Differential effects of tumor necrosis factor and asbestos fibers on manganese superoxide dismutase induction and oxidant-induced cytotoxicity in human mesothelial cells

We compared induction of manganese superoxide dismutase (MnSOD) by asbestos fibers and tumor necrosis factor (TNF) using cultures human mesothelial cells. Transformed pleural mesothelial cells (MET 5A) were exposed for 48 h to amosite asbestos fibers (2 g/cm²), to TNF (10 Ng/ml), and to the combination of these two. TNF and amosite+TNF caused significant MnSOD mRNA upregulation. Similarly MnSOD specific activity was increased by TNF (290% increase) and the amosite+TNF combination (313% increase) but not by amosite alone. In cell injury experiments, amosite and amosite+TNF exposures caused significant cell membrane injury when assessed by lactate dehydrogenase release, which was 31% and 57% higher than in the unexposed cells. However, only the amosite+TNF combination caused significant depletion of cellular high-energy nucleotide when expressed as percentage of [¹⁴C]denine labeling in cellular high-energy nucleotides. The nucleotide levels were 91.5 ± 2.0% in the unexposed cells, 89.9 ± 3.9% in amosite-exposed cells, 90.1 ± 2.2% in TNF-exposed cells, and 79.8 ± 9.4% in amosite+TNF-exposed Amosite+TNF-exposed cells were also most sensitive to menadione (20 mol/L, 2 h), a compound which generates superoxide radicals intracellularly. In conclusion, our data suggests that in human mesothelial cells inflammatory cytokines but not asbestos fibers alone can cause MnSOD induction. In this study, however amosite asbestos+TNF treatment rendered these cells more vulnerable to oxidant-induced cell damage despite elevated MnSOD activity.Abbreviations MnSOD manganese superoxide dismutase - TNF tumor necrosis factor - LDH lactate dehydrogenase     

Expression of manganese superoxide dismutase is not altered in transgenic mice with elevated level of copper-zinc superoxide dismutase

In evaluating the relative expression of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in vivo in states like Down syndrome in which one dismutase is present at increased levels, we measured activities of both enzymes, in tissues of control and transgenic mice constitutively expressing increased levels of CuZnSOD, during exposure to normal and elevated oxygen tensions. Using SOD gel electrophoresis assay, CuZnSOD and MnSOD activities of brain, lung, heart, kidney, and liver from mice exposed to either normal (21%) or elevated (>99% oxygen, 630 torr) oxygen tensions for 120 h were compared. Whereas CuZnSOD activity was elevated in tissues of transgenic relative to control mice under both normoxic or hyperoxic conditions, MnSOD activities in organs of transgenic mice were remarkably similar to those of controls under both conditions. To confirm the accuracy of this method in quantitating MnSOD relative to CuZnSOD expression, two other methods were utilized. In lung, which is the organ exposed to the highest oxygen tension during ambient hyperoxia, a sensitive, specific ELISA for MnSOD was used. Again, MnSOD protein was not different in transgenic relative to control mice during exposure to air or hyperoxia. In addition, lung MnSOD protein was not changed significantly by exposure to hyperoxia in either group. In kidney, a mitochondrion-rich organ, SOD assay, before and after inactivation of CuZnSOD with diethyldithiocarbamate, was used. MnSOD activity was not different in organs from air-exposed transgenic relative to control mice. The data indicated that expression of MnSOD in vivo was not affected by overexpression of the CuZnSOD and, therefore, the two enzymes are probably regulated independently.     

The effects of superoxide dismutase on H2O2 formation

Numerous reports of the effects of overproduction of SODs have been explained on the basis of increased H2O2 production by the catalyzed dismutation of O2-. In this review we consider the effects of increasing [SOD] on H2O2 formation and question this explanation.     

Neuronal uptake of nanoformulated superoxide dismutase and attenuation of angiotensin II-dependent hypertension after central administration

Excessive production of superoxide (O₂⁻) in the central nervous system has been widely implicated in the pathogenesis of cardiovascular diseases, including chronic heart failure and hypertension. In an attempt to overcome the failed therapeutic impact of currently available antioxidants in cardiovascular disease, we developed a nanomedicine-based delivery system for the O₂⁻-scavenging enzyme copper/zinc superoxide dismutase (CuZnSOD), in which CuZnSOD protein is electrostatically bound to a poly-l-lysine (PLL₅₀)–polyethylene glycol (PEG) block copolymer to form a CuZnSOD nanozyme. Various formulations of CuZnSOD nanozyme are covalently stabilized by either reducible or nonreducible crosslinked bonds between the PLL₅₀–PEG polymers. Herein, we tested the hypothesis that PLL₅₀–PEG CuZnSOD nanozyme delivers active CuZnSOD protein to neurons and decreases blood pressure in a mouse model of angiotensin II (AngII)-dependent hypertension. As determined by electron paramagnetic resonance spectroscopy, nanozymes retain full SOD enzymatic activity compared to native CuZnSOD protein. Nonreducible CuZnSOD nanozyme delivers active CuZnSOD protein to central neurons in culture (CATH.a neurons) without inducing significant neuronal toxicity. Furthermore, in vivo studies conducted in adult male C57BL/6 mice demonstrate that hypertension established by chronic subcutaneous infusion of AngII is significantly attenuated for up to 7 days after a single intracerebroventricular injection of nonreducible nanozyme. These data indicate the efficacy of nonreducible PLL₅₀–PEG CuZnSOD nanozyme in counteracting excessive O₂⁻ and decreasing blood pressure in AngII-dependent hypertensive mice after central administration. Additionally, this study supports the further development of PLL₅₀–PEG CuZnSOD nanozyme as an antioxidant-based therapeutic option for hypertension.     

Effects of a carotene-deficient diet on measures of oxidative susceptibility and superoxide dismutase activity in adult women

The effect of consuming a low carotene diet (≈60 μg carotene/day) on oxidative susceptibility and superoxide dismutase (SOD) activity in women living in a metabolic research unit was evaluated. The diet had sufficient vitamins A, E, and C. The women ate the diet supplemented with 1500 μg/day β-carotene for 4 days (baseline), then the unsupplemented diet for 68 days (depletion), followed by the diet supplemented with > 15,000 μg/day carotene for 28 days (repletion). Production of hexanal, pentanal, and pentane by copper-oxidased plasma low density lipoproteins from carotene-depleted women was greater than their production of these compounds when repleted with carotene. Erythrocyte SOD activity was depressed in carotene-depleted women; it recovered with repletion. Thiobarbituric acid reactive substances in plasma of carotene-depleted women were elevated and diminished with repletion. Dietary carotene seems to be needed, not only as a precursor of vitamin A, but also to inhibit oxidative damage and decrease oxidation susceptibility.     

Yeast superoxide dismutase mutants reveal a pro-oxidant action of weak organic acid food preservatives

Saccharomyces cerevisiae could provide a simple experimental system for testing the antioxidant or pro-oxidant actions of chemicals, because it has the capacity for aerobic and anaerobic growth and can readily lose its mitochondrial electron transport chain (the major endogenous source of reactive oxygen species [ROS]). This study showed that yeast superoxide dismutase mutants, in a simple petri dish test, readily distinguish a compound that enhances the detrimental effects of endogenous ROS production by the mitochondrial respiratory chain from another chemical that generates oxidative stress by redox cycling. Using this system, weak organic acid food preservatives are shown to exert a strong pro-oxidant action on aerobic yeast cells. In addition these acids are mutagenic toward the yeast mitochondrial genome, even at levels that are subinhibitory to growth. This raises the concern that the large-scale consumption of these preservatives in the human diet may generate oxidative stress within the epithelia of the gastrointestinal tract.     

Effects of catechin and epicatechin on superoxide dismutase and glutathione peroxidase activity,in vivo

Abstract

Objectives

The objective of this study was to investigate the effects of catechin and epicatechin on the activity of the endogenous antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) (as well as the total antioxidant capacity (TAC)) of rats after intra-peritoneal (i.p.) administration.

Methods

Twenty-four Wistar rats were randomly divided into two groups: the experimental group which was administered daily with a 1:1 mixture of epicatechin and catechin at a concentration of 23 mg/kg body weight for 10 days and the control group which was injected daily with an equal amount of saline. Blood and urine samples were collected before and after the administration period, as well as 10 days after (follow-up).

Results

Intra-peritoneal administration of catechins led to a potent decrease in GPx levels and a significant increase in SOD levels. TAC was significantly increased in plasma and urine. Malonaldehyde levels in urine remained stable. In the animals treated with catechins, SOD activity showed a moderate negative correlation with GPx activity.

Discussion

Boosting the activity of the antioxidant enzymes could be a potential adjuvant approach for the treatment of the oxidative stress-related diseases.     

Mutations by near-ultraviolet radiation in Escherichia coli strains lacking superoxide dismutase

Om wild-type Escherichia coli, near-ultraviolet radiation (NUV) was only weakly mutagenic. However, in an allelic mutant strain (sodA sodB) that lacks both Mn- and Fe-superoxide dismutase (SOD) and assumed to have excess superoxide anion (O₂⁻), NUV induced a 9-fold increase in mutation above the level that normally occurs in this double mutant. When a sodA sodB double mutant contained a plasmid carrying katG⁺ HP-I catalase), mutation by NUV was reduced to wild-type (sodA⁺ sodB⁺) levels. Also, in the sodA sodB xthA triple mutant, which lacks exonuclease III (exoIII) in addition to SOD, the mutations frequency by NUV was reduced to wild-type levels. This synergistic action of NUV and O₂⁻ suggested that pre-mutational lesions occur, with exoIII converting these lesions to stable mutants. Exposure to H₂O₂ induced a 2.8 fold increase in mutations in sodA sodB double mutants, but was reduced to control levels when a plasmid carrying katG⁺ was introduced. These results suggest that NUV, in addition to its other effects on cells, increases mutations indirectly by increasing the flux of OH^. radicals, possibly by generating excess H₂O₂.     

Screening of thermotolerant yeasts as producers of superoxide dismutase

Abstract A screening procedure for highly thermostable yeast superoxide dismutase was developed. Growth yields at various temperatures were estimated for ten mesophilic and thermotolerant strains, belonging to the genera Saccharomyces, Kluyveromyces and Pichia . Higher yields at 45°C were obtained for K. lactis 90-3 and 90-4. A correlation between the ability to grow at higher temperature and the thermostability of the superoxide dismutase enzyme synthesized was observed. A comparison of the operational stability of the superoxide dismutase of all tested strains suggests that the enzyme of K. lactis strains was more thermostable than that of the other tested microorganisms.     

Mutations by near-ultraviolet radiation in Escherichia coli strains lacking superoxide dismutase

Om wild-type Escherichia coli, near-ultraviolet radiation (NUV) was only weakly mutagenic. However, in an allelic mutant strain (sodA sodB) that lacks both Mn- and Fe-superoxide dismutase (SOD) and assumed to have excess superoxide anion (O₂⁻), NUV induced a 9-fold increase in mutation above the level that normally occurs in this double mutant. When a sodA sodB double mutant contained a plasmid carrying katG⁺ HP-I catalase), mutation by NUV was reduced to wild-type (sodA⁺sodB⁺) levels. Also, in the sodA sodB xthA triple mutant, which lacks exonuclease III (exoIII) in addition to SOD, the mutations frequency by NUV was reduced to wild-type levels. This synergistic action of NUV and O₂⁻ suggested that pre-mutational lesions occur, with exoIII converting these lesions to stable mutants. Exposure to H₂O₂ induced a 2.8 fold increase in mutations in sodA sodB double mutants, but was reduced to control levels when a plasmid carrying katG⁺ was introduced. These results suggest that NUV, in addition to its other effects on cells, increases mutations indirectly by increasing the flux of OH^. radicals, possibly by generating excess H₂O₂.     

Metalloantibiotic Mn(II)-bacitracin complex mimicking manganese superoxide dismutase

Superoxide dismutase (SOD) activities of various metallobacitracin complexes were evaluated using the riboflavin-methionine-nitro blue tetrazolium assay. The radical scavenging activity of various metallobacitracin complexes was shown to be higher than those of the negative controls, e.g., free transition metal ions and metal-free bacitracin. The SOD activity of the complex was found to be in the order of Mn(II)>Cu(II)>Co(II)>Ni(II). Furthermore, the effect of bacitracin and their complexation to metals on various microorganisms was assessed by antibiotic susceptibility testing. Moreover, molecular modeling and quantum chemical calculation of the metallobacitracin complex was performed to evaluate the correlation of electrostatic charge of transition metal ions on the SOD activity.     

The effects of hypochlorous acid and neutrophil proteases on the structure and function of extracellular superoxide dismutase

Extracellular superoxide dismutase (EC-SOD) is expressed by both macrophages and neutrophils and is known to influence the inflammatory response. Upon activation, neutrophils generate hypochlorous acid (HOCl) and secrete proteases to combat invading microorganisms. This produces a hostile environment in which enzymatic activity in general is challenged. In this study, we show that EC-SOD exposed to physiologically relevant concentrations of HOCl remains enzymatically active and retains the heparin-binding capacity, although HOCl exposure established oxidative modification of the N-terminal region (Met32) and the formation of an intermolecular cross-link in a fraction of the molecules. The cross-linking was also induced by activated neutrophils. Moreover, we show that the neutrophil-derived proteases human neutrophil elastase and cathepsin G cleaved the N-terminal region of EC-SOD irrespective of HOCl oxidation. Although the cleavage by elastase did not affect the quaternary structure, the cleavage by cathepsin G dissociated the molecule to produce EC-SOD monomers. The present data suggest that EC-SOD is stable and active at the site of inflammation and that neutrophils have the capacity to modulate the biodistribution of the protein by generating EC-SOD monomers that can diffuse into tissue.     

Conformational stability of recombinant manganese superoxide dismutase from the filamentous fungus Trichoderma reesei

Superoxide dismutases (SODs; EC 1.15.1.1) are part of the antioxidant system of aerobic organisms and are used as a defense against oxidative injury caused by reactive oxygen species (ROS). The cloning and sequencing of the 788-bp genomic DNA from Trichoderma reesei strain QM9414 (anamorph of Hypocrea jecorina) revealed an open reading frame encoding a protein of 212 amino acid residues, with 65-90% similarity to manganese superoxide dismutase from other filamentous fungi. The TrMnSOD was purified and shown to be stable from 20 to 90 °C for 1 h at pH from 8 to 11.5, while maintaining its biological activity.     

Temperature dependence of superoxide dismutase activity in plankton

The effect of temperature (from 1 to 37 °C) on in vitro effective superoxide dismutase (SOD) activity of several organisms was investigated and compared. Antarctic plankton, cultures of the alga Nannochloropsis sp., and the cyanobacterium Synechococcus strain WH 7803, and pure bovine erythrocyte SOD was studied. It was found that in all cases SOD activity increased with decreasing temperature within the temperature range assayed, in the Polar as well as the temperate plankton cells. This behavior of SOD is counterintuitive in terms of our experience when looking at enzyme activity or any other chemical reaction. We suggest a theoretical explanation for this apparently odd behavior. The advantage of such behavior is that the same amount of antioxidant will act better under low temperatures when reactive oxygen species (ROS) increase. Moreover, this protective process would act in vivo at a faster pace than the ex novo enzyme synthesis.     

Isolation and characterization of superoxide dismutase: A personal history and tribute to Joe McCord and Irwin Fridovich

The discovery of superoxide dismutase twenty years ago gave new meaning to work on erythrocuprein. This tribute to the achievement of Joe McCord and Irwin Fridovich is an account of experience of superoxide dismutase from old obscure copper protein of red blood cells to new exciting enzyme of oxygen free-radical metabolism, and an affirmation of the superoxide theory of oxygen toxicity.     

Design and characterization of enzymosomes with surface-exposed superoxide dismutase

Superoxide dismutase (SOD) was chemically modified by covalent linkage of fatty acid chains to the accessible ε-amino groups of the enzyme. This acylation method gave rise to a different enzyme entity (Ac-SOD) as evidenced by different physicochemical properties such as octanol/water partition coefficient and isoelectric point (pI) as compared to SOD. Ac-SOD was incorporated in conventional and long-circulating liposomes (LCL) and characterized in terms of incorporation efficiency, protein to lipid ratio (Prot/Lip), enzymatic activity retention and zeta potential. The observation that Ac-SOD liposomes present enzymatic activity on their external surface indicates that these formulations can act independent of rate and extent of enzyme release as required in case of SOD liposomes. The decrease of superficial charge of liposomal formulations containing Ac-SOD, as compared to SOD liposomes, may be related to the negatively charged enzyme molecules localized on the liposome surface. The comparative characterization of Ac-SOD and SOD liposomal formulations evidenced that the two enzyme forms differ substantially regarding their intraliposomal location: SOD tends to be localized in the internal aqueous spaces, whereas Ac-SOD is expected to be localized in the lipid bilayers of the liposomes, partially buried into the outer surface and exposed to the external medium. These liposomal structures with surface-exposed SOD were designated as Ac-SOD enzymosomes. The properties of these enzymosomes may influence the therapeutic effect, as the release of the enzyme from extravasated vesicles is no longer a necessary requirement for achieving dismutating activity within the inflamed target site.     

In vivo real-time measurement of superoxide anion radical with a novel electrochemical sensor

The dynamics of superoxide anion (O₂⁻) in vivo remain to be clarified because no appropriate method exists to directly and continuously monitor and evaluate O₂⁻ in vivo. Here, we establish an in vivo method using a novel electrochemical O₂⁻ sensor. O₂⁻ generated is measured as a current and evaluated as a quantified partial value of electricity (Q_part), which is calculated by integration of the difference between the baseline and the actual reacted current. The accuracy and efficacy of this method were confirmed by dose-dependent O₂⁻ generation in xanthine–xanthine oxidase in vitro in phosphate-buffered saline and human blood. It was then applied to endotoxemic rats in vivo. O₂⁻ current began to increase 1 h after lipopolysaccharide, and Q_part increased significantly for 6 h in endotoxemic rats, in comparison to sham-treated rats. These values were attenuated by superoxide dismutase. The generation and attenuation of O₂⁻ were indirectly confirmed by plasma lipid peroxidation with malondialdehyde, endothelial injury with soluble intercellular adhesion molecule-1, and microcirculatory dysfunction. This is a novel method for measuring O₂⁻ in vivo and could be used to monitor and treat the pathophysiology caused by excessive O₂⁻ generation in animals and humans.     

纯化超氧化物歧化酶的新工艺

为探索简便实用纯化ＳＯＤ的工艺路线,以人或猪血红细胞溶血上清液,经铜胺中空纤维透析器（分子量截留值为１５ｋＤ）透析和超滤,收集分子量大于１５．０ｋＤ的物质,再加热６０℃１０ｍｉｎ,离心取上清即得。Ｃｕ、ＺｎＳＯＤ和ＭｎＳＯＤ分子量分别为３２．０ｋＤ和８０．０ｋＤ。人血和猪血纯化的ＳＯＤ总收率分别为８８．２％和８９．２％,比活性分别为１７４２９Ｕ／ｍｇ和１８２２８Ｕ／ｍｇ。工艺简便实用,适于工业纯化生产。     

Alterations in manganese, copper, and zinc contents, and intracellular status of the metal-containing superoxide dismutase in human mesothelioma cells

BACKGROUND AND AIM: Molecular diagnostics and therapeutics of human mesothelioma using disease-related markers present major challenges in clinical practice. To identify biochemical alternations that would be markers of human mesothelioma, we measured the intracellular steady-state levels of biologically important trace metals such as manganese (Mn), copper (Cu), and zinc (Zn) in a human mesothelial cell line, MeT-5A, and in five human mesothelioma cell lines (MSTO-211H, NCI-H226, NCI-H2052, NCI-H2452, ACC-MESO-1) by inductively coupled plasma-mass spectrometry (ICP-MS). We also aimed to investigate whether the alterations were related to the intracellular status of metal-containing superoxide dismutase (SOD). RESULTS: There were no significant differences in the contents of the trace metals among MeT-5A, MSTO-211H, and ACC-MESO-1 cells. However, each of the other three mesothelioma cell lines had a unique characteristic in terms of the intracellular amounts of the metals; NCI-H226 contained an extremely high level of Mn, an amount 7.3-fold higher than that in MeT-5A. NCI-H2052 had significantly higher amounts of Cu (3.4-fold) and Zn (1.3-fold) compared with MeT-5A. NCI-H2452 contained about 5.8-fold the amount of Cu and 2.5-fold that of Mn compared with MeT-5A. As for the intracellular levels of copper/zinc-SOD (Cu/Zn-SOD) and manganese-SOD (Mn-SOD), those of Cu/Zn-SOD were relatively unchanged among the cells tested, and no notable correlation with Cu or Zn contents was observed. On the other hand, all mesothelioma cells highly expressed Mn-SOD compared with MeT-5A, and a very high expression of the enzyme with a robust activity was observed in the two mesothelioma cells (NCI-H226, NCI-H2452) containing a large amount of Mn. CONCLUSIONS: In comparison with MeT-5A human mesothelial cells, some human mesothelioma cells had significantly higher amounts of Mn or Cu and one mesothelioma cell had a significantly higher amount of Zn. Interestingly, all mesothelioma cells overexpressed Mn-SOD compared with MeT-5A, and the cells whose Mn-SOD activity was increased contained higher amounts of Mn. It seemed that intracellular Mn content was positively correlated with Mn-SOD, suggesting that the intracellular Mn level is associated with Mn-SOD activity. These biochemical signatures could be potential disease-related markers of mesothelioma.