New conjugates of superoxide dismutase/catalase mimetics with cyclodestrins

Mimetics of antioxidant enzymes such as superoxide dismutases (SOD) or catalases are reported as potential new drugs able to reduce oxidative stress damage. In particular, manganese(III) complexes of salen-type ligands have been studied as both SOD and catalase mimetics. In this paper, we report the synthesis of two novel conjugates of salen-type ligands with the β-cyclodextrin, the 6-deoxy-6-[(S-cysteamidopropyl(1,2-diamino)N,N′-bis(salicylidene))]-β-cyclodextrin and the 6-deoxy-6-[(S-cysteamidopropyl(1,2-diamino)N,N′-bis(3-methoxysalicylidene))]-β-cyclodextrin, their spectroscopic characterization, and the synthesis and the characterization of their manganese(III) complexes. The SOD-like activity of the metal complexes was investigated by the indirect Fridovich method. The catalase like activity was tested using a Clark-type oxygen electrode. The peroxidase activity was tested using the ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) assay. The glycoconjugation of salen-manganese(III) complexes yields compounds with enhanced SOD activity. These complexes also show catalase and peroxidase activities higher than the simple salen complexes (EUK 113 and EUK 108).     

Oxidative stress in Caenorhabditis elegans: protective effects of superoxide dismutase/catalase mimetics

The lifespan of Caenorhabditis elegans can be extended by the administration of synthetic superoxide dismutase/catalase mimetics (SCMs) without any effects on development or fertility. Here we demonstrate that the mimetics, Euk-134 and Euk-8, confer resistance to the oxidative stress-inducing agent, paraquat and to thermal stress. The protective effects of the compounds are apparent with treatments either during development or during adulthood and are independent of an insulin/IGF-I-like signalling pathway also known to affect thermal and oxidative stress resistance. Worms exposed to the compounds do not induce a cellular stress response and no detrimental effects are observed.     

Effects of superoxide dismutase/catalase mimetics on life span and oxidative stress resistance in the housefly,Musca domestica

The purpose of this study was to determine whether superoxide dismutase/catalase mimetics lengthen the life span of the housefly, Musca domestica, as previously demonstrated for the nematode Caenorhabditis elegans. Various concentrations of Eukarion-8 or Eukarion-134 were administered via the drinking water and the effects on the life span of the flies and amounts of protein carbonyls were determined under normoxic and hyperoxic conditions. These SOD/catalase mimetics neither extended the life span of the flies nor attenuated the protein carbonyl content under normoxic conditions and shortened life span under hyperoxic conditions. Thus, the effect of these SOD/catalase mimetics on the life span of animals seem to be species-specific.     

Stabilization of L-ascorbic acid by superoxide dismutase and catalase

The effects of superoxide dismutase (SOD) and catalase on the autoxidation rate of L-ascorbic acid (ASA) in the absence of metal ion catalysts were examined. The stabilization of ASA by SOD was confirmed, and the enzyme activity of SOD, which scavenges the superoxide anion formed during the autoxidation of ASA, contributed strongly to this stabilization. The stabilization of ASA by catalase was observed for the first time; however, the specific enzyme ability of catalase would not have been involved in the stabilization of ASA. Such proteins as bovine serum albumin (BSA) and ovalbumin also inhibited the autoxidation of ASA, therefore it seems that non-specific interaction between ASA and such proteins as catalase and BSA might stabilize ASA and that the non-enzymatic superoxide anion scavenging ability of proteins might be involved.     

Modulation of oxidant lung injury by using liposome-entrapped superoxide dismutase and catalase

Increased cellular generation of partially reduced species of oxygen mediates the toxicity of hyperoxia to cultured endothelial cells and rats exposed to 95-100% oxygen. Liposomal entrapment and intracellular delivery of superoxide dismutase (SOD) to cultured porcine aortic endothelial cells increased the specific activity of cellular SOD up to 15-fold. The liposome-mediated augmentation of SOD activity persisted in cell monolayers and rendered these cells resistant to oxygen-induced injury in a cell SOD activity-dependent manner. Addition of free SOD to culture medium had no effect on cell SOD activity or resistance to oxygen toxicity. SOD and catalase-containing liposomes injected i.v. into rats increased lung-associated enzyme specific activities two- to fourfold. Liposome entrapment of both SOD and catalase significantly increased the circulating half-lives of these enzymes and was critical for prevention of in vivo oxygen toxicity. Free SOD and catalase injected i.v. in the absence or presence of control liposomes did not increase corresponding lung enzyme activities or survival time in 100% oxygen. These studies show that O2- and H2O2 are important mediators of oxygen toxicity and that intracellular delivery of oxygen protective enzymes can reduce tissue injury owing to overproduction of partially reduced oxygen species.     

Effects of growth hormone on hypothalamic catalase and Cu/Zn superoxide dismutase

Age-associated changes in hypothalamic catalase activity and level, and Cu/Zn superoxide dismutase (Cu/Zn SOD) activity were examined in Ames dwarf mice with growth hormone (GH) deficiency and prolonged lifespan, in PEPCK-hGH transgenic mice with overexpression of GH and reduced lifespan, and compared to values measured in normal controls. Hypothalami from young (3-4 months), middle-aged (9-10 months), and old (19-23 months) male mice were examined using spectrophotometric assay and Western blot. In dwarf mice, Cu/Zn SOD and catalase activities declined with age, and were higher than the corresponding normal values in young and middle-aged groups. Catalase levels also declined with age, but were similar to values in normal controls. In GH transgenic mice, age-associated decline of both catalase and Cu/Zn SOD occurred earlier than in normal animals. Catalase levels and activities in transgenic animals were similar to controls, whereas Cu/Zn SOD activity was higher in transgenics than in normal mice. The present results suggest that dwarf mice, during early life, have enhanced hypothalamic free radical defenses, which may contribute to their extended lifespan. However, from the present results in GH transgenic mice, it is impossible to conclude whether early decline of hypothalamic catalase and Cu/Zn SOD in these animals represents a correlate of accelerated aging, or contributes to their reduced lifespan.     

Induction and inactivation of catalase and superoxide dismutase of Escherichia coli by ozone

Oxyradicals have been implicated in ozone (O3) toxicity and in other oxidant stress. In this study, we investigated the effects of O3 on the biosynthesis of the antioxidant enzymes catalase and superoxide dismutase in Escherichia coli to determine their role in the defense against ozone toxicity. Inhibition of growth and loss of viability were observed in cultures exposed to ozone. Results also showed an increase in the activities of catalase and superoxide dismutase in cultures exposed to ozone, which was shown to be due to true induction rather than activation of preexisting apoproteins. Cessation of O3 exposure resulted in 30 min of continual high rate of catalase biosynthesis followed by a gradual decrease in the level of the enzyme approaching that of control cultures. This decrease was attributed to a concomitant cessation of de novo enzyme synthesis and dilution of preexisting enzyme by cellular growth. Ozonation of cell-free extracts showed that superoxide dismutase and catalase are subject to oxidative inactivation by ozone. In vivo induction of these enzymes may represent an adaptive response evolved to protect cells against ozone toxicity.     

Inactivation of catalase and superoxide dismutase by singlet oxygen derived from photoactivated dye

Both superoxide dismutase (SOD) and catalase are key enzymes in the antioxidant system of the cells that work to maintain low steady-state concentrations of the reactive oxygen species. When exposed to a singlet oxygen-producing system composed of dye, such as methylene blue or rose bengal, and visible light both SOD and catalase were susceptible to oxidative modification and damage as indicated by the loss of activity, fragmentation and aggregation of peptide as well as by the formation of carbonyl groups. Histidine, a powerful quenching agent for singlet oxygen, and the polyamines, such as spermine and spermidine, were effective at protecting the activity loss mediated by illuminated dye, whereas spin traps were only mildly effective. The structural alterations of modified enzymes were indicated by the increase in susceptibility to proteases, the change in absorption spectra and in fluorescence spectra. The singlet oxygen-mediated damage to SOD and catalase may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

Simultaneous determination of superoxide dismutase and catalase in biological materials by polarography.

The polarographic method of catalytic currents applied to a wave of oxygen permits the simultaneous assay of superoxide dismutase and catalase in biological materials with high speed and reproducibility and minimal manipulation of tissues. Washed red blood cells and tissue homogenates give rise to a strong polarographic maximum, apparently due to heme proteins, which interferes with the measurement. This maximum is suppressed by addition of approximately 0.2% plasma. Therefore, the determination of the two enzymes in red blood cells can be carried out by direct addition of whole blood to the polarographic solution. Thirty microliters of blood are enough for optimal determination of both enzymes. The method can determine superoxide dismutase and catalase at concentrations as low as 2 × 10⁻¹¹m and 5 × 10⁻¹⁰m, respectively, and shows a linear correlation between measured activity and enzyme levels. The average values of the two enzymes in human red blood cells was found by this method to be 2.6 × 10⁻⁶m for catalase and 1.8 × 10⁻⁶m for superoxide dismutase, which agree with previously reported values.     

6,6'-Bis(2-hydroxyphenyl)-2,2'-bipyridine manganese(III) complexes: a novel series of superoxide dismutase and catalase mimetics

A series of novel manganese(III) complexes is described based on a 6,6'-bis(2-hydroxyphenyl)-2,2'-bipyridine template. These complexes show superoxide dismutase and catalase activity. The effect of the aromatic substitution pattern on the SAR is described.     

Mitigation of cocaine-mediated mitochondrial damage,defective mitophagy and microglial activation by superoxide dismutase mimetics

ABSTRACT

Although cocaine exposure has been shown to potentiate neuroinflammation by upregulating glial activation in the brain, the role of mitophagy in this process remains an enigma. In the present study, we sought to examine the role of impaired mitophagy in cocaine-mediated activation of microglia and to determine the ameliorative potential of superoxide dismutase mimetics in this context. Our findings demonstrated that exposure of mouse primary microglial cells (mPMs) to cocaine resulted in decreased mitochondrial membrane potential, that was accompanied by increased expression of mitophagy markers, PINK1 and PRKN. Exposure of microglia to cocaine also resulted in increased expression of DNM1L and OPTN with a concomitant decrease in the rate of mitochondrial oxygen consumption as well as impaired mitochondrial functioning. Additionally, in the presence of cocaine, microglia also exhibited upregulated expression of autophagosome markers, BECN1, MAP1LC3B-II, and SQSTM1. Taken together, these findings suggested diminished mitophagy flux and accumulation of mitophagosomes in the presence of cocaine. These findings were further confirmed by imaging techniques such as transmission electron microscopy and confocal microscopy. Cocaine-mediated activation of microglia was further monitored by assessing the expression of the microglial marker (ITGAM) and the inflammatory cytokine (Tnf, Il1b, and Il6) mRNAs. Pharmacological, as well as gene-silencing approaches aimed at blocking both the autophagy/mitophagy and SIGMAR1 expression, underscored the role of impaired mitophagy in cocaine-mediated activation of microglia. Furthermore, superoxide dismutase mimetics such as TEMPOL and MitoTEMPO were shown to alleviate cocaine-mediated impaired mitophagy as well as microglial activation.

Abbreviations: 3-MA: 3-methyladenine; Δψm: mitochondrial membrane potential; ACTB: actin, beta; AIF1: allograft inflammatory factor 1; ATP: adenosine triphosphate; BAF: bafilomycin A₁; BECN1: beclin 1, autophagy related; CNS: central nervous system; DNM1L: dynamin 1 like; DMEM: Dulbecco modified Eagle medium; DAPI: 4,6-Diamidino-2-phenylindole; DRD2: dopamine receptor D2; ECAR: extracellular acidification rate; FBS: fetal bovine serum; FCCP: Trifluoromethoxy carbonylcyanide phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IL1B: interleukin 1, beta; IL6: interleukin 6; ITGAM: integrin subunit alpha M; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mPMs: mouse primary microglial cells; MRC: maximal respiratory capacity; NFKB: nuclear factor kappa B; NLRP3: NLR family pyrin domain containing 3; NTRK2: neurotrophic receptor tyrosine kinase 2; OCR: oxygen consumption rate; OPTN: optineurin; PBS: phosphate buffered saline; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TNF: tumor necrosis factor     

Purification and characterization of an iron superoxide dismutase and a catalase from the sulfate-reducing bacterium Desulfovibrio gigas

The iron-containing superoxide dismutase (FeSOD; EC 1.15.1.1) and catalase (EC 1.11.1.6) enzymes constitutively expressed by the strictly anaerobic bacterium Desulfovibrio gigas were purified and characterized. The FeSOD, isolated as a homodimer of 22-kDa subunits, has a specific activity of 1,900 U/mg and exhibits an electron paramagnetic resonance (EPR) spectrum characteristic of high-spin ferric iron in a rhombically distorted ligand field. Like other FeSODs from different organisms, D. gigas FeSOD is sensitive to H(2)O(2) and azide but not to cyanide. The N-terminal amino acid sequence shows a high degree of homology with other SODs from different sources. On the other hand, D. gigas catalase has an estimated molecular mass of 186 +/- 8 kDa, consisting of three subunits of 61 kDa, and shows no peroxidase activity. This enzyme is very sensitive to H(2)O(2) and cyanide and only slightly sensitive to sulfide. The native enzyme contains one heme per molecule and exhibits a characteristic high-spin ferric-heme EPR spectrum (g(y,x) = 6.4, 5.4); it has a specific activity of 4,200 U/mg, which is unusually low for this class of enzyme. The importance of these two enzymes in the context of oxygen utilization by this anaerobic organism is discussed.     

Induction of apoptosis in fetal pulmonary arterial smooth muscle cells by a combined superoxide dismutase/catalase mimetic

Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are known to play an important role in the proliferation and viability of vascular smooth muscle cells. In this study, we determined the effects of increased superoxide dismutase and catalase activity on fetal pulmonary arterial smooth muscle cell (FPASMC) proliferation and viability using EUK-134, a superoxide dismutase/catalase mimetic. Treatment of FPASMC with EUK-134 or with a combination of superoxide dismutase and catalase enzymes decreased superoxide and hydrogen peroxide levels as detected by the fluorescent dyes dihydroethidium and dichlorodihydrofluorescein diacetate, respectively. EUK-134 (5 microM) attenuated serum-induced FPASMC proliferation, whereas 50 microM EUK-134 decreased the number of viable cells, suggesting cell death. Conversely, combined superoxide dismutase and catalase enzyme activity equivalent to 50 microM EUK-134 prevented proliferation but did not reduce the number of viable FPASMC. The loss of mitochondrial membrane potential after 18 h, an increase in caspase-9 and caspase-3 activity after 24 h, and the subsequent appearance of TdT-mediated dUTP nick end labeling-positive nuclei were detected in FPASMC after treatment with 50 microM EUK-134. This indicates an induction of programmed rather than necrotic cell death and suggests that prolonged removal of ROS is required to stimulate apoptosis. Compounds such as EUK-134 may, therefore, prove more effective than enzymic antioxidants over longer periods, especially when the aim is to decrease the number of smooth muscle cells in diseases resulting from excessive muscularization.     

A superoxide dismutase from the archaeon Sulfolobus solfataricus is an extracellular enzyme and prevents the deactivation by superoxide of cell-bound proteins.

An oxygen-induced iron superoxide dismutase was found in the culture fluid of the thermoacidophilic crenarchaeon Sulfolobus solfataricus during growth on glucose-rich media. This protein was also identified as being associated with the cell-surface, with the amount of the released and cell-bound protein fractions depending on the growth phase of the cells. The steady decrease in cell-associated superoxide dismutase during continued growth correlated with the increase of free superoxide dismutase in the medium. Both enzyme fractions were purified to homogeneity and found to be active with different catalytic efficiency, with the released superoxide dismutase showing a fourfold lower specific activity. Characterization in comparison with the cytosolic superoxide dismutase revealed identical N-terminal sequences, electrophoretic mobility, isoelectric point, and molecular mass for all three differently located enzymes. In order to clarify the physiological role of the cell-associated superoxide dismutase, the prevention of cell-bound protein deactivation by oxyradicals was also investigated. Glucose dehydrogenase, which was chosen as a model enzyme, was demonstrated to be located on the cell surface and to be inactivated by potassium superoxide by in vivo assays. The direct protective effect of superoxide dismutase on glucose dehydrogenase was demonstrated by in vitro assays on the free released enzyme. Similarly, the prevention of deactivation by potassium superoxide was also demonstrated for the integral membrane protein succinate dehydrogenase by intact cell assay. Superoxide dismutase added to cells was shown to moderately reduce the critical damaging peroxidation and hence play a major role in maintaining the integrity of the outer cell envelope components.     

Photoinactivation in vivo of superoxide dismutase and catalase in the cyanobacterium Microcystis aeruginosa

Abstract The planktonic cyanobacterium Microcystis aeruginosa is particularly sensitive to photoinhibition by visible light, Photosystem II and ribulose 1,5-bisphosphate (RuBP) carboxylase activities being affected. Although the organism contains superoxide dismutase (SOD) and catalase, these protective enzymes are also photoinactivated during the illumination of whole cells by visible light.     

Ontogeny of human fetal catalase superoxide dismutase and lipid peroxidation: A comparative study

A comparative study on the activity profile of catalase and superoxide dismutase, the two scavenging enzymes, as well as the developmental profile of lipid peroxidation in the human fetal brain, liver and kidney have been done for gestation periods ranging from 12 weeks to 28 weeks and beyond. The activity of the scavenging enzymes increase gradually in_all the tissues with the advancement of pregnancy. Brain is an exception in case of catalase where the activity remains more or less same throughout the developmental period except in the case of fetuses, 28 weeks and above where significant decrease in the catalase activity is observed. A high level of lipid peroxidation is observed during early stages of development which declines thereafter.