Low Molecular Weight Superoxide Dismutase Research Articles

TEMPOL, a membrane-permeable radical scavenger, attenuates gastric mucosal damage induced by ischemia/reperfusion: A key role for superoxide anion

The pathogenesis of gastric mucosal injury is a multifaceted process involving reactive oxygen and nitrogen species, both of which play a crucial role in the ischemia/reperfusion model of gastric damage. Hence, several studies have evaluated the anti-ulcerogenic effect of metal chelators, antioxidative enzymes, and low-molecular-weight antioxidants. Low molecular weight superoxide dismutase (SOD) mimetics have been shown to play a protective role against oxidative damage. Therefore, the aim of the current study was to investigate the modulatory effect of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl [TEMPOL (50 mg/kg)], a SOD mimetic, and the SOD inhibitor, diethyldithiocarbamate [DETCA (100 mg/kg)] on gastric lesions induced by ischemia/reperfusion. This insult produced typical gastric lesions, a significant fall in the gastric mucosal glutathione (GSH) and nitric oxide (NO) levels, accompanied by an increase in malondialdehyde (MDA) content and myeloperoxidase (MPO) activity. TEMPOL markedly minimized gastric ulceration and restored MDA, NO, and MPO levels, but did not alter GSH level, which dropped drastically in DETCA treated group, an effect that was not reflected on gross lesions induced by ischemia/reperfusion. In conclusion, TEMPOL can confer protection from gastric ischemia/reperfusion injury possibly by reducing the level of superoxide anion (O 2 •−), replenishing NO, and minimizing neutrophil infiltration. Therefore, specific SOD mimetics could be beneficial as complementary agents in the management of gastric ulceration.

The Magnetic Resonance Imaging Contrast Agent Mangafodipir Exerts Antitumor Activity via a Previously Described Superoxide Dismutase Mimetic Activity

To the Editor: It was recently described in Cancer Research by Laurent et al. ( [1][1]) how low molecular weight superoxide dismutase (SOD) mimetics increase H2O2 levels, which in turn killed colon (CT26) and liver (Hepa 1-6) tumor cells. In contrast, the SOD mimetics stimulated proliferation of

Superoxide-mediated nitration of spinal manganese superoxide dismutase: a novel pathway in N-methyl- d-aspartate-mediated hyperalgesia

N-methyl- d-aspartate (NMDA) receptors serve prominent roles in vast physio-pathological conditions including hyperalgesia (defined as augmented pain intensity in response to painful stimuli) associated with central sensitization. Using M40403 a synthetic low molecular weight superoxide dismutase mimetic that removes superoxide we show for the first time that this radical plays a key role in NMDA-mediated hyperalgesia. Intrathecal administration of NMDA in rats led to a time-dependent development of thermal hyperalgesia. Removal of superoxide with M40403 abolished NMDA-mediated hyperalgesia, while its inactive congener had no effect. Thus NMDA-mediated hyperalgesic response to heat is mediated through spinal release of superoxide. At time of near-to-maximal hyperalgesia, we observed that spinal endogenous manganese superoxide dismutase (MnSOD), the enzyme that normally keeps superoxide under well-controlled condition was nitrated, as shown by immunoprecipitation. Subsequently and as determined by biochemical analysis, nitration of MnSOD led to its deactivation as shown by the loss of the enzyme's ability to dismute and hence remove superoxide. M40403 by preventing MnSOD nitration restored its activity and inhibited the hyperalgesic response to intrathecal NMDA. Thus, superoxide-mediated nitration and deactivation of spinal MnSOD is a novel pathway of NMDA-mediated spinal hyperalgesia and hence central sensitization since it helps to maintain high levels of superoxide that in turn maintains nociceptive signaling. The broader implication of our findings is that superoxide may contribute to various forms of pain events that are driven by NMDA-receptor activation.

Superoxide and post-ischemic liver injury: potential therapeutic target for liver transplantation.

Cessation of blood flow to the liver is required during liver transplantation and resectional surgery. A growing body of experimental evidence suggests that restoration of blood flow to the ischemic liver initiates hepatocellular injury which may lead, in some cases, to severe liver injury and graft failure. A large number of studies have implicated reactive oxygen species as potential mediators of post-ischemic tissue injury. Recent developments in genetic engineering as well as chemical modeling, have allowed for the production of novel free radical scavengers including mutated forms of superoxide dismutase (SOD) and low molecular weight SOD mimics with extended circulating half-lives and/or significant membrane permeability's. Application of these newly developed free radical scavengers show promising results in animal models of liver I/R and may become powerful tools in the treatment of post-ischemic liver injury that occurs in liver transplantation.

The protective effect of M40401, a superoxide dismutase mimetic, on post-ischemic brain damage in Mongolian gerbils

BackgroundOverproduction of free radical species has been shown to occur in brain tissues after ischemia-reperfusion injury. However, most of free radical scavengers known to antagonize oxidative damage (e.g. superoxide dismutase, catalase), are unable to protect against ischemia-reperfusion brain injury when given in vivo, an effect mainly due to their difficulty to gain access to brain tissues. Here we studied the effect of a low molecular weight superoxide dismutase mimetic (M40401) in brain damage subsequent to ischemia-reperfusion injury in Mongolian gerbils.ResultsIn animals undergoing ischemia-reperfusion injury, neuropathological and ultrastructural changes were monitored for 1–7 days either in the presence or in the absence of M40401 after bilateral common carotid artery occlusion (BCCO). Administration of M40401 (1–40 mg/kg, given i.p. 1 h after BCCO) protected against post-ischemic, ultrastructural and neuropathological changes occurring within the hippocampal CA1 area. The protective effect of M40401 was associated with a significant reduction of the levels of malondialdehyde (MDA; a marker of lipid peroxidation) in ischemic brain tissues after ischemia-reperfusion.ConclusionTaken together, these results demonstrate that M40401 provides protective effects when given early after the induction of ischemia-reperfusion of brain tissues and suggest the possible use of such compounds in the treatment of neurological dysfunction subsequent to cerebral flow disturbances.

Modification of inflammatory response to implanted biomedical materials in vivo by surface bound superoxide dismutase mimics.

The healing response to implanted biomedical materials involves varying degrees and stages of inflammation and healing which in some cases leads to device failure. In this article, we describe synthetic methods and in vivo results of a novel surface treatment for biomedical materials involving covalent conjugation of a low molecular weight superoxide dismutase mimic (SODm), which imparts anti-inflammatory character to the material. SODm investigated in this study are a new class of anti-inflammatory drugs consisting of a Mn(II) complex of a macrocyclic polyamine ring that catalyze the dismutation of superoxide at rates equivalent to that of native enzyme. The SODms were covalently linked to small disks of ultra-high molecular weight polyethylene, poly(etherurethane urea), and tantalum metal at two concentrations and implanted in a subcutaneous rat implant model for 3, 7, 14, and 28 days. Histological examination of the implant tissue performed at 3 and 28 days revealed striking anti-inflammatory effects on both acute and chronic inflammatory responses. At 3 days, the formation of a neutrophil-rich acute inflammatory infiltrate seen in control implants was inhibited for all three materials treated with SODm. At 28 days, foreign body giant cell formation (number of FBGCs per field) and fibrous capsule formation (mean thickness of implant capsule) were also significantly inhibited over untreated control implants. A mechanism based on our current understanding of superoxide as an inflammatory mediator at implanted biomedical materials is proposed.

Manganese-based Superoxide Dismutase Mimetics Inhibit Neutrophil Infiltration in Vivo

In a previous study (Hardy et al. (1994) J. Biol. Chem. 269, 18535-18540), we observed that the manganese-based superoxide dismutase mimetic Mn(II)-dichloro(1,4,7,10,13-pentaazacyclopentadecane) (MnPAM) inhibited neutrophil-mediated cell injury in vitro. We have extended these studies with the low molecular weight superoxide dismutase mimic to evaluate the role of superoxide in neutrophil-mediated tissue injury in vivo. In a dose-dependent manner, MnPAM inhibited colonic tissue injury and neutrophil accumulation into the colonic tissue induced by the intracolonic instillation of dilute aqueous acetic acid in mice. Tissue injury was assessed by visual and histological analysis. Neutrophil infiltration was determined by tissue myeloperoxidase activity and confirmed by histological analysis. Two novel Mn(II) dichloro complexes of the carbon-substituted macrocycles 2-methyl-1,4,7,10,13-pentaazacyclopentadecane (MnMAM) and 2-(2-methylpropyl)-1,4,7,10,13-pentaazacyclopentadecane (MnBAM) effectively catalyzed the dismutation of superoxide with catalytic rate constants (kcat) of 3. 31 x 10(7) M-1 s-1 and 1.91 x 10(7) M-1 s-1, respectively, as determined by stopped-flow kinetic analysis at pH 8.1 and 21 degrees C. The superoxide dismutase mimetics MnMAM and MnBAM also attenuated dilute aqueous acetic acid-induced tissue injury and neutrophil infiltration into colonic tissue; however, two Mn(II) complexes that had little or no detectable SOD activity (kcat </= 0.1 x 10(7) M-1 s-1), specifically the Mn(II) dichloro complexes of 1,4,7,10,13-pentaazacyclohexadecane and 1,4,7,11,14-pentaazacycloheptadecane, failed to inhibit the colonic tissue injury or infiltration of neutrophils in mice treated intracolonically with dilute aqueous acetic acid. These results are consistent with a proinflammatory role for superoxide in the mediation of neutrophil infiltration in vivo.

Synthesis, Characterization, and Stability of Manganese(II) C-Substituted 1,4,7,10,13-Pentaazacyclopentadecane Complexes Exhibiting Superoxide Dismutase Activity

Superoxide radical anion has been demonstrated to be a mediator of many disease states, including inflammatory, autoimmune, and cancerous diseases. As a consequence, we have developed a program to design, synthesize, and test synthetic low-molecular weight superoxide dismutase (SOD) mimics as potential pharmaceutical agents. A critical feature of the design of metal-based drugs is not only high activity as an enzyme mimic but also chemical stability. In this report we describe the synthesis and characterization of a series of C-substituted 1,4,7,10,13-pentaazacyclopentadecane, [15]aneN5, ligands, 1, and their corresponding Mn(II) complexes, 2 and 3, as their dichloro complexes, [Mn([15]aneN5)Cl2]. The purpose of the work is to probe the role that substituent (methyl and fused cycloalkyl) groups exert on the catalytic activity and stability of the complexes. All of the 18 new complexes described here are catalysts for the dismutation of superoxide, and substantial substituent effects are observed with kcat values at pH = 7.4 varying in the range (1.4−9.1) × 107 M-1 s-1. The kinetic and thermodynamic stabilities of these new complexes were studied by a Cu(II) ion competition spectrophotometric assay and by potentiometric titrations, respectively. It is observed that increasing the number of carbon substituents increases the kinetic stability of the complexes, while the thermodynamic stability increases, but less dramatically. The crystal structure for one of these new complexes, [Mn(2,2,3,3-Me4[15]aneN5)Cl2], 2i, was determined, and reveals that the Mn(II) ion is coordinated by a pentagonal bipyramid array of five macrocyclic derived nitrogen atoms in a plane with the Mn(II) center and capped by trans-dichloro ligands. Crystal data for MnC14H33N5Cl2·CH3CH2OH: triclinic, P1̄-Ci1 (#2), a = 9.941(2) Å, b = 11.190(2) Å, c = 11.613(2) Å, V = 1125.4(4) Å3, Z = 2, T = 20 ± 1 °C, for 3357 independent absorption-corrected reflections having 2θ(Cu Kα) < 120.0° and I > 3σ(Ι), with Rint = 0.027.

Protective effect of 4-hydroxy-TEMPO, a low molecular weight superoxide dismutase mimic, on free radical toxicity in experimental pancreatitis

Rats develop acute pancreatitis when infused iv for 3 h with cerulein (10 micrograms/kg/h). Autopsies of the pancreas seen by light microscope show interstitial edema, acinar cells vacuolization, and leukocyte margination in pancreatic capillaries; under electron microscope, severe damage concerning mitochondrial and zymogen granules structures are apparent. Particularly, swelling of the mitochondria and disruption of mitochondrial cristae was observed as well as formation of large vacuoles arising from zymogen granules and liposome fusion. A significant increase of lipid hydroperoxide level in the pancreatic tissue was observed. The purpose of this study was to evaluate the effect of 4-hydroxy-TEMPO--a low-mol-wt superoxide dismutase mimic--in a rat cerulein model of acute pancreatitis, with the expectation that free radical mediated hydroperoxide formation and tissue damage may be reduced significantly. Twenty-one male Wistar rats were divided into three groups: Group 1 (n = 5) served as a control and was infused iv for 3 h with physiologic saline; Group 2 (n = 8) was infused i.v. for 3 h with cerulein 10 micrograms/kg/h; and Group 3 (n = 8) infused i.v. both with cerulein and 4-hydroxy-TEMPO 22.6 mg/kg/h. Pancreatic tissue damage was quantified by measuring lipid hydroperoxide (LOOH) level, the weight of the organ, and by light and electron microscopic examination. 4-hydroxy-TEMPO penetration across cellular membrane barriers was quantified by ESR spectrometric measurements of 4-hydroxy-TEMPO concentration in pancreatic tissue samples and pancreatic juice as well. Administering 4-hydroxy-TEMPO to rats resulted in preventing both lipid hydroperoxide formation and severe morphological damage. 4-hydroxy-TEMPO crossed cellular membrane barriers and was excreted to pancreatic juice. Infusion of 4-hydroxy-TEMPO appears to prevent pancreatic injury caused by free radicals in experimental cerulein pancreatitis.

Protective Effects of the SOD-mimetic SC-52608 Against Ischemia/Reperfusion Damage in the Rabbit Isolated Heart

An experimental model of myocardial ischemia/reperfusion injury was used to assess the cardioprotective effects of SC-52608, a low molecular weight superoxide dismutase mimetic. Langendorff perfused rabbit isolated hearts were subjected to 30 min of global ischemia followed by 45 min of reperfusion. Hearts perfused in the presence of 20 μM SC-52608 exhibited a decrease in the release of creatine kinase and intracellular potassium compared to hearts receiving vehicle (control). A progressive increase in left ventricular end-diastolic pressure developed upon reperfusion in all hearts, but was significantly greater in control hearts when compared to hearts with SC-52608 (P < 0.05). In addition, results obtained with a radiolabeled monoclonal antibody to the intracellular protein myosin, indicate an increased degree of irreversible damage in vehicle-treated hearts. Myocardial protection was not significant in an additional group of hearts with 10 μM SC-52608. The hemodynamic, biochemical, morphological, as well as the antimyosin binding data, demonstrate that pretreatment with SC-52608 protects the myocardium from damage associated with global ischemia and reperfusion. The mechanism by which SC-52608 mediates the observed protective effect is most likely related to its ability to scavenge superoxide.

Antimutagenicity of a low molecular weight superoxide dismutase mimic against oxidative mutagens.

A set of stable nitroxide free radicals that are used as spin labels have been shown to possess metal-independent superoxide dismutase-like activity. Unlike superoxide dismutase (SOD), these compounds are low molecular weight, and readily penetrate into the cell. A representative nitroxide, 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (Tempol), was investigated for antimutagenic activity in the XPRT forward mutation assay in CHO AS52 cells. AS52 cells were exposed to hydrogen peroxide, or the hypoxanthine/xanthine oxidase superoxide generating system, in the presence or absence of 10 mM Tempol. Tempol itself was not mutagenic or toxic to AS52 cells. Tempol protected cells nearly completely from the cytotoxic and mutagenic effects of hydrogen peroxide and hypoxanthine/xanthine oxidase. We have previously shown that nitroxides do not alter the extracellular concentration of hydrogen peroxide, and that they are taken up by mammalian cells, suggesting that the antimutagenic activity of Tempol is an intracellular phenomenon.

A novel metal-free low molecular weight superoxide dismutase mimic.

2-Ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH), the one-electron reduction product of the stable nitroxide radical, 2-ethyl-2,5,5-trimethyl-3-oxazolidinoxyl (OXANO), is reportedly oxidized by superoxide, and its oxidation has been proposed as a method for assaying superoxide. We find that superoxide can both reduce OXANO and oxidize OXANOH. The respective rate constants, k1 and k2, were determined using two superoxide-generating systems (xanthine oxidase/xanthine as well as ionizing radiation). OXANOH oxidation and OXANO reduction are both inhibitable by superoxide dismutase, pH-dependent (4.5-9.3), and result in a steady state distribution of [OXANO] and [OXANOH], independent of their initial concentrations, i.e. the OXANO/OXANOH couple exhibits a metal-independent superoxide dismutase-like function. Thus it provides a prototype for future development of improved low molecular weight superoxide dismutase mimics which will also function in cellular hydrophobic (aprotic) compartments such as membranes.