Abstract

M

S

, H

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Superoxide Dismutase as a Potential Therapeutic Agent*

Dysmutaza ponadtlenkowa jako potencjalny czynnik terapeutyczny

Department of Biochemistry Medical University of Warsaw, Poland Adv Clin Exp Med 2007, 16, 4, 561–568

ISSN 1230−025X

REVIEWS

© Copyright by Silesian Piasts University of Medicine in Wrocław

Abstract

Superoxide dismutase (SOD) is a major antioxidative enzyme and the only one that neutralizes superoxide ion, a precursor of reactive oxygen species (ROS) and their reactive derivatives. ROS can cause considerable damage to biological systems and are implicated in many pathologies, e.g. neurodegenerative diseases, tumors, atheroscle− rosis, hypertension, and diabetes. This paper presents a history of the discovery of superoxide dismutase and the functions of particular SOD isoforms in the organism. It also describes the possibilities of their use in the treat− ment of free−radical−based diseases (Adv Clin Exp Med 2007, 16, 4, 561–568).

Key words: superoxide dismutase, oxidative stress, enzymatic therapy, SOD mimetics.

Streszczenie

Dysmutaza ponadtlenkowa (SOD) jest głównym i jedynym enzymem antyoksydacyjnym, który unieczynnia w ko− mórkach anionorodnik ponadtlenkowy – prekursor wolnych rodników tlenowych (w.r.t.) i ich reaktywnych po− chodnych. Skutki działania w.r.t. prowadzą do rozwoju wielu patologii (chorób neurodegeneracyjnych, nowotwo− rów, miażdżycy, nadciśnienia, cukrzycy i innych). W artykule przedstawiono historię odkrycia dysmutazy ponad− tlenkowej i funkcje, jakie pełnią poszczególne izoenzymy SOD w organizmie, omówiono także możliwości ich zastosowania w terapii chorób o podłożu wolnorodnikowym (Adv Clin Exp Med 2007, 16, 4, 561–568).

Słowa kluczowe:dysmutaza ponadtlenkowa, stres oksydacyjny, terapia enzymatyczna, mimetyki SOD.

* This work was supported by grants nos. 1WK/W2/06, 1WK/W1/07, and 1WK/WB1/06.

The copper−containing superoxide dismutase (SOD) was first isolated from bovine erythrocytes in 1938 [1]. In the 1960s, SOD was also isolated from brain and liver and it was observed that under inflam− matory conditions, SOD protects cells against inflammatory injury, which indicated the anti−inflam− matory properties of this protein [2, 3]. The exact mechanism of the reaction catalyzed by SOD was finally explained in 1968–1969 by McCord and Fridovich. They showed that superoxide dismutase catalyzes the superoxide ion dismutation to hydrogen peroxide and molecular oxygen [4]. Thus just 30 years after the isolation of SOD protein by Mann and Keilin and the discovery of superoxide ion by Pauling, McCord and Fridovich showed that this pro− tein in vivocatalyzes the two−stage reaction of super− oxide ion dismutation to hydrogen peroxide [1, 5].

Although SOD’s reaction mechanism was understood, the physiological role of O2

–

was still unknown, and it was not known why it is neutral− ized by SOD. A discovery by Babior et al. linked together all the known observations concerning SOD [6]. They found that phagocytosing neu− trophils produce large amounts of superoxide ion, which might indicate its bactericidal function. Soon it was shown in work by McCord that super− oxide ion produced by neutrophils is responsible for the oxidative damage to tissue associated with the inflammatory process, and SOD could protect cells from this damage [7].

SOD1). In 1970, Keele et al. isolated a superoxide dismutase that contained a manganese ion in its active center from E. colicells and they called it MnSOD(SOD2) [8]. Soon thereafter, MnSOD was found in the mitochondria of mammalian cells [9]. In 1973, ferric dismutase (FeSOD), a homologue of MnSOD, was isolated from E. coli cells. It exists mainly in prokaryotic and in several lower eukaryotic organisms, e.g. yeasts [10]. The last SOD isoenzyme was discovered relatively late, in 1982: extracellular superoxide dismutase (EC− SOD, SOD3). Its presence was revealed in extra− cellular fluids such as serum, lymph, synovial fluid, and cerebrospinal fluid. This enzyme also exists on extracellular matrix and the outer surface of cells because of the strong affinity to proteogly− cans commonly found on the outer cell surface. EC−SOD contains copper and zinc ions in its active center; however, it does not have the immunospecificity typical for CuZnSOD and it has a larger molecular mass. It is the only enzyme that removes superoxide ion from extracellular spaces [11]. Following years have been a time of intensive research on SOD isoenzymes, character− izing their structures, properties, and functions.

Functions of SOD

Superoxide dismutase (SOD, EC 1.15.1.1.) is found in all aerobic organisms, both pro− and eukaryotic. It is a metalloprotein, containing metal ions (Fe, Ni, Cu, Zn, Mn) in its active center [12]. This enzyme catalyzes the two−stage reaction of superoxide ion (O2–) dismutation to hydrogen per− oxide (H2O2).

2O2 –

+ 2H+→_H

2O2+ O2

k = 2 × 109_M–1_s–1

In the human organism, SOD functions in dif− ferent forms which together form the system of superoxide ion neutralization in the mitochondria, nucleus, cytosol, and extracellular space. There are three dismutase isoenzymes: copper−zinc (CuZnSOD, SOD1), found in the cytosol and nucleus; manganese (MnSOD, SOD2), found almost exclusively in mitochondria; and the extra− cellular superoxide dismutase (EC−SOD, SOD3) in the extracellular space and fluids [12, 13]. Available scientific data indicates that superoxide dismutase is a key enzyme which scavenges the ROS precursor superoxide ion both in vitroand in vivo. Under conditions of oxidative stress, which often accompany many pathologies (atherosclero− sis, hypertension, tumor, liver cirrhosis, and autoimmune and neurodegenerative diseases), SOD has particular significance.

Among the SOD isoenzymes, the mitochondr− ial superoxide dismutase (i.e. MnSOD) is the first and most important line of defense against toxic superoxide ion [14, 12]. The second line of defense is CuZnSOD in the cytosol. This isoen− zyme supports the protective function of MnSOD by neutralizing the O2– generated in the cytosol and leaking from the mitochondria. Such coopera− tion provides efficient elimination of superoxide ion excess [15]. EC−SOD scavenges O2–on outer surfaces of cells and in the extracellular matrix and fluids. Besides protecting cell surface, it removes O2–from the vasculature. It has great importance in preserving the bioactivity of NO in the cardio− vascular circulation and endothelial tissue [16]. Scientific data indicate that superoxide dismutase, by maintaining a balance between O2

–

removal and H2O2generation in cells, participates in the regula− tion of the most important life processes, such as proliferation and apoptosis [17].

The functions of the SOD isoenzymes in cells indicate that they play a significant role in the pathologies of many diseases, e.g. atherosclerosis, diabetes, neurodegenerative diseases, acute rheumatoid arthritis, and tumors [18–21].

Capabilities of SOD’s

Therapeutic Application

Growing scientific data point to the great importance of both ROS and SOD in numerous physiological processes. ROS participate in an unusually wide range of processes, which suggests that free radicals might contribute to many dis− eases of unknown origin or mechanism. There are also many suggestions for the direct application of SOD in the therapy of free−radical−based diseases. As a precursor of all ROS, superoxide ion in large measure contributes to the damage of biolog− ically important molecules (DNA, proteins, lipids) in cells and, consequently to tissue injury. The accumulation of structural damage could be the cause of many diseases (coronary artery disease, tumors, arthritis, Crohn`s disease, autoimmune and neurodegenerative diseases, diabetes, and others) [14, 22, 23]. Many scientific publications indicate the possibility of applying antioxidants in the treat− ment of free−radical−based diseases. By neutraliz− ing excess ROS, antioxidants might efficiently pre− vent or palliate disease symptoms. Therefore it is reasonable that superoxide dismutase, a superoxide ion scavenger, should demonstrate exceptionally beneficial therapeutic effects.

mainly used, and introducing the alien protein to body fluids (blood or synovial fluid) induced a strong immunological response and allergic reaction in the organism. Applying human recom− binant CuZnSOD was also inconvenient. Due to its low molecular weight, CuZnSOD undergoes rapid renal clearance and the duration halftime of this protein in the vasculature (6–10 minutes) is too short for effective therapy. In addition, this protein penetrates cells from body fluids weakly.

Still, the possibilities of the therapeutic use of SOD hold promise and attempts are underway to solve problems which appear. It was shown that covalent binding of CuZnSOD to a large polymer− ic molecule, e.g. polyethylene glycol, inhibits CuZnSOD renal clearance and extends its halftime in the vasculature to up to 30 hours and decreases its immunogenicity. Covalent binding of CuZnSOD with hydrophobic anions or fatty acids could be another solution which leads this modi− fied protein to bind with albumins or anchoring in cell membranes. Linking CuZnSOD with hialuro− nic acid also allows its adherence to cell surfaces and increases its halftime in the vasculature [24, 25]. CuZnSOD protein could also be packed in liposomes and introduced in this form to the blood circulation. This method extends CuZnSOD’s half− time in the vasculature and facilitates its penetra− tion to cells because the liposomes are absorbed by cells as entire particles. Introducing SOD protein absorbed by special carriers, i.e. hydrogels, to the organism yielded interesting results. Recent research showed that this network of polymers containing great amounts of water can very effi− ciently absorb SOD molecules and release them after 72 hours from introduction to the organism [26]. Increased growth of fibroblast cultures was observed after the addition of hydrogels containing SOD, demonstrating its significance in the therapy and regeneration of injured tissues [24]. Due to the greater stability of MnSOD compared with CuZnSOD in the circulation (5–6 hours), its appli− cation might bring much better therapeutic effects. The particular mechanisms of the activation and function of SOD isoenzymes and their role in the pathogenesis of many diseases are the subjects of numerous studies in molecular biology. These stud− ies, besides explaining the details of the develop− ment of free−radical−based diseases, also contribute to introducing new therapeutic strategies. At the cur− rent stage of research, SOD isoenzymes are applied as potential therapeutic agents using the following strategies: 1) enhancing cells’ abilities to remove superoxide ion [27], 2) application of SOD mimetics [28, 29], and 3) supplementary enzyme therapy [30]. The first strategy is aimed at increasing native SOD activity and/or gene expression. This may be

with drugs that are already in common use, such as the anti−hypertensive (candesartan, an angiotensin receptor antagonist) and inhibitors of angiotensin I to angiotensin II converting enzymes (ACE), such as ramipril. Upregulation of CuZnSOD activity in heart tissue was shown as well as prevention against the downregulation of EC−SOD expression in the vascular endothelium, and the functioning of the vascular endothelial smooth muscles was also improved [31–33]. These results found confirmation in large−scale studies conducted recently involving the application of selected drugs (ramipril and vita− min E) to decrease cardiovascular disease risk and outcome (HOPE, The Heart Outcomes Prevention Evaluation). The HOPE studies showed that vitamin E did not decreased cardiovascular disease risk. However, ramipril decreased blood pressure, signif− icantly reduced the risks of stroke, myocardial infraction, atherosclerosis, and diabetic nephropathy [34–36]. Since ramipril is an inhibitor of angiotensin I−converting enzymes, it protects against a decrease in EC−SOD expression in the endothelium, provid− ing better antioxidant protection to blood vessels and increasing the bioavailability of nitric oxide.

In the case of SOD mimetic drugs, compounds such as a porphyrin−based drug containing Mn(III) (MnTBAP), salen−manganese complexes (EUK−8), nitroxide derivatives (tempol), and low−weight manganese or iron chelators, that can mimic either SOD or catalase activity (or both), are adminis− tered over long periods at protective doses [37].

Supplementary enzyme therapy relies on com− plementation of SOD activity by other antioxidant enzymes. An interesting approach was the applica− tion of a SOD−catalase conjugate. Both enzymes in this conjugate are linked by a glutaryl dialde− hyde molecule. The halftime of this conjugate in the blood of rats was about 4 hours. The SOD part present in the conjugate was protected by the cata− lase part against inactivation by H2O2[38, 39].

Recently, a so−called “super−SOD” was con− structed. This chimeric protein contains an MnSOD (SOD2) primary structure plus the 26−amino−acid C−terminus of EC−SOD (SOD3). This tail allows SOD2/3 adherence to endothelial surfaces. As the chimera is less positively charged than SOD3, it does not bind as strongly to cell sur− faces, allowing it successful intravascular admin− istration [40, 41].

Clinical Trials of SOD

Application

numerous free−radical−based diseases in many experiments. Initially, before clinical trials of SOD application, studies of its use in animal models were conducted. The administration of CuZnSOD, catalase, and allopurinol showed protective effects in experimental rat acute pancreatitis [42]. CuZnSOD or catalase also suppressed experimen− tal autoimmune neuritis symptoms in rats [43]. The administration of polyethylene glycol−conju− gated CuZnSOD and desferroxyamine reduced oxidative injuries in rat brains in experimental brain ischemia and experimental meningitis [44, 45]. Similarly, CuZnSOD showed strong anti− inflammatory effects in experimental ischemia in island skin flaps. Infusion of CuZnSOD also pre− vented against skin injuries during ischemia−reper− fusion [46]. An increase in rat resistance to daily exposure to 100% oxygen was achieved by inject− ing polyethylene glycol−conjugated CuZnSOD to their lungs [47]. CuZnSOD conjugated with poly− ethylene glycol showed its effectiveness in the therapy of heart muscle ischemia and reperfusion arrhythmia, protected lungs against damage caused by E. coli, and decreased ischemia−reperfu− sion injuries in liver and kidneys [48].

Besides conjugation with polyethylene glycol, SOD may also be linked to a polyanionic com− pound such as divinyl ether and maleic anhydride, known as DIVEMA. Administration of the SOD− DIVEMA conjugate to rats with liver fibrosis led to decreased ROS generation in liver cells and inhi− bition of liver inflammation [49]. SOD−DIVEMA protected rat lungs also against lung edema and inflammation changes [50].

Protective and palliative activities of SOD were shown also in the case of skin burns. The administration of CuZnSOD packed to liposomes to rabbits with skin burns or to blood in the form of a gel reduced edema, decreased the area of tis− sue injury and necrosis, and accelerated wound healing [51].

Because EC−SOD scavenges superoxide ion from cell surfaces and vasculature lumen, this enzyme functions as a protector of NO bioactivity in the cardiovascular circulation and endothelial tis− sue. Therefore its application is possible in the ther− apy of coronary artery disease, hypertension, dia− betes, and in lung degenerative diseases. This found confirmation in experiments in which EC−SOD administration protected rats against brain vessel injuries induced by acute hypertension [52].

During organ transplantation, ischemia−reper− fusion is a frequent phenomenon. It is a source of large amounts of ROS generated after the fresh influx of oxygenated blood to tissues. These ROS contribute to transplant tissue injuries. Therefore, the application of SOD during transplantation

should demonstrate positive effects. Indeed, the addition of CuZnSOD and catalase to the perfu− sion fluid during rat liver transplantation increased the survival rate of the transplanted organs [53]. Recently it was shown that the application of SOD linked with lecithin protects endothelial cells against ischemia−reperfusion injury [54].

Superoxide dismutase is also utilized in pro− tecting cell cultures in vitro against toxic ROS. Addition of SOD to the medium in cultured cell lines increased their survival rates [25]. It was observed that ROS in in vitro cultured mouse embryos blocked the first zygote division, and the presence of SOD in the culture medium allowed their further normal development [55]. Positive effects were shown in studies on the overexpres− sion of MnSOD in cell cultures. The human melanoma cell line A375 and the human histiocyt− ic lymphoma cell line U937, with MnSOD over− expression achieved by gene transduction, showed greater resistance to doxorubicin (an antitumor drug that induces the generation of large amounts of ROS). It could ensure greater viability of nor− mal cells during chemotherapy and, on the other hand, increase the sensitivity of tumor cells to dox− orubicin [56].

The positive results of SOD application in cell cultures and animal models opened the way to applying SOD isoenzymes in clinical trials.

Palliative effects of SOD application were shown in patients with diseases such as rheuma− toid arthritis, bladder inflammation resulting from irradiation, Crohn’s disease, and lupus erythe− matosus [57, 58]. Application of a topical gel con− taining liposomally encapsulated recombinant SOD in patients with painful Peyronie`s disease allowed a reduction in pain in as much as 89% of cases [59]. Lecithin−bound SOD decreased the sizes of defect in the cornea caused by keratiniza− tion, thermal or chemical burns, and corneal surgery. This has particular significance for pa− tients who do not respond to conventional therapy [60]. In premature infants, intratracheal treatment with recombinant human CuZnSOD partially pre− vented pulmonary injuries, which eventually lead to bronchopulmonary dysplasia. Additional bene− fits of this therapy were decreased incidence and symptoms of lung disease over the first year of life of premature infants [61, 62].

At the present moment, CuZnSOD is most widely applied in transplantology, where it is employed as one of the components in some per− fusion fluids together with other antioxidant com− pounds, e.g. reduced glutathione (GSH), allopuri− nol, and desferroxyamine mezylan (Carolin’s fluid) [25].

EC−SOD

as a Therapeutic Agent

Due to its function in the human organism, the most promising therapeutic agent seems to be EC−SOD. The decreased activity of this enzyme observed in many diseases indicates it as a poten− tial target in antioxidant therapy. There are a lot of experimental data concerning the application of EC−SOD. EC−SOD’s duration halftime in blood vessels (20 h) and strong affinity to heparan sulfate in vessel walls both endorse the application of EC−SOD in therapy. Due to its properties, the administration of a synthetic EC−SOD (recombi− nant rEC−SOD) might efficiently prevent distur− bances related to exaggerated production of super− oxide ion [64, 65].

Numerous studies (most of all on animal mod− els) have shown the usefulness of EC−SOD in the therapy and prevention of such diseases as arthri− tis, liver intoxication, coronary disease, pathology of brain blood vessels, and others [66–68]. The usefulness of EC−SOD in genomic therapy was also examined, as it is a factor protecting cells against oxidative damage [67]. The ability of EC−SOD to bind to the cell surface allows better protection of heart and blood vessels walls than other SOD isoenzymes administered intravascu− lary because they cannot penetrate to the intracel− lular space. Whereas EC−SOD, administered in the form of a viral vector, is the only SOD isoenzyme which is naturally secreted from cells, it is excep− tionally well adapted to production in the liver and further transport in the organism.

Genomic therapy of rabbits using EC−SOD enhances the possibilities of vasculature protection against atherosclerosis and particularly against myocardial infraction. It has great importance in the development of new cardioprotection strate− gies [69]. It was also shown that rat intravascular injections of adenoviruses containing EC−SOD decreased impaired relaxation of vascular endo− thelium smooth muscles caused by LPS (lipopoly− saccharides) [70]. In addition, EC−SOD gene transfer to mice liver cells distinctly contributed to a decrease in liver cell injury and necrosis res− ulting from paracetamol [71]. Application of EC−SOD genomic therapy in the treatment of murine collagen−induced arthritis reduced numer− ous symptoms of the disease [67].

The possibility of modifying EC−SOD activity and protein level in the organism could be useful in the treatment of many pathologies. Therefore, a better understanding of EC−SOD’s role in the pathogenesis of disease should provide knowledge about its potential therapeutic capabilities.

The authors conclude that since the discovery of superoxide dismutase 68 years ago, research into the function, structure, and physiological role of SOD has been conducted persistently. This research yielded many unexpected discoveries and provided massive knowledge about superoxide dismutase. An exact understanding of the func− tions and activity mechanisms of SOD isoenzymes in significant living cell processes allows us to understand the molecular and biochemical bases of the initiation and development of free−radical− based diseases. Understanding these processes allows for better prophylaxis, diagnostics, and therapy of these diseases. Research conducted in this field has already proved the efficiency of SOD, particularly EC−SOD, as a therapeutic factor and has overcome the initial problems related to its application in therapy. Although most of the stud− ies were conducted on animal models, the possi− bilities of applying SOD are unusually wide and give hope for more effective treatment of diseases in which reactive oxygen species participate.

References

[1] Mann T, Keilin D:Haemocuprein and hepatocuprein, copper protein compounds of blood and liver in mammals. Proc R Soc Ser B 1938, 126, 303–315.

[2] Carrico RJ, Deutsch HF: Isolation of human hepatocuprein and cerebrocuprein. Their identity with ery− trocuprein. J Biol Chem 1969, 244, 6087–6093.

[3] Huber W, Schule TL, Carson S, Goldhamer RE, Vogin EE:Some chemical and pharmacologic properties of a novel anti−inflammatory protein. Toxicol Appl Pharmacol 1968, 12, 308.

[4] McCord JM, Fridovich I:Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J Biol Chem 1969, 244, 6049–6055.

[5] Pauling L:The discovery of superoxide radical. Trends Biochem Sci 1979, 4, N270–N271.

[7] McCord JM: Free radicals and inflammation: Protection of synovial fluid by superoxide dismutase. Science 1974, 185, 529–531.

[8] Keele BB, McCord JM, Fridovich I:Superoxide dismutase from Escherichia coliB. A new manganese−con− taining enzyme. J Biol Chem 1970, 245, 6176–6181.

[9] Weisiger RA, Fridovich I:Mitochondrial superoxide simutase. Site of synthesis and intramitochondrial local− ization. J Biol Chem 1973, 248, 4793–4796.

[10] Yost FJ, Fridovich I:An iron−containing superoxide dismutase from Escherichia coli.J Biol Chem 1973, 248, 4905–4908.

[11] Marklund SL, Holme E, Hellner L:Superoxide dismutase in extracellular fluids. Clin Chim Acta 1982, 126, 41–51.

[12] Fridovich I:Superoxide anion radical (O2 –

), superoxide dismutases, and related matters. J Biol Chem 1997, 272, 18515–18517.

[13] Zelko IN, Mariani TJ, Folz RJ:Superoxide dismutase multigene family: A comparison of the Cu,ZnSOD (sod1), Mn−SOD (sod2), and EC−SOD (sod3) gene structures, evolution, and expression. Free Radic Biol Med 2002, 33, 337–349.

[14] Beckman KB, Ames BN:Endogenous oxidative damage of mtDNA. Mutat Res 1999, 424, 51–58.

[15] Isoherranen K, Peltola V, Laurikainen L, Punnonen J, Laihia J, Ahotupa M, Punnonen K:Regulation of copper/zinc and manganese superoxide dismutase by UVB irradiation, oxidative stress and cytokines. J Photochem Photobiol 1997, B 40, 288–293.

[16] Fattman CL, Schafter M, Oury TD:Extracellular superoxide dismutase in biology and medicine. Free Radic Biol Med 2003, 35, 226–256.

[17] Antunes F, Cadenas E:Cellular titration of apoptosis with steady state concentrations of H2O2: submicromolar

levels of H2O2induce apoptosis through Fenton chemistry independent of the cellular thiol state. Free Radic Biol

Med 2001, 30, 1008–1018.

[18] Szatrowski TP, Nathan CF:Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res 1991, 51, 794–798.

[19] Casado A, de laTorre R, Lopez−Fernandez EM, Carrascosa D, Casado CM, Ramirez VM:Superoxide dis− mutase and catalase blood levels in patients with malignant diseases. Cancer Lett 1995, 93, 187–192.

[20] Oberley TD, Oberley LW:Antioxidant enzyme levels in cancer. Histol Histopathol 1997, 12, 525–535.

[21] Gate L, Paul J, Nguyen−Ba G, Tew KD, Tapiero H:Oxidative stress induced in pathologies: the role of antiox− idants. Biomed Pharmacother 1999, 53, 169–180.

[22] Ciolino HP, Levine RL:Modification of proteins in endothelial cell death during oxidative stress. Free Radic Biol Med 1997, 22, 1277–1282.

[23] Halliwell B, Chirico S: Lipid peroxidation: its mechanism, measurement, and significance. Am J Clin Nutr 1993, 57 (Suppl.), 715–724.

[24] Dominguez A: Modifying superoxide dismutase for improved biopharmaceutical properties. Biotechnol Apl 2006, 23, 17–21.

[25] Bartosz G:Druga twarz tlenu. Wolne rodniki w przyrodzie. Wydawnictwo Naukowe PWN, Warszawa 2003, wyd. 2.

[26] Nguyen KT, West JL:Photopolymerizable hydrogels for tissue engineering applications. Biomaterials 2002, 23, 4307–4314.

[27] Ng TB, Gao W, Li L, Niu SM, Zhao L, Liu J, Shi LS, Fu M, Liu F:Rose (Rosa rugosa)−flower extract increas− es the activities of antioxidant enzymes and their gene expression and reduces lipid peroxidation. Biochem Cell Biol 2005, 83 (1), 78–85.

[28] Izumi M, McDonald MC, Sharpe MA, Chatterjee PK, Thiemermann C:Superoxide dismutase mimetics with catalase activity reduce the organ injury in hemorrhagic shock. Shock 2002, 18, 230–235.

[29] Pong K, Rong Y, Doctrow SR, Baudry M:Attenuation of zinc−induced intracellular dysfunction and neurotox− icity by a synthetic superoxide dismutase/catalase mimetic, in cultured cortical neurons. Brain Res 2002, 950, 218–230.

[30] Johnson F, Giulivi C:Superoxide dismutases and their impact upon human health. Mol Asp Med 2005, 26, 340–352.

[31] Umemoto S, Tanaka M, Kawahara S, Kubo M, Umeji K, Hashimoto R, Matsuzaki M:Calcium antagonist reduces oxidative stress by upregulating Cu/Zn superoxide dismutase in stroke−prone spontaneously hypertensive rats. Hypertens Res 2004, 27, 877–885.

[32] Buter H, Navis GY, Woittiez AJ, de Zeeuw D, de Jong PE: Pharmacokinetics and pharmacodynamics of can− desartan cilexetil in patients with normal to severely impaired renal function. Eur J Clin Pharmacol 1999, 54, 953–958.

[33] Pizzi C, Manfrini O, Fontana F, Bugiardini R: Angiotensin−converting enzyme inhibitors and 3−hydroxy−3− −methylglutaryl coenzyme A reductase in cardiac Syndrome X: role of superoxide dismutase activity. Circulation 2004, 109, 53–58.

[34] Sleight PJ:The HOPE Study (Heart Outcomes Prevention Evaluation). J Renin Angiotensin Aldosterone Syst 2000, (1), 18–20.

[36] Hoogwerf BJ, Young JB:The HOPE study. Ramipril lowered cardiovascular risk, but vitamin E did not. Cleve Clin J Med 2000, 67, 287–293.

[37] Muscoli C, Cuzzocrea S, Riley DP, Zweier JL, Thiemermann C, Wang ZQ, Salvemini D:On the selectivity of superoxide dismutase mimetics and its importance in pharmacological studies. Br J Pharmacol 2003, 140, 445–460.

[38] Maksimienko AV, Bezrukavnikova LM, Grigorieva EL, Yaglov VV, Torchilin VP:Effect of native and mod− ified forms of superoxide dismutase and catalase on experimental silicosis in rats. Ann NY Acad Sci 1992, 672, 118–125.

[39] Mao GD, Thomas PD, Lopaschuk GD, Poznansky MJ:Superoxide dismutase (SOD)−catalase conjugates. Role of hydrogen peroxide and the Fenton reaction in SOD toxicity. J Biol Chem 1993, 268, 416–420.

[40] Gao B, Flores SC, Leff JA, Bose SK, McCord JM:Synthesis and anti−inflammatory activity of a chimeric recombinant superoxide dismutase: SOD2/3. Am J Physiol Lung Cell Mol Physiol 2003, 284, L917–L925.

[41] Hernandez−Saavedra D, Zhou H, McCord JM:Anti−inflammatory properties of a chimeric recombinant super− oxide dismutase: SOD2/3. Biomed Pharmacoter 2005, 59, 204–208.

[42] Closa D, Bulbena O, Rosello−Catafau J, Fernandez−Cruz L, Gelpi E: Effect of prostaglandin and superoxide dismutase administration on oxygen free radical production in experimental acute pancreatitis. Inflammation 1993, 17, 563–571.

[43] Hartung HP, Schafer B, Heininger K, Toyka KV:Suppression of experimental autoimmune neuritis by the oxy− gen radical scavengers superoxide dismutase and catalase. Ann Neurol 1988, 23, 453–460.

[44] Liu TH, Beckman JS, Freeman BA, Hogan EL, Hsu CY:Polyethylene glycol−conjugated superoxide dismu− tase and catalase reduce ischemic brain injury. Am J Physiol 1989, 256, H589–H593.

[45] Pfister HW, Koedel U, Lorenzl S, Tomasz A:Antioxidants attenuate microvascular changes in the early phase of experimental pneumococcal meningitis in rats. Stroke 1992, 23, 1798–1804.

[46] Manson PN, Anthenelli RM, Im MJ, Bulkley GB, Hoopes JE:The role of oxygen−free radicals in ischemic tis− sue injury in island skin flaps. Ann Surg 1983, 198, 87–90.

[47] Tang G, White JE, Gordon RJ, Lumb PD, Tsan MF:Polyethylene glycol−conjugated superoxide dismutase protects rats against oxygen toxicity. J Appl Physiol 1993, 74, 1425–1431.

[48] Veronese FM, Caliceti P, Schiavon O, Sergi M:Polyethylene glycol−superoxide dismutase, a conjugate in search of exploitation. Adv Drug Deliv Rev 2002, 54, 587–606.

[49] Swart PJ, Hirano T, Kuipers ME, Ito Y, Smit C, Hashida M, Nishikawa M, Beljaars K, Meijer DK, Poelstra K:

Targeting of superoxide dismutase to the liver results in antiinflammatory effects in rats with fibrotic livers. J Hepatol 1999, 31, 1034–1043.

[50] Hirano T, Todoroki T, Morita R, Kato S, Ito Y, Kim K, Shukla PG, Veronese F, Maeda H, Ohashi S:Anti− inflammatory effect of the conjugate of superoxide dismutase with the copolymer of divinyl ether and maleic anhydride against rat re−expansion pulmonary edema. J Control Release 1997, 48, 131–139.

[51] Vorauer−Uhl K, Furnschlief E, Wagner A, Ferko B, Katinger H:Topically applied liposome encapsulated superoxide dismutase reduces postburn wound size and edema formation. Eur J Pharm Sci 2001, 14, 63–67.

[52] McIntyre M, Bohr DF, Dominiczak AF:Endothelial function in hypertension: the role of superoxide anion. Hypertension 1999, 34, 539–545.

[53] Connor HD, Gao W, Nukina S, Lemasters JJ, Mason RP, Thurman RG: Evidence that free radicals are involved in graft failure following orthotopic liver transplantation in the rat – an electron paramagnetic resonance spin trapping study. Transplantation 1992, 54, 199–204.

[54] Koo DD, Welsh KI, West NE, Channon KM, Penington AJ, Roake JA, Morris PJ, Fuggle SV:Endothelial cell protection against ischemia/reperfusion injury by lecithinized superoxide dismutase. Kidney Int 2001, 60, 786–796.

[55] Orsi NM, Leese HJ:Protection against reactive oxygen species during mouse preimplantation embryo development: role of EDTA, oxygen tension, catalase, superoxide dismutase and pyruvate. Mol Reprod Dev 2001, 59, 44–53.

[56] Suresh A, Guedez L, Moreb J, Zucali J: Overexpression of manganese superoxide dismutase promotes survival in cell lines after doxorubicin treatment. Br J Haematol 2003, 120, 457–463.

[57] Flohe L:Superoxide dismutase for therapeutic use: clinical experience, dead ends and hopes. Mol Cell Biochem 1988, 84, 123–131.

[58] Emerit J, Pelletier S, Likforman J, Pasquier C, Thuilier A:Phase II trial of copper zinc superoxide dismutase (CuZnSOD) in the treatment of Crohn’s disease. Free Radic Res Commun 1991, 12–13, 563–569.

[59] Riedl CR, Sternig P, Galle G, Langmann F, Vcelar B, Vorauer K, Wagner A, Katinger H, Pfluger H:

Liposomal recombinant human superoxide dismutase for the treatment of Peyronie’s disease: a randomized place− bo−controlled double−blind prospective clinical study. Eur Urol 2005, 48, 656–661.

[60] Shimmura S, Igarashi R, Yaguchi H, Ohashi Y, Shimazaki J, Tsubota K:Lecithin−bound superoxide dismu− tase in the treatment of noninfectious corneal ulcers. Am J Ophthalmol 2003, 135, 613–619.

[61] Davis JM, Parad RB, Michele T, Allred E, Price A, Rosenfeld W; North American Recombinant Human CuZnSOD Study Group: Pulmonary outcome at 1 year corrected age in premature infants treated at birth with recombinant human CuZn superoxide dismutase. Pediatrics 2003, 111, 469–476.

[63] Xia XG, Zhou H, Zhou S, Yu Y, Wu R, Xu Z:An RNAi strategy for treatment of amyotrophic lateral sclerosis caused by mutant Cu,Zn superoxide dismutase. J Neurochem. 2005, 92, 362–367.

[64] Karlsson K, Sandstrom J, Edlund A, Marklund SL:Turn−over of extracellular superoxide dismutase in tissues. Lab Invest 1994, 70, 705–710.

[65] Stromqvist M, Houdebine M, Andersson JO, Edlund A, Johansson T, Viglietta C, Puissant C, Hansson L:

Recombinant human extracellular superoxide dismutase produced in milk of transgenic rabbits. Transgenic Res 1997, 6, 271–278.

[66] Li Q, Bolli R, Qiu Y, Tang XL, Murphree SS, French BA:Gene therapy with extracellular superoxide dismu− tase attenuates myocardial stunning in conscious rabbits. Circulation 1998, 98, 1438–1448.

[67] Iyama S, Okamoto T, Sato T, Yamauchi N, Sato Y, Sasaki K, Takahashi M, Tanaka M, Adachi T, Kogawa K, Kato J, Sakamaki S, Niitsu Y:Treatment of murine collagen−induced arthritis by ex vivoextracellular superox− ide dismutase gene transfer. Arthritis Rheum 2001, 44, 2160–2167.

[68] Nakane H, Chu Y, Faraci FM, Oberley LW, Heistad DD:Gene transfer of extracellular superoxide dismutase increases superoxide dismutase activity in cerebrospinal fluid. Stroke 2001, 32, 184–189.

[69] Li Q, Bolli R., Yumin Qiu, Xian−Liang Tang, Yiru Guo, French BA:Gene therapy with extracellular superox− ide dismutase protects conscious rabbits against myocardial infraction. Circulation 2001, 103, 1893–1898.

[70] Lund DD, Gunnett CA, Chu Y, Brooks RM, Faraci FM, Heistad DD:Gene transfer of extracellular superox− ide dismutase improves relaxation of aorta after treatment with endotoxin. Am J Physiol Heart Circ Physiol 2004, 287, H805–H811.

[71] Laukkanen MO, Leppanen P, Turunen P, Tuomisto T, Naarala J, Yla−Herttuala S:EC−SOD gene therapy reduces paracetamol−induced liver damage in mice. J Gene Med 2001, 3, 321–325.

Address for correspondence:

Hanna Czeczot

Department of Biochemistry Medical University of Warsaw Banacha 1

02−097 Warsaw Poland

Tel.: +48 22 572 06 93 E−mail: [email protected]

Conflict of interest: None declared