High Concentrations Of Superoxide Dismutase Research Articles

Coating shiitake mushrooms (Lentinus edodes) with a polysaccharide from Oudemansiella radicata improves product quality and flavor during postharvest storage

In this work, we investigated whether coating fresh shiitake mushrooms with a polysaccharide isolated from Oudemansiella radicata (ORWP) would impact key quality characteristics after 18 d of storage at 4 °C. We found that ORWP-coated mushrooms had significant improvements in many qualities during storage, including reduced weight loss, improved firmness, reduced browning, decreased malondialdehyde content, and an improved physical microstructure. Further, ORWP-coated mushrooms had higher contents of nutritional and cell wall compounds compared to control samples. ORWP-coated mushrooms had reduced activities of the following enzymes: protease, polyphenol oxidase, peroxidase, phenylalanine ammonia lyase, cellulase, and chitinase, relative to control samples. However, mushrooms coated with ORWP had higher concentrations of superoxide dismutase and catalase, as well as higher contents of certain key monosodium glutamate-resembling amino acids, umami 5′-nucleotides and 1-octen-3-ol. These findings suggest that ORWP coatings have potential value as a method to improve the postharvest quality of shiitake mushrooms.

Watermelon and l-arginine consumption improve serum lipid profile and reduce inflammation and oxidative stress by altering gene expression in rats fed an atherogenic diet

Watermelon (Citrullus lanatus) is rich in l-citrulline, an l-arginine precursor that may reduce cardiovascular disease risk. The purpose of this study was to compare the effects of watermelon powder and l-arginine on lipid profiles, antioxidant capacity, and inflammation in rats fed an atherogenic diet. We hypothesized that watermelon and l-arginine would increase antioxidant capacity and reduce blood lipids and inflammation by modulating hepatic gene expression. Male Sprague-Dawley rats aged 21 days (N = 32) were assigned to 3 groups and fed diets containing watermelon powder (0.5% wt/wt), l-arginine (0.3% as 0.36% l-arginine HCl wt/wt), or a control diet for 9 weeks. Watermelon and l-arginine supplementation improved lipid profiles by lowering serum concentrations of triglycerides, total cholesterol, and low-density lipoprotein cholesterol (P < .050). Serum concentrations of C-reactive protein were significantly lower (P < .050) in the watermelon and l-arginine groups. Rats in the watermelon and l-arginine groups showed reduced oxidative stress, increased total antioxidant capacity, and higher concentrations of superoxide dismutase and glutathione S-transferase (P < .050). Concentrations of aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase were lower (P < .050) in the watermelon and l-arginine groups. Watermelon and l-arginine consumption upregulated hepatic gene expression of endothelial nitric oxide synthase and downregulated expression of fatty acid synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, sterol regulatory element-binding protein 1, sterol regulatory element-binding protein 2, cyclooxygenase-2, and nuclear factor–κB p65 (P < .050). The results support the hypothesis that watermelon and arginine improve cardiovascular disease risk factors including lipid profile, antioxidant capacity, and inflammation by altering relevant gene expression.

Oxidative stress in drug-naïve first episode patients with schizophrenia and major depression: effects of disease acuity and potential confounders.

Oxidative stress and immune dysregulation have been linked to schizophrenia and depression. However, it is unknown whether these factors are related to the pathophysiology or whether they are an epiphenomenon. Inconsistent oxidative stress-related findings in previous studies may have resulted from the use of different biomarkers which show disparate aspects of oxidative stress. Additionally, disease severity, medication, smoking, endocrine stress axis activation and obesity are potential confounders. In order to address some of these shortcomings, we have analyzed a broader set of oxidative stress biomarkers in our exploratory study, including urinary 8-iso-prostaglandin F2α (8-iso-PGF2α), 8-OH-2-deoyxguanosine (8-OH-2-dG), and blood levels of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione S-transferase (GST) in acutely ill drug-naïve first episode patients with schizophrenia (n=22), major depression (n=18), and controls (n=43). Possible confounding factors were considered, and patients were followed-up after 6weeks of treatment. No differences were observed regarding 8-OH-2-dG, MDA and GST. At baseline, 8-iso-PGF2α levels were higher in patients with schizophrenia (p=0.004) and major depression (p=0.037), with a trend toward higher SOD concentrations in schizophrenia (p=0.053). After treatment, schizophrenia patients showed a further increase in 8-iso-PGF2α (p=0.016). These results were not related to age, sex, disease severity, medication or adipose tissue mass. However, 8-iso-PGF2α was associated with smoking, endocrine stress axis activation, C-reactive protein levels and low plasma concentrations of brain-derived neurotrophic factor. This study suggests a role of lipid peroxidation particularly in drug-naïve acutely ill schizophrenia patients and highlights the importance of taking into account other confounding factors in biomarker studies.

Antioxidants and biomarkers of oxidative damage in the saliva of patients with Down's syndrome

BackgroundThe aim of this study was to investigate enzymatic and non-enzymatic antioxidant systems and levels of biomarker levels of oxidative damage in the saliva of patients with Down's syndrome (DS). MethodsSaliva samples were collected from 30 patients with DS and control group (age: 14–24 years). Subsequently, the concentrations of superoxide dismutase, concentration of malondialdehyde, carbonylated proteins, uric acid, vitamin C and total protein, peroxidase activity and total antioxidant capacity were analyzed. ResultsPatients with DS presented significantly higher concentrations of superoxide dismutase, higher levels of malondialdehyde and salivary total protein content than controls (p<0.05). Conversely, no difference in carbonylated proteins or antioxidants (uric acid, vitamin C, peroxidase, and total antioxidant capacity) was observed between DS patients and controls (p>0.05). ConclusionPatients with DS are more vulnerable to oxidative stress in saliva as indicated by the significant increase in malondialdehyde and superoxide dismutase concentrations found in this study.

Human colostral phagocytes eliminate enterotoxigenic Escherichia coli opsonized by colostrum supernatant

Several elements in colostrum and human milk, including antibodies and nonspecific factors with bactericidal and antiviral activity, may play an important anti-infectious and protective role. In developing countries, enterotoxigenic Escherichia coli (ETEC) is the main etiological agent of diarrhea in low-socioeconomic level children. In the present work, we studied the functional activity of mononuclear (MN) and polymorphonuclear (PMN) phagocytes of human colostrum against ETEC, as well as the interactions between these cells and colostral or serum opsonins. Colostrum samples were collected from 33 clinically healthy women between 48 and 72 hours postpartum. We verified superoxide release in colostral MN and PMN using cytochrome C reduction methods, phagocytosis, and bactericidal activity using acridine orange methods and superoxide dismutase (SOD) in the colostrum supernatants. Colostral MN and PMN phagocytes exposed to ETEC opsonized with colostrum supernatants caused a significant increase (p<0.05) in superoxide release. Phagocytosis by colostral PMN cells increased significantly (p<0.5) when the phagocytes were incubated with both sources of opsonins (sera and colostrum). Increases in superoxide release in the presence of opsonized bacteria triggered the bactericidal activity of the phagocytes. Phagocyte treatment with SOD decreased their ability to eliminate ETEC. Colostrum supernatant had higher SOD concentrations (p<0.05) compared with normal human sera. These results suggest that the ability of phagocytes to eliminate ETEC depends on the activation of cellular oxidative metabolism; moreover, activation of colostral phagocytes is likely an additional breast-feeding protection mechanism against intestinal infections in infants.

Hemodynamic Forces-Induced Biochemical Changes in Aortic wall: Effect on Redox State of the Tissue

Shear stress and hydrostatic pressure-induced stretch are known to enhance the production of free radicals, causing oxidative stress in the vascular wall and have been implicated in endothelial inflammation and vascular lesions. Production of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and catalase probably constitute a key factor in maintaining the redox state of arterial wall in response to hemodynamic forces. In the present study, redox state of arterial wall at constant pulsatile/laminar shear and varying hydrostatic pressure (70 and 150 cm water) was evaluated. The aim was to study the pressure, in combination with different flow patterns (laminar/pulsatile shear stress), induced oxidative stress which presumably cause arterial wall inflammation leading to vascular legions. The level of reduced glutathione (GSH), lipid peroxidation (LPO) and enzymes known to contribute to the redox status of the cell/tissue, have been measured. Present study quantitatively evaluates the hemodynamic forces induced vascular oxidative stress. We individually assessed the activity of Superoxide Dismutase (SOD), catalase, glutathione peroxidase (GPx), xanthine oxidase (XO), and glutathione-s-transferase (GST) under hemodynamic stress. Secondary antioxidant enzymes like glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) were also measured. The increased oxidative stress under pulsatile shear stress and high hydrostatic pressure potentially suggests that vascular inflammation is the key to understand initiation of vascular lesions. Higher concentrations of SOD in arterial wall ought to be considered since this explains arterial wall antioxidant defense in vascular pathologies, which potentially involve oxidative stress. In conclusion, pulsatile shear stress in combination with hydrostatic pressure is a weak inducer of antioxidant defense in blood vessels, hence, considered to be atherogenic. The finding is relevant to both the normal and pathophysiologically relevant hemodynamically stress rabbit thoracic aorta.

Limb Ischemic Preconditioning Reduces Heart and Lung Injury After an Open Heart Operation in Infants

Open heart surgery supported by cardiopulmonary bypass is associated with heart and lung ischemia-reperfusion injury (IRI). Limb remote ischemic preconditioning (RIPC) reduces injury caused by ischemia-reperfusion in multiple distant organs. We conducted a prospective clinical trial (randomized and controlled) to test the feasibility and safety of limb RIPC, as well as its protective effects against myocardial and pulmonary IRI for infants undergoing repair of simple congenital heart defects. Infants undergoing repair of ventricular septal defects were enrolled in our study and randomly assigned to one of two treatment groups: limb RIPC or control. RIPC was induced twice (24 h and 1 h preoperatively) via three 5-min cycles of ischemia and reperfusion on the left upper arm using a blood pressure cuff. Lung compliance, respiratory index (RI), and cardiac inotropic score (IS) were calculated for each patient. Serum concentrations of the following factors were measured perioperatively: interleukin (IL)-6, IL-8, IL-10, and tumor necrosis factor (TNF)-alpha; lactate dehydrogenase (LDH), creatine kinase (CK), and its isoenzyme (CK-MB), and troponin I (TnI); malondialdehyde (MDA) and superoxide dismutase (SOD). The expression of heat shock protein 70 (HSP 70) in cardiomyocytes was analyzed by Western blot. Surgical outcomes, including limb movement and sensory function, were recorded in detail. Sixty infants weighting less than 7 kg were studied, with 30 patients in the RIPC group and 30 in the control group. Within 6 months of discharge from the hospital, no limb disability, sensory disturbance, or other surgical complications were found in any patient. Compared with the control group, patients in the RIPC group had higher Cs and Cd, along with lower RI and IS at various postoperative phases. At the beginning of the operation, serum concentrations of IL-6, IL-8, IL-10, TNF-alpha, LDH, CK, and TnI were higher in the RIPC group than the control group. Postoperatively, release of cytokines and leakage of heart enzymes were attenuated in the RIPC group; serum concentrations of cytokines and heart enzymes were lower in the RIPC group at some, but not all, postoperative time points. Furthermore, the RIPC group had lower coronary sinus venous concentrations of MDA and higher concentrations of SOD. Similarly, the expression of HSP 70 was upregulated in cardiomyocytes from the RIPC group. Limb RIPC can be applied safely and easily in infants, can attenuate systemic inflammatory response syndrome, and can increase systemic tolerance to IRI, imparting a protective effect against myocardial and pulmonary IRI. The expression of HSP 70 has an important role in the mechanism of action for RIPC.

Comparison of the effects of the superoxide dismutase mimetics EUK-134 and tempol on paraquat-induced nephrotoxicity

Paraquat-induced nephrotoxicity involves severe renal cell damage caused by reactive oxygen species (ROS), specifically via increasing concentrations of superoxide anions in the kidney. Recently, superoxide dismutase (SOD) mimetics (SODm) have been developed that display safe SOD activities but which also possess additional antioxidant enzyme (e.g., catalase) or ROS-scavenging activities. The aim of this study was to compare the effects of two such SODm, specifically, EUK-134, a SODm with catalase activity, and tempol, a SODm with ROS-scavenging properties, on paraquat-induced nephrotoxicity of renal NRK-52E cells. Incubation with paraquat (1 mM) for 24 h reduced cell viability and increased necrosis significantly. Paraquat also generated significant quantities of superoxide anions and hydroxyl radicals. Both EUK-134 (10–300 μM) and tempol (0.3–1.0 mM) were able to improve cell viability and reduced paraquat-induced cell death significantly via dismutation or scavenging of superoxide anions and reduced hydroxyl radical generation. The data presented here suggest that SODm such as EUK-134 and tempol, which possess additional catalase and/or ROS-scavenging activities, can significantly reduce renal cell damage caused by paraquat. These effects were evident at concentrations which avoid the pro-oxidant activities associated with higher concentrations of SOD. Such SODm could therefore prove to be beneficial as therapies for paraquat nephrotoxicity.

Alternative pathways might mediate toxicity of high concentrations of superoxide dismutase.

One of the most important antioxidant enzymes is superoxide dismutase (SOD), which catalyzes the dismutation of superoxide radicals to peroxide. The gene for CuZnSOD lies in humans on chromosome 21, and its activity is increased in patients with Down syndrome. However, instead of being beneficial, increased lipid peroxidation is associated with this increased expression, and also studies on bacteria and transgenic animals show that high levels of SOD actually lead to increased lipid peroxidation and hypersensitivity to oxidative stress. Using mathematical models, we investigated the question of how overexpression of SOD can lead to increased oxidative stress, although it is an antioxidant enzyme. We considered several possibilities that have been proposed in the literature, such as CuZnSOD-catalyzed hydroxyl radical formation, superoxide-mediated inhibition of membrane peroxidation, and short-circuiting of the Cu(I)ZnSOD/Cu(II)ZnSOD redox cycle. We found that one of the proposed mechanisms under certain circumstances is able to explain the increased oxidative stress caused by SOD. Furthermore, we identified an additional mechanism that agrees well with experimental observations. We call it the "alternative pathway" mechanism, because it depends on superoxide radicals having alternative pathways besides their reaction with SOD. The alternative pathway mechanism is a very general explanation for SOD-associated oxidative stress, because it does not depend on the specific type of SOD, nor on the redox status of the cell. We therefore think that it might be the common mechanism for the detrimental effects seen in cells and organisms with increased levels of the different forms of superoxide dismutase.

Turning a Daf Ear to Stress

NOVATO, CALIFORNIA --In the life-extension Olympics, nematodes have bragging rights. Unlike the oldest rodents, which can survive only about 50% longer than normal, some mutant worms have doubled life-spans. Many observations have hinted that mammals can benefit from perturbations in the same genes that confer exceptionally long life on worms, but no one had directly tested that idea before. Now, a study presented here 25 September 2002 at the Buck Institute Symposium on Neuroendocrine Systems and Lifespan Determination reveals that a life-extending worm mutation helps mice fight stress. Unfortunately for the resilient mice, the mutation also elicits the beginning stages of diabetes. Some long-lived nematodes harbor mutations that quench a molecular cascade that resembles the insulin and insulin-like growth factor-1 (IGF-1) pathways in mammals. One such mutation is in the daf-2 gene, which encodes the cell surface receptor that transmits the worm's hormonal signal. Some studies suggest that crippling the IGF-1 pathway could delay the demise of rodents as well: Dwarf mice with mutations that stem the production of growth hormone--which sparks the production of IGF-1--have extended life-spans (see Bartke Viewpoint ). But the effects of insulin and IGF-1 on mammalian longevity remain fuzzy, because other hormone pathways are perturbed in the dwarf mice and because insulin and IGF-1 modulate so many processes, such as metabolism and bone and muscle growth. To elucidate the similarities between the putative aging pathways in worms and mammals, Shirasawa and colleagues engineered mice to carry a mutation in the insulin-receptor gene that is equivalent to the life-extending mutation in daf-2 . Animals carrying two altered copies of the insulin receptor died shortly after birth from a diabetes-like imbalance in fat metabolism; those with only one altered copy survived, although these rodents carried elevated concentrations of insulin in their blood, which is an early sign of diabetes. The animals didn't develop the full-blown disease, however: Their blood glucose concentrations remained normal. Long-lived daf-2 mutant worms withstand stress better than normal worms do, so the team investigated whether the mutant mice had similar powers. In 80% oxygen--an environment that inflicts oxidative damage on tissues--mice with the daf-2 -like mutation survived longer than control animals did. The altered mice also held up unusually well to bleomycin, a chemical that normally induces an age-related disease called lung fibrosis. Compared with controls, the lungs of the chemically treated mutants carried few inflammatory cells and high concentrations of superoxide dismutase (SOD), an enzyme that defuses potentially damaging oxygen-containing compounds. Curiously, amounts of inflammation-stimulating compounds called interleukins rose in the lungs of the mutants; why these animals show fewer signs of inflammation despite having elevated quantities of interleukin isn't clear. "It's an important study," says physiologist Andrzej Bartke of Southern Illinois University in Springfield. It strengthens the idea that "controlling insulin signaling may be critical in controlling aging" in mice and perhaps humans, he says. If researchers can tinker with the pathway without interfering with insulin's many beneficial contributions, perhaps worms won't be the only ones bragging. --R. John Davenport T. Shirasawa, Insulin signal pathways and resistance for oxidative stress in mammals. Buck Institute Symposium 2002: Neuroendocrine Systems and Lifespan Determination, 24 to 27 September 2002, Novato, California. [Symposium Home Page]

Electron-paramagnetic resonance spectroscopy using N-methyl- d-glucamine dithiocarbamate iron cannot discriminate between nitric oxide and nitroxyl: implications for the detection of reaction products for nitric oxide synthase

Purified neuronal nitric oxide synthase (NOS) does not produce nitric oxide (NO) unless high concentrations of superoxide dismutase (SOD) are added, suggesting that nitroxyl (NO −) or a related molecule is the principal reaction product of NOS, which is SOD-dependently converted to NO. This hypothesis was questioned by experiments using electron paramagnetic resonance spectroscopy and iron N-methyl- d-glucamine dithiocarbamate (Fe-MGD) as a trap for NO. Although NOS and the NO donor S-nitroso-N-acetyl-penicillamine produced an electron paramagnetic resonance signal, the NO − donor, Angeli’s salt (AS) did not. AS is a labile compound that rapidly hydrolyzes to nitrite, and important positive control experiments showing that AS was intact were lacking. On reinvestigating this crucial experiment, we find identical MGD 2-Fe-NO complexes both from S-nitroso-N-acetyl-penicillamine and AS but not from nitrite. Moreover, the yield of MGD 2-Fe-NO complex from AS was stoichiometric even in the absence of SOD. Thus, MGD 2-Fe directly detects NO −, and any conclusions drawn from MGD 2-Fe-NO complexes with respect to the nature of the primary NOS product (NO, NO −, or a related N-oxide) are invalid. Thus, NOS may form NO − or related N-oxides instead of NO.

Synergistic induction of DNA strand breakage by cigarette tar and nitric oxide.

Cigarette smoking is a major cause of human cancer at a variety of sites, although its carcinogenic mechanisms remains unestablished. Cigarette smoke can be divided into two phases, gas phase and particulate matter (tar). Both phases contain high concentrations of oxidants and free radicals, especially nitric oxide (NO) and nitrogen oxides in the gas phase and quinone/hydroquinone complex in the tar. We have found that incubation of pBR322 plasmid DNA with aqueous extracts of cigarette tar and a NO-releasing compound (diethylamine NONOate) caused synergistic induction of DNA single-strand breakage, whereas either cigarette tar alone or NO alone induced much less strand breakage. This synergistic effect of cigarette tar and NO on DNA strand breakage was prevented by high concentrations of superoxide dismutase, carboxy-PTIO (an NO-trapping agent) or N-acetylcysteine, whereas hydroxyl radical scavengers such as dimethylsulfoxide, ethanol and D-mannitol did not show inhibitory effects. Possible mechanisms for this synergistic effect mediated by cigarette tar and NO are proposed, including involvement of peroxynitrite, which is a strong oxidant and nitrating agent formed rapidly by the reaction between NO and O2.-. NO is present in the gas phase of smoke and may be formed by a constitutive or inducible NO synthase in the lung, whereas O2.- is generated by auto-oxidation of polyhydroxyaromatic compounds such as catechol and 1,4-hydroquinone present in cigarette tar. Thus, potent reactive species including peroxynitrite formed by the interaction between cigarette tar and NO may play an important role in smoking-related diseases including lung cancer.

Evidence for sulphite oxidase activity in spinach leaves

The present paper provides evidence that spinach chloroplasts possess a sulphite oxidase activity coupled with oxygen consumption and reduction of ferricyanide. This activity is associated with thylakoids and solubilized by non-ionic biological detergents. The pH and temperature dependencies of sulphite oxidase activity solubilized by Triton X-100 from spinach thylakoids were consistent with those of an intrinsic membrane protein. This isolated activity was insensitive towards radical scavengers (mannitol, mannose and fructose) and catalase, and was inhibited only with very high concentrations of superoxide dismutase. Thus, observed sulphite oxidation was not induced through the photosynthetic electron transport system, but achieved via a thylakoid membrane enzymic system showing a sulphite oxidase activity. Kinetic parameters of thylakoid sulphite oxidase were measured and compared with those of other sulphite oxidases.

The differential degradation of two cytosolic proteins as a tool to monitor autophagy in hepatocytes by immunocytochemistry.

The major pathway for cytosolic constituents to enter lysosomes is by autophagy. We used two cytosolic proteins, CuZn superoxide dismutase (SOD) and carbonic anhydrase III (CAIII), as autophagic markers in male rat hepatocytes. We took advantage of the differential presence of the two proteins in autophagic vacuoles because of the high resistance of SOD to lysosomal degradation as compared with CAIII. This allows us to determine the sequence of autophagic vacuole formation. We have double immunogold-labeled SOD and CAIII in cryosections of fasted rat liver and calculated the ratios of SOD over CAIII labeling densities (SOD/CAIII) in autophagic vacuoles (AV), as compared with the cytoplasm. Different classes of AV were defined according to their SOD/CAIII, their morphology, and their additional immunolabeling for the lysosomal markers lgp120 and cathepsin D. Of all AV, 15% exhibited a cytosol-like SOD/CAIII, indicating that degradation had not yet begun. Most of these initial AV (AVi) showed two enveloping membranes. The formation of AVi was prevented by 3-methyladenine, a potent inhibitor of autophagy. Of all AV, 85% showed a SOD/CAIII that exceeded the cytosolic ratio. These single membrane-bound vacuoles were called degradative AV (AVd). Labeling for lysosomal markers allowed the characterization of AV that shared features with both AVi and AVd. These AVi/d had a cytosol-like SOD/CAIII and a double membrane, but showed some labeling for lysosomal markers. Probably these AVi/d represent the recipient compartment for lysosomal components. AVd were positive for cathepsin D and lgp120. We discerned two AVd subclasses. Early AVd with cytosol-like SOD labeling density while CAIII labeling density was consistently lower than in the cytosol. Their size was similar to AVi and AVi/d. Late AVd contained higher SOD concentrations and were mostly larger. Our findings suggest that AV acquire lysosomal constituents by fusion with small nonautophagic structures and that after subsequent elimination of the inner membrane of AVi, degradation starts resulting in the formation of early AVd and late AVd.

Effects of superoxide dismutase on development following the implantation stage in mice.

To examine the effects of superoxide dismutase on embryonic development after the implantation stage, mouse blastocysts were cultured under low-(5%), standard- (20%), and high-oxygen (40%) conditions with or without superoxide dismutase (SOD), which is a potent scavenger of superoxide radicals. Under 5% O2 conditions, the rates of formation of two germ cell layers and egg cylinder were 61.5% and 43.6%, respectively. These rates did not differ significantly from values determined in embryos cultured under 20% O2 conditions (69.7% and 51.3%, respectively). However, under 40% O2 conditions, the respective two germ cell layers and egg cylinder rates of formation were significantly lower than those under 5% and 20% O2 conditions (31.7% and 27.2%). Addition of 5μg/ml SOD resulted in no significant difference in formation rates of two germ cell layers (71.3%) and egg cylinder (52.9%) from those of the SOD-free group (61.3% and 45.2%). However, both rates were significantly decreased at SOD concentrations of 10μg/ml or greater. Microscopic examination of the morphology of cultured embryos showed that the degeneration of the inner cell mass (ICM) occurred follwing its migration from the trophetoderm and direct exposure to medium supplemented with 500μg/ml SOD. These results indicate that excessive oxidative stress exerts adverse effects on embryonic development after implantation. Embryos which are exposed to such stress after the implantation stage are in turns more resistant to these effects than those observed at the pronuclear stage, which is known to proliferate under 5% O2 conditions. Moreover, it is suggested that higher concentrations of SOD in the medium may diminish the concentration of superoxide anion radicals in ICM during differentiation, thus causing the degeneration of ICM.

Superoxide dismutase is preferentially degraded by a proteolytic system from red blood cells following oxidative modification by hydrogen peroxide

The cupro-zinc enzyme superoxide dismutase (SOD) undergoes an irreversible (oxidative) inactivation when exposed to its product, hydrogen peroxide (H 2O 2). Recent studies have shown that several oxidatively modified proteins (e.g., hemoglobin, albumin, catalase, etc.) are preferentially degraded by a novel proteolytic pathway in the red blood cell. We report that bovine SOD is oxidatively inactivated by exposure to H 2O 2, and that the inactivated enzyme is selectively degraded by proteolytic enzymes in cell-free extracts of bovine erythrocytes. For example, 95% inactivation of SOD by 1.5 mM H 2O 2 was accompanied by a 106 fold increase in the proteolytic susceptibility of the enzyme during (a subsequent) incubation with red cell extract. Both SOD inactivation and proteolytic susceptibility increased with H 2O 2 concentration and/or time of exposure to H 2O 2. Pre-incubation of red cell extracts with metal chelators, serine reagents, or sulfhydryl reagents inhibited the (subsequent) preferential degradation of H 2O 2-modified SOD. Furthermore, a slight inhibition of degradation was observed with the addition of ATP. We suggest that H 2O 2-inactivated SOD is recognized and preferentially degraded by the same. ATP-independent, metallo- serine- and sulfyhydryl- proteinase pathway which degrades other oxidatively denatured red cell proteins. Related work in this laboratory suggests that this novel proteolytic pathway may actually consist of a 700 kDa enzyme complex of proteolytic activities. Mature red cells have no capacity for de novo proteins synthesis but do have extremely high concentrations of SOD. Red cell SOD generates (and is, therefore, exposed to) H 2O 2 on a continuous basis, by dismutation of superoxide (from hemoglobin autooxidation and the interaction of hemoglobin with numerous xenobiotics). We suggest that preferential degradation of H 2O 2-modified SOD may protect erythrocytes against the accumulation of oxidatively denatured SOD aggregates, and potentially toxic protein fragments.

2,6-Dichlorophenolindophenol-reducing activity of phagocytosis-associated NADPH oxidase.

The 2,6-dichlorophenolindophenol (DCIP)-reducing activity of the phagocytosis-associated NADPH oxidase was investigated using homogenates and a membrane fraction (F2) of elicited guinea pig peritoneal macrophages stimulated by phorbol myristate acetate. Essentially all of the stimulation-specific DCIP reduction under aerobic conditions could be inhibited when high concentrations of superoxide dismutase (SOD), about 10 times those usually used to inhibit the superoxide (O-2)-mediated cytochrome c reduction, were used. SOD inhibited the DCIP reduction by chemically generated O2- in the same manner as the stimulation-specific DCIP reduction by the macrophage F2, and the concentration of SOD necessary for 50% inhibition was about 10 times that for the reduction of cytochrome c. Under anaerobic conditions, however, the NADPH oxidase could reduce DCIP, though the rate was slow because we could not use a sufficiently high DCIP concentration. The observations indicate that the NADPH oxidase preferentially reduces oxygen under aerobic conditions, though the oxidase can reduce DCIP in the anaerobic state.

The superoxide dismutase activity of myeloperoxidase; formation of Compound III

The reaction of superoxide anions with myeloperoxidase (donor:hdyrogen-peroxide oxidoreductase, EC 1.11.1.7), which results in the formation of Compound III of myeloperoxidase, was investigated. It is shown that myeloperoxidase has a high affinity for superoxide anions because formation of Compound III was only partially inhibited by high concentrations of superoxide dismutase. Furthermore, when superoxide anions were generated in a mixture of both cytochrome c and mycloperoxidase in the absence of Cl −, only Compound III was formed and reduction of cytochrome c was not observed. In the presence of Cl −, Compound III was also formed and reduction of cytochrome c was inhibited. From the results described in this paper we conclude that Compound III is able to react with superoxide anions, probably resulting in formation of an intermediate (Compound I) which is catalytically active in the oxidation of Cl − to yield hypochlorous acid (HOCl). Because Compound III of myeloperoxidase is formed in phagocytosing neutrophils (Winterbourn, C.C., Garcia, R.C., and Segal, A.W. (1985) Biochem. J. 228, 583–592) we propose that, in vivo, myeloperoxidase also acts as a superoxide dismutase, and via formation of Compound I uses superoxide anions in the formation of HOCl.

Human vascular smooth muscle cells and endothelial cells lack catalase activity and are susceptible to hydrogen peroxide.

51Cr release as lytic and cell detachment as nonlytic injury were employed to estimate neutrophil-mediated injury of cultured human vascular smooth muscle cells and endothelial cells. The reagents hydrogen peroxide or hypoxanthine-xanthine oxidase produced dose-dependent killing and nonlytic cell detachment, which were specifically inhibited by catalase but not by superoxide dismutase. The concentration of hydrogen peroxide or xanthine oxidase to induce cell detachment was less than lytic dose, suggesting that cell detachment was a much more sensitive assay of injury. Neutrophil-mediated cell lysis averaged 15% at most and was mostly dependent on hydrogen peroxide, while neutrophil-mediated cell detachment was nearly 100% and its dependency on hydrogen peroxide varied from 46% to 60%. These results suggest that vascular smooth muscle cells and endothelial cells in neutrophil-mediated events are destroyed by a hydrogen peroxide-dependent process, mainly via a nonlytic cell detachment mechanism. There was no striking difference of sensitivity to hydrogen peroxide between vascular smooth muscle cells and endothelial cells. Vascular smooth muscle cells and endothelial cells contained fairly high concentrations of superoxide dismutase, but not catalase, activity. The sensitivity of these cells to hydrogen peroxide but not to superoxide may arise from the fact that these cells lack intracellular catalase activity. The injury of vascular cells, which constitute important components of blood vessels, may lead to vascular injury and subsequent tissue damage.

Role of free radicals in the initiation and promotion of radiation transformation in vitro.

We have studied the effects of superoxide dismutase (SOD), catalase, Cu(II) (3,5-diisopropylsalicylate)2 (CuDIPS) and other copper compounds on radiation transformation in vitro using C3H 10T1/2 cells. When present only during irradiation, high concentrations of SOD in the medium enhanced transformation, while catalase, inactivated SOD (autoclaved), CuDIPS, cupric chloride and cuprous chloride inhibited the initiation phase of radiation transformation. SOD, catalase and CuDIPS did not affect the expression phase of radiation transformation. Suppression of the TPA enhancement of transformation by catalase was a highly significant effect, while the suppression by SOD was not of statistical significance. Our results suggest that hydrogen peroxide (H2O2) may be important in the cellular damage leading to malignant transformation.