change 6h after CCl4 treatment, but they all increased at 12h and further increased at 24h. An increase in the concentration of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, and decreases in reduced glutathione concentration and Cu, Zn-superoxide dismutase (SOD) activity, but not Mn-SOD activity, were observed in the liver at 6h after CCl4 treatment and these changes were further enhanced at 24h. When allopurinol (50mg/kg), an XOD inhibitor, was administered to CCl4-treated rats 6h after CCl4 treatment, liver injury progression was prevented with attenuation of increased hepatic and serum XOD activities, serum uric acid concentration, and hepatic TBARS concentration and decreased hepatic reduced glutathione concentration and Cu, Zn-SOD activity at 24h after CCl4 treatment. These results indicate that XOD-derived oxygen free radicals contribute to the progression of CCl4-induced acute liver injury in rats both by stimulating hepatic lipid peroxidation and by disrupting hepatic antioxidant defense system.

Red blood cell copper, zinc superoxide dismutase activity is higher in Alzheimer's disease and is decreased by d-penicillamine

Copper, zinc superoxide dismutase (Cu, Zn SOD) activity was measured in red blood cells (RBC) of 32 patients affected by Alzheimer's disease (AD), eight other AD patients treated with the copper-chelating agent d-penicillamine, 13 first-degree relatives and 22 controls. All AD patients enrolled in our study showed a higher level of Cu, Zn SOD activity early in the disease. No correlation between apolipoprotein E genotype and SOD activity was found in AD patients. d-penicillamine treatment of AD patients for 24 weeks lowered the enzyme activity even below the control value. These results support the hypothesis that a higher level of Cu, Zn SOD activity in RBC can be an early diagnostic peripheral marker of this disease and a sensor to monitor treatments with copper-chelating drugs.

The effects of acute melatonin and ethanol treatment on antioxidant enzyme activities in rat testes

The pineal hormone melatonin (N -acetyl, 5-methoxytryptamine) was recently accepted to act as an antioxidant under both in vivo and in vitro conditions. In this study, we examined the possible preventive effect of melatonin on ethanol-induced lipid peroxidation in rat testes. Thirty-seven male Wistar albino rats, 5.5–6 months old, were randomly divided into four groups (9–10 animals in each). The first group (control animals) received 4% ethanol at similar intervals to the experimental groups to equalize the stress effect. The second group received only melatonin i.p. 7 mg kg−1bw three times over 1.5 h intervals. The third group received only 30% alcohol 3 g kg−1bw twice daily. The fourth group were treated with melatonin and ethanol according to the above protocole, melatonin injections preceeding ethanol treatments. The product of lipid peroxidation, malondialdehyde (MDA) and antioxidant enzymes, superoxide dismutase (Cu–Zn SOD), glutathione peroxidase (GPx) and catalase (CAT) were measured in the post-mitochondrial fraction of the testes. MDA levels were significantly increased due to acute ethanol intoxication. GPx activity was higher in the three experimental groups than the control levels. The activity of CAT was increased significantly in the melatonin plus ethanol-treated group but the other groups appeared not to be influenced by acute ethanol treatment. Cu–Zn SOD activity remained unaltered. These results suggest that antioxidants may be a protective agent for the testicular injury caused by ethanol consumption.

UPREGULATION OF ANTIOXIDANT CAPACITY BY ENDURANCE TRAINING IN PIGS FOLLOWING CHRONIC CORONARY ARTERY OCCLUSION

Growing evidence indicates that oxidative stress is associated with muscle fatigue and weakness in skeletal muscle of heart disease patients. Previously, our laboratory and others demonstrated that reactive oxygen species (ROS) and reactive nitrogen species (RNS) have the ability to inhibit key antioxidant enzymes in skeletal muscle, thus increasing susceptibility to oxidative stress. Endurance training has been shown to elevate skeletal muscle mantioxidant enzyme activities in healthy animal models. Chronic coronary artery occlusion in pigs was used to model heart disease and to test the following hypothesis: twelve weeks of treadmill endurance training will increase the antioxidant capacity in skeletal muscle in pigs with chronic coronary occlusion. Miniature Yucatan swine were divided into three groups: sedentary animals (n = 6) with coronary artery occlusion (SO); endurance trained animals (n = 6) with coronary occlusion (EO); and sedentary (n = 3) without coronary occlusion (CON). Skeletal muscle samples (triceps brachii) were extracted after a 12 week training period, and frozen in liquid nitrogen until analysis. Superoxide dismutase (total SOD, Mn-SOD isoform, and Cu, Zn-SOD isoform), glutathione peroxidase (GPX), and catalase (CAT) activities were measured. The quenching of ABTS+ radical was used as a marker of non-enzymatic antioxidant scavenging capacity (TASC). Total SOD, Mn-SOD, and Cu, Zn-SOD activities were significantly higher in EO than SO by 82.6%, 72.0%, and 112.0%, respectively. GPX activity was 176.6% greater in EO than in SO. CAT trended higher (P = 0.059) in EO when compared to SO. TASC was also significantly improved (12.7%) by exercise training over SO. All antioxidant measurements for EO were not different from CON, while SO values were lower than CON. These data indicate that endurance exercise training is critical for the protection of antioxidant capacity in pigs with chronic coronary occlusion. Grant support by NIH PO1-HL52490 and HL64931.

Flow injection-chemiluminescent assay for the determination of superoxide dismutase activity

Superoxide dismutase (SOD) activity was measured using a flow injection-chemiluminescent assay (FI-CLA) based on xanthine–xanthine oxidase dependent superoxide (O2.–) formation. The mobile phase consists of 50 mM potassium phosphate buffer (pH 7.4) containing lucigenin (5 µM) and xanthine (0.3 mM). Under optimum conditions, bovine albumin did not affect chemiluminescence at concentrations of 1–1000 µg mL–1and KCN inhibited 100% of the Cu, Zn-SOD activity using 5 µL of a 0.23 mM concentration. The analysis of one sample was done in less than 30 s with a relative standard deviation of ±3.1%. SOD activity in the biological samples was correlated to the amount of exogenously applied SOD. The FI-CLA method reported here appears to be one of the faster and more useful tools used to assay SOD activity.Key words: superoxide dismutase, flow injection-chemiluminescent assay, superoxide, xanthine oxidase, lucigenin.

Cu–Zn SUPEROXIDE DISMUTASE INHIBITS LACTATE DEHYDROGENASE RELEASE AND PROTECTS AGAINST CELL DEATH IN MURINE FIBROBLASTS PRETREATED WITH ULTRAVIOLET RADIATION

The effects of adding Cu–Zn superoxide dismutase (Cu–Zn SOD) to culture medium of the murine fibroblast cell line, L-929, pretreated with UV-B (312nm, 480mJ/cm2) have been investigated. Cell injury was monitored by the release of lactate dehydrogenase (LDH) into the medium, and cell death by the trypan blue exclusion test. UV-B radiation induced cell death by apoptosis, as demonstrated by DNA fragmentation. Over the range 0.1–0.3μm Cu–Zn SOD, a significant dose-dependent protection against cell death was obtained of the UV-B exposed cells. Cell death correlated with the amount of LDH released into the medium, and Cu–Zn SOD treatment inhibited this. Heat-denatured Cu–Zn SOD did not affect either cell viability or the release of LDH from the cells. Endogenous Cu–Zn SOD activity, monitored by chemiluminescence, decreased by 20% in UV-B-irradiated cells; the addition of 0.3μm exogenous Cu–Zn SOD to the medium did not affect intracellular Cu–Zn SOD activity. These results establish that Cu–Zn SOD added to extracellular medium can protect cells against injury caused by UV-B exposure.

Free radicals antioxidant enzymes and lipid peroxidation in different types of leukemias

Free radicals are highly reactive species that have been implicated in the pathogenesis of many diseases. Reactive oxygen species can initiate lipid peroxidation and DNA damage leading to mutagenesis, carcinogenesis and cell death, if the antioxidant system is impaired. This study was undertaken to examine the prevalence of oxidative stress and the role of antioxidant defence in untreated leukemia patients. The generation of superoxide anion and hydrogen peroxide by leukocytes, plasma malondialdehyde levels, red cell copper zinc superoxide dismutase (Cu–Zn SOD) and glutathione peroxidase (GSH-PX) activities were determined in 30 patients with different types of leukemias prior to therapy. The superoxide anion generation by polymorphonuclear leukocytes was found to be significantly increased in leukemia patients especially those with acute lymphocytic and nonlymphocytic leukemias, while the hydrogen peroxide levels were comparable to the control values. Plasma lipid peroxidation products in untreated leukemia patients were in the normal range. Red cell Cu–Zn SOD and GSH-PX activities were significantly increased and showed no correlation with the hemoglobin content. Although superoxide generation was high, lipid peroxide levels were normal in these patients. This might be due to the increased activities of the antioxidant enzymes (SOD, GSH-PX) which counteract lipid peroxidation. Increased free radical generation, especially superoxide anion in leukemia patients and increased antioxidant defence enzymes, which is an adaptive protective response, are indicative of mild oxidative stress. There were no significant differences for the parameters cited above between different types of leukemias, suggesting that the changes are not specific to the type of leukemia.