Superoxide dismutase: pharmacological developments and applications.

Superoxide Dismutase: Pharmacological Developments and Applications ~~~~~~~~~~~

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Bassam A. Omar, Sonia C. Flores, and Joe M. McCord Webb-Waring Lung Institiitv University of Colorado Health Science3 Center Denver. Colorado 80262

I. Introduction 11. The Protein Chemistry and Enzymology of the Superoxide Disniutascs 111. The Superoxide Kdical: Toxicity versus Reactivity tv. The Pathological Production of Superoxide Radical A . Heart B. Central Nervous System C. Gastrointestinal Tract D. Liver and Pancreas E. Lung F. Kidney V . The Pharmacodynaniics and Pharmacokinetics of the Superoxide Dismutases V I . Chemical Modification of Superoxide Dismutases VII. Genetic Modification of Superoxide Dismutases VIII. Down Syndrome and the Concept of "Oxidant-Antioxidant Balance" IX . Concluding Remarks References

1. Introduction The superoxide dismutases represent a unique family of enzymes, the sole purpose of which appears to be the rapid elimination of an already evaneAduon5 x lo4 M-sec-') (Armstrong and Buchanan, 1978). The rate constant for the reaction of superoxide with creatine phosphokinase is approximately equal to that for the reaction of superoxide with ferricytochrome c (McCord and Fridovich, 1969). The latter reaction is commonly used as an indicator (or even as a trapping reaction) for the radical, and is considered by most to be quite avid. Purified creatine phosphokinase has been shown to be inactivated on exposure to superoxide in the test tube, but the phenomenon may also be demonstrated in an intact organ. Postischemic reoxygenation of the isolated perfused rat heart (see below) results in the generation of superoxide radicals that inactivate creatine phosphokinase in situ. Hearts perfused with 20 pglml SOD contained 26% more creatine phosphokinase activity

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than untreated hearts after 10 min of reperfusion, representing a total preservation of the activity (McCord and Russell, 1988). The questions of whether superoxide is toxic and whether the true function of the SODS is to eliminate 0; have been elegantly addressed by Touati and co-workers (Carlioz and Touati, 1986; Natvig et ul., 1987). Over the years, some have suggested that superoxide is an innocuous metabolite (Sawyer and Valentine, 1981) and have entertained the possibility that “superoxide dismutase activity is a trivial property of metalloproteins, inherent to the metal ion, and the real biological functions have not yet been discovered” (Fee, 1982). Carlioz and Touati (1986) described the production of a mutant of Escherichirx coli devoid of both the Mn-SOD (sodA) and the Fe-SOD (sodl?).This mutant showed normal anaerobic growth but could not grow aerobically in minimal medium. This does not prove that SOD is necessary for the dismutation of superoxide-one could argue that other “unknown functions” of the two proteins could be vital for aerobic growth. The coup de grcice, however, was the demonstration that the ability to grow in oxygen could be restored to the sodA sodB mutant by expression of a plasmid encoding the evolutionarily unrelated human Cu,Zn-SOD (Natvig rt al., 1987).That is, the only obvious thing in common between the missing E. coli gene products and the supplied human gene product is the ability to dismute superoxide. The likelihood that the human Cu,Zn-SOD would serendipitously possess the other “unknown activities” of the bacterial proteins seems extraordinarily remote.

IV. The Pathological Production of Superoxide Radical The amount of evidence implicating the superoxide anion in various pathologies is overwhelming. SOD has been used as the primary tool for collecting such evidence in various experimental models. While the generation of free radicals in general, and superoxide in particular, has been unquestionably demonstrated in almost every organ in the body, the ability of SOD to protect in certain specific models is still unclear. The following sections will address the studies done with SOD, surveying the various models examined in the major organs, and pointing out controversies. The sources of oxyradicals will also be addressed briefly for each organ or experimental model discussed.

A. Heart Recent studies with spin-trapping probes confirm that a variety of free radicals are generated in the reperfused heart (Blasig ei af.,1986; Arroyo et

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al., 1987; Garlick et al., 1987; Bolli, 1988; Weglicki et al., 1988). Moreover, if hearts are perfused with SOD, all trapped radical signals are attenuated, suggesting that all are derived secondarily from superoxide (Zweier et al., 1987, 1989). Three major models of myocardial oxyradical damage have been examined in the literature, namely arrhythmias, stunning, and infarction. While the role of SOD in ameliorating arrhythmias and stunning has been well established, its protection against myocardial tissue necrosis is still a matter of intense debate. Each one of these models will be discussed separately. A number of studies examined the effect of SOD on reperfusion-induced arrhythmias. Riva et al. (1987) obtained a dose-response curve using SOD and demonstrated a bell-shaped curve with a reduction of the incidence of ventricular fibrillation and mortality at lower doses and loss of protection at higher doses. Watanabe et a / . (1989a,b) demonstrated protection against arrhythmias in rats using an SOD derivative that circulates bound to albumin (SMA-SOD), which had a prolonged half-life of 6 hr and accumulated in tissues with a decreased local pH. These investigators found no protection with native SOD, at 5 and 20 mg/kg, two doses that were previously employed in the dose-response curve of Riva et ul. (1987), and also reported to have no effect on the incidence of arrhythmias. Mehta et al. (1990) induced thrombi in dogs and found SOD to potentiate the protective effect of thrombolysis with tissue plasminogen activator on the incidence of premature ventricular contractions. Several in uitro studies using isolated heart preparations have also demonstrated a protective effect of SOD on reperfusion arrhythmias. Woodward and Zakaria (1985) showed that SOD at concentrations of 5 , 10, and 20 U/ml decreased the incidence of ventricular fibrillation in isolated rat hearts by 38, 64, and 76%, respectively. Bernier et al. (1989) carried out a doseresponse curve using SOD and showed that the protective effect obtained at lower doses was lost at higher doses. Yamakawa et al. (1989) found a significant reduction in the incidence of arrhythmias using polyethyleneglycol-modified SOD (PEG-SOD),but not native SOD, in the isolated rat and guinea pig hearts. Although the source of free radicals causing arrhythmias is not known, some studies (Hearse et af., 1986; Manning et a/., 1988) show that allopurinol protects against arrhythmias in the rat heart, therefore implicating xanthine oxidase as a possible source of these radicals. The role of SOD in protecting the stunned myocardium has been somewhat controversial. M. L. Myers et al. (1983, Przyklenk and Kloner (1986), Gross et al. (1986), and Hatori et al. (1989) all reported positive effects of a combination of SOD and catalase on the stunned canine myocardium. Mehta et al. (1990) administered SOD with tissue plasmino-

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gen activator in dogs and also reported a positive effect. Buchwald et al. (1989) used SOD alone and found a mild but significant improvement of the recovery of myocardial segment shortening. All of the above-mentioned studies, except the study by Hatori et al., employed less than 20 min of ischemia. Nejima et al. (1989) and Przyklenk and Kloner (1989) reported negative effects of SOD plus catalase on the functional recovery of dog hearts subjected to 90 and 120 min of ischemia, respectively. Recently, Jeroudi et al. (1990) reported modest, but nonsignificant, effects of SOD and catalase on the recovery of regional function of the canine myocardium when given separately, but a significant improvement when given together. This implicates other cytotoxic radical species, besides superoxide, in the genesis of postischemic myocardial dysfunction. Xanthine oxidase has been suggested to be a primary source of free radicals causing myocardial stunning in the dog heart, based on the protection obtained with allopurinol (Charlat et a l . , 1987; Bolli et ul., 1988). Neutrophils have also been suggested to contribute to the postischemic dysfunction in the myocardium (Engler and Covell, 1987). The reduction of infarct size by SOD has been a matter of intense controversy for the past decade. A number of reviews concentrated on trying to resolve some of the controversies regarding the reduction of infarct size by SOD (Engler and Gilpin, 1989;Downey et al., 1991)or other 1989). Jolly et al. (1984) found smaller radical scavengers (Reimer et d., infarct size in dogs that received SOD and catalase at the time of reperfusion. Chambers et al. (1985)later reported a positive effect in open chest, nephrectomized dogs receiving SOD only. Werns et al. (1985)and Ambrosio et al. (1986) confirmed that SOD alone can reduce canine infarct size. Nakazawa et al. (1988) and Hatori rt al. (1989) also confirmed that SOD plus catalase can reduce canine infarct size. SOD plus catalase was reported to limit infarct size in a rabbit (Downey et al., 1987) and a porcine model (Naslund et al., 1986)of ischemia-reperfusion, and later SOD alone in a porcine (Hatori et d.,1987)and a rabbit (Omar et al., 1989) model as well. PEG-SOD alone has been reported to be protective in three studies (Tamura et a l . , 1988; Chi et u l . , 1989; Ohkubo et al., 1990). In the majority of these studies infarct size was evaluated by tetrazolium staining 24 hr or less following reperfusion with no collateral flow measurement in the dog studies. Unfortunately it takes at least 24 hr, preferably several days, of coronary artery reperfusion before changes become apparent at the light microscopic level. In the tetrazolium-staining method, dehydrogenase enzymes and NADH in surviving cells react with tetrazolium salts to produce a highly colored and insoluble formazan pigment (Klein et al., 1981; Nachlas and Schnitka, 1963). Because reperfusion quickly washes these components out of dead cells, it has been found that

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tetrazolium staining only several hours after reperfusion yields essentially the same infarct size as seen with histology several days after reperfusion (Horneffer et al., 1987). Gallagher er al. (1986), Uraizee e t a / . (1987), and Patel et al. (1990) found no protective effect with SOD on the canine infarct size following ischemia and reperfusion. Richard et al. (1988), Nejima et al. (1989), and Przyklenk and Kloner (1989) found no reduction in infarct size using SOD plus catalase in dogs subjected to coronary artery ligation and reperfusion. Klein et al. (1988) and Matsuda et (11. (1988) failed to show any myocardial salvage of SOD in pigs. Finally, Ooiwa et al. (1989) and Tanaka et al. (1990) failed to show protection with PEG-SOD against infarct size in the rabbit and dog, respectively. One possible explanation for the conflict in findings among the studies is that the earlier studies did not take an important determinant of infarct size, collateral flow, into account (Reimer e f a/., 1977; Miura er al., 1987). The importance of measuring collateral flow is illustrated in the study by Ambrosio et al. (1986), who found significantly smaller infarcts in the SOD-treated group when infarct size as a fraction of the risk zone was compared. Nevertheless, a plot of percentage infarction against collateral flow shows that the SOD-treated animals tended to have higher collateral flows. If a one-way analysis of variance with collateral flow as a covariate is performed on their published data, no significance is found. The collateral flow analysis argument, however, can hardly be applied to rabbit (Downey et al., 1987; Omar et al., 1989) or pig trials (Naslund et a/., 1986; Hatori et al., 1987) since neither of these species has significant coronary collateral circulation (Maestro et al., 1981; Maxwell rt al., 1987). Furthermore, the dog study of Werns et al. (1988) does use the collateral flow analysis. A second possible source of the discrepancy could be the method used to estimate the infarct size. All of the positive studies involved tetrazolium staining 24 hr or less after reperfusion. The most logical explanations are that protection afforded by SOD is simply not sustained, or that a second, slowly progressing, nonradical-mediated component of injury is revealed, once the radical-mediated component has been suppressed. Shirato er al. (1988) recently examined SOD alone in a rabbit model with three different reperfusion times. After 3 and 24 hr of reperfusion, tetrazolium indicated very small infarcts, but at 72 hr no differences were found by tetrazolium. One disturbing feature of that work was the direct comparison of infarcts by histology to those by tetrazolium in the 24-hr reperfusion group. SOD treatment seems to retard the rate of loss of dehydrogenase enzyme and cofactor from necrotic tissue, perhaps by preserving capillary integrity (Granger et al., 1986). Much of the evidence supporting the involvement of xanthine oxidase in

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myocardial reperfusion injury has been obtained indirectly from the protection afforded by allopurinol to the reperfused heart. The effect of allopurinol on canine infarct size has been discrepant (Reimer and Jennings, 1985; Werns et al., 1986). The causes of this discrepancy are not clear, but one point is that the pretreatment period, which has been longer in the positive studies, appears to be important for allopurinol to be converted into the noncompetitive inhibitor, oxypurinol (Werns, 1990). Indeed, when both allopurinol and oxypurinol were administered acutely with no pretreatment, only oxypurinol was protective (Cross. 1990). The cardiac xanthine oxidase data are further complicated by important species differences. Both the rat and the dog contain at least several orders of magnitude higher amounts of xanthine oxidase in their hearts than rabbit, pig, or man (Grum et u!., 1986; Parks and Granger, 1986; Eddy er ul., 1987). We and others have used the rabbit heart as a model of the xanthine oxidase-deficient human heart. Enzyme-inhibiting doses of allopurinol have not been found to be protective in rabbit heart (McCord et al., 1989; Downey rt ul., 1986). The involvement of neutrophils in myocardial reperfusion injury has been examined by several investigators. Administration of anti-Mo- 1 antibody (Simpson t t ul., 1988b), a reagent that prevents neutrophils from interacting with vascular and myocardial cells during the phase of reperfusion (Simon er al., 1986), or neutrophil depletion (Simpson rt ul., 1988a) resulted in a significant decrease of infarct size in the canine heart subjected to 90 min of ischemia and reperfusion.

B. Central Nervous System Brain ischemia in experimental models is associated with distinct free radical pathology that affects the predominant membrane lipids in the ischemic tissues. Kirsch et ul. (1987) recently demonstrated an electron spin resonance (ESR) signal using the spin adduct phenyl-r-butyl nitrone (PBN) in rat brains exposed to global ischemia followed by reperfusion. Yusa et al. (1984) used liposome-encapsulated SOD and catalase to protect rats against convulsions caused by hyperbaric oxygen exposure. They found this treatment to significantly enhance the levels of these enzymes in brain tissue and to increase the time to convulsions. Schettini et al. (1989) found SOD to increase the recovery of cerebral blood flow, brain elastic response (a measure of brain edema), and survival in dogs exposed to cerebral compression for 15 min followed by decompression. Ando ef al. (1989) found SMA-SOD to protect against edema in rat brains caused by applying a liquid nitrogen-cold probe over the bony skull for 20 sec. Using a model of concussive brain injury to cats, Wei et al. (1981) demonstrated

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inhibition of the vasodilation and the reduced responsiveness to the vasoconstrictor effects of hypocapnia by SOD after brain injury. Wei et al. (1985) and Zhang and Ellis (1990) demonstrated that norepinephnneinduced acute hypertension in cats resulted in the generation of superoxide in the brain, causing sustained vasodilation that was inhibited by SOD. Cerchiari et al. (1987, 1990) examined a model of cerebral damage after cardiac arrest in dogs and found a combination of SOD plus deferoxamine to significantly improve cerebral blood flow and recovery of somatosensory and brainstem auditory evoked potentials. Block (1977) and Hilton et al. (1980) administered SOD intraperitoneally or subcutaneously to rats or mice. respectively, exposed to hyperbaric oxygen. They found no effect of this treatment on the time to convulsions and the time to death. Several studies examined the protective effect of SOD on central nervous system ischemia and reperfusion. Lim et d.(1986) found SOD to enhance the recovery of somatosensory evoked potential of the spinal cord of dogs subjected to a 10-min cross-clamping and release of the descending thoracic artery. Snelling et ul. (1987) and Davis et al. (1987) showed significant improvement in postischemic cerebral blood flow and somatosensory evoked potential recovery on SOD administration in models of reversible global and focal ischemia in the cat. Forsman et al. (1988) subjected dogs to 48 hr of cerebral ischemia without reperfusion by occluding the ascending aorta and found a combination of SOD and catalase to have no effect on the neurologic outcome. This suggests that oxyradicals are probably not a major source of damage in chronic complete brain ischemia or that native SOD was not able to cross the blood-brain barrier and protect the brain. Kitagawa et ul. (1989), on the other hand, subjected gerbils to bilateral common carotid artery occlusion for only 5 min and found pyran-SOD (SOD conjugated to divinyl ether-maleic acid copolymer, half-life 20 rnin), but not native SOD, to protect against immunohistochemical ischemic lesions of the hippocampus. Liu et al. (1989) found that a combination of PEG-SOD and PEG-catalase attenuates brain infarct volume in rats subjected to 90 min of right middle cerebral artery occlusion and 24 hr of reperfusion. Imaizumi et ul. (1990) used a rat model of acute focal cerebral ischemia and found liposome-entrapped SOD (which elevated the SOD activities of the blood and brain) to significantly decrease infarct size in brain slices. Inoue et al. (1990b)found SMA-SOD to significantly inhibit the increase in the intracranial pressure, vascular permeability, and brain edema in dogs subjected to 18 min of common carotid and vertebral artery occlusion followed by reperfusion. Phelan and Lange (1990) found intravenous liposomal SOD, but not free SOD, to augment brain SOD activity and protect it against ischemiah-eperfusion-induced membrane damage.

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The source of free radicals in the injured central nervous system is not clear. The prevalence of lipid radical pathology in such injury, however, implicates arachidonate metabolism as an important contributor of oxyradicals. A beneficial effect of steroidal and nonsteroidal antiinflammatory therapy has been documented in several models of brain ischemia (Hall and Travis, 1988; Kontos er al., 1985). Nonglucocorticoid 21-aminosteroids have been demonstrated to inhibit lipid peroxidation in the CNS (Braughler et al., 1987). One such agent, U74006F, has been reported to improve postischemic neuronal survival in gerbils (Berry el ul., 1987) and reduce infarction and improve glucose utilization in cat brain following cerebral ischemia (Silvia et al., 1987; Hall and Yonkers, 1988). The biochemical, physiological, and pharmacological evidence for the involvement of oxygen radicals and lipid peroxidation in central nervous system trauma and stroke has been recently reviewed (Braughler and Hall, 1989; Hall and Braughler, 1989). In the feline regional cerebral ischemia model, wherein one middle cerebral artery is occluded, there is a period of approximately 2 hr during which the situation is reversible; reperfusion before 2 hr in this model results in no discernable infarction and no clinical deficits (Flamm er a/., 1980). Three hours of occlusion, however, will result in progressive decline in blood flow (90-95%), which appears to coincide with irreversibility and amplification of morphologic damage (Ransohoff et al., 1980). The mechanism behind this hypoperfusion and its consequent damage is unknown, but there is some evidence that it may be free radical mediated. It has been postulated (Demopoulos er al., 1980) that lipid peroxides produced from lipid radical reactions selectively inhibit the endothelial synthesis of PGIz, which counteracts the proaggregating properties of thromboxane A2 in platelets. This will result in platelet aggregation and numerous platelet-induced microocclusions that exacerbate hypoperfusion.

C. Gastrointestinal Tract Reperfusion of the ischemic intestine results in an accelerated tissue damage that adds considerably to the already existing ischemic insult (Haglund and Lundgren, 1978). Granger ef al. (1980) have shown that 1 hr of local arterial hypotension (30 mmHg arterial pressure), followed by normotensive reperfusion, causes a significant increase in intestinal capillary permeability. In other studies, SOD (Granger er d . ,1981; Parks et d., 1982)and, to a lesser extent, catalase (Parks and Granger, 1984) significantly diminished the increase in capillary permeability following 1 hr of partial ischemia followed by reperfusion in the cat intestine, thereby implicating free radicals. Further experiments showed that the infusion of xanthine and xan-

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thine oxidase (a superoxide-generating system) caused an increase in capillary permeability in normal intestine comparable to that seen following ischemia (Grogaard et a/., 1982), which was also abolished by SOD pretreatment. Oshima et al. (1984) used a model of neonatal necrotizing enterocolitis in weanling rats and found a combination of SOD and catalase to limit the progression of intestinal necrosis and to increase viability. Perry et al. (1986) reported a protective effect of SOD on the ischemiainduced increase in vascular permeability of the small intestine. Younes et a/. (1987) observed a significant increase in lipid peroxidation products following reperfusion of the ischemic intestine, which was attenuated by SOD. Yoshikawa (1989) also demonstrated protection with SOD against reperfusion-induced gastric mucosal lesions in rats subjected to a 30-min clamping of the celiac artery. Droy-Lefaix et u/. (1989)subjected rats to 30 min of mesenteric artery occlusion followed by 24 hr of reperfusion and found SOD to significantly reduce jejunal and colonic lesions. Watanabe and co-workers (Kimura et al., 1989: Ozasa et a / . , 1990) subjected dogs to total small bowel ischemia by occlusion of the superior mesenteric artery, vein, and all collaterals for 60 min, followed by reperfusion for 14 or 28 days and found SOD to preserve ultrastructure and D-xylose absorption. The effect of SOD on ulcer-producing stress has also been examined. Dalsing et al. (1983) showed that SOD reduces mortality due to bowel infarction and perforation from 63 to 25% in rats receiving aminophylline intraperitoneally. Yoshida et a / . (1989) reported that SOD, alone or in combination with catalase, protects the gastric mucosa against platelet activating factor induced mucosal damage. Takemura et a / . (1989)induced acute gastric lesions in rats by 42°C hyperthermia for 40 min and found SOD, with or without catalase, to decrease the total area of gastric lesions. Oka et al. (1989)and Shibuya et af. (1989) used a water immersion restraint stress ulcer model in rats and guinea pigs respectively, and found SOD to significantly reduce the ulcer index. The protective effect of SOD has also been examined in models of hypotension and hemorrhagic shock. Schoenberg et al. (1984) found SOD to improve both morphological and biochemical intestinal variables in cats subjected to partial superior mesenteric artery occlusion (25-30 mmHg) for 2 hr followed by reperfusion for 1 hr. Yoshimura et al. (1989) bled rats for 30 min and found SOD to inhibit intestinal mucosal damage 30 min after reinfusion of the removed blood. Models of sepsis and peritonitis have also been examined. Limm et al. (1988) infected rats with an intraperitoneal injection of E. coli and found a combination of SOD and catalase to reduce mortality from 80 to 30%. Muramoto er al. (1990) found SOD to potentiate the effect of ceftizoxime, an antimicrobial, in a peritonitis model in the rat. The last model to be discussed here is that of colitis. Fretland et al. (1990)


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used acetic acid-induced colitis as a model of inflammatory bowel disease in mice and found SOD to significantly reduce colonic inflammation. Emerit et al. (1989, 1990) reported limited improvement of gastrointestinal symptoms in patients with Crohn's disease receiving SOD treatment. One source of reactive oxygen species in the reperfused intestine has been suggested to be xanthine oxidase. I t has been shown that allopurinol and oxypurinol are almost as effective as SOD and catalase in ameliorating the increase in vascular permeability (Granger et a/., 1981) and morphologic changes (Morris et ( I / . , 1987) seen with intestinal ischemia/ reperfusion. Furthermore, a low-molybdenum, high-tungsten diet which leads to production of inactive demolybdo-xanthine oxidase, protects against the increased postichemic intestinal microvascular permeability (Parks et al., 1986). Although the conversion of xanthine dehydrogenase to xanthine oxidase in the ischemic intestine was originally reported to occur within 1 min (Roy and McCord. 1983). more recent reports suggest a much slower conversion rate (Parks r t ul., 1988). Conversion, however, might not be necessary since the fresh intestine is already rich in xanthine oxidase (Parks and Granger, 1986). Another potential source of oxygen radicals in the ischemic intestine is the NADPH oxidase of neutrophils. Grisham et ul. (1986) reported an increase in neutrophil infiltration of the ischemic intestine on reperfusion, which was attenuated by SOD and allopurinol. Otamiri (1989) has also shown that either SOD plus allopurinol or hydrocortisone pretreatment resulted in the attenuation of the postischemic increase of mucosal permeability, malondialdehyde content, and myeloperoxidase activity (a neutrophi1 marker enzyme) in the mucosa. Hernandez el u/. (1987) demonstrated an important role for neutrophils in ischemia-reperfusion-induced microvascular intestinal injury. Using anti-neutrophil serum and Mo-1 antibody (which prevents neutrophil adhesion) these investigators showed a nearcomplete attenuation of microvascular permeability caused by ischemia and reperfusion of the small intestine. The fact that oxygen radical scavengers and allopurinol also provided a comparable extent of protection to that of neutrophil depletion led these investigators to suggest that xanthine oxidase-derived reactive oxygen metabolites play an important role in eliciting ischemia-reperfusion-induced neutrophil infiltration.

D. Liver and Pancreas Several models of liver injury have been studied in the literature. Atalla et al. (1985) induced hepatic ischemia in dogs in uiuo by cross-clamping the portal vein and hepatic artery for 40 min followed by isolation of the liver and perfusion with crystalloid. They found SOD, alone or plus catalase, to

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significantly attenuate enzyme release, enhance reperfusion flow rate, and improve histological findings at the end of 3 hr of reperfusion. Adkison et af. (1986) subjected rats to abdominal aorta ligation and found a combination of SOD and catalase to decrease liver enzyme release and improve oxygen consumption after reperfusion. McEnroe et af.(1986), on the other hand, reported a modest, but nonsignificant, improvement in the recovery of gluconeogenesis of isolated rat liver subjected to 55 min of ischemia and reperfusion by a combination of SOD and catalase. Flye and Yu (1987) found SOD plus catalase to reduce the mortality of rats from 1 hr of hepatic ischemia and reperfusion by 25% of controls. Kawamoto et af. (1989) subjected rats to a 20-min portal vein occlusion and reperfusion and found SMA-SOD to enhance the recovery of plasma clearance of bromosulfophthalein and indocyanine green. Hasuoka et af. (1990) performed an orthotopic liver transplantation in pigs following 30 min of warm ischemia and found SMA-SOD, but not native SOD, to significantly improve survival and the rate of indocyanine green disappearance. Many investigators have studied the effect of SOD on isolated hepatocytes in vivo. Kyle et al. (1988) found SOD to be internalized by hepatocytes and to significantly increase their viability on exposure to hydrogen peroxide. Togashi et al. (1989) examined rat liver slices incubated with paraquat and found liposome-entrapped SOD to decrease the release of enzymes in the culture medium. Seto et af. (1989) incubated rat hepatocytes with bile acids (lithocholic acid and deoxycholic acid) and found SOD to attenuate the reduction in viability and the release of lipid hydroperoxides. Tiegs et af. (1989) induced hepatitis in mice by an intraperitoneal injection of galactosamine and endotoxin and found SOD to protect against liver enzyme release. A number of studies examined the effect of SOD on pancreatic pathology. Sanfey et af. (1984, 1985, 1986) examined ischemia-, gallstone-, and alcohol-induced pancreatis models and reported that SOD plus catalase significantly inhibited pancreatic amylase secretion and weight gain in all models. Morita et al. (1989) found SOD plus catalase to protect the rat pancreas against amylase loss induced by cerulein given subcutaneously. Allopurinol has been found to be as effective as oxyradical scavengers in ameliorating the reperfusion injury of the ischemic liver (Adkison et af., 1986; Atalla et af., 19851, suggesting xanthine oxidase as an important source of these radicals. The role of neutrophils in hepatic ischemia/ reperfusion injury is not well defined yet.

E. Lung

Several lines of evidence indicate that reperfusion of the ischemic lung results in some free radical generation. Fisher and Block (1977) showed

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that SOD ameliorates the reduction in serotonin clearance from rat lungs exposed to 100% oxygen. Turrens et a / . (1984) found that intravenous liposome-encapsulated, but not free, SOD and catalase augmented the level of these enzymes in the lungs and protected against hypoxia-induced lung edema. Padmanabhan et d.(1985) reported that liposomal SOD alone, but not free SOD, resulted in improved survival and better lung morphology in rats exposed to >957% hyperoxia. Tanswell and Freeman (1987) also demonstrated protection with liposome-entrapped SOD and catalase on survival of rat pups exposed to hyperoxia. White et al. (1989) found PEG-SOD to increase the survival of rats exposed to 100% oxygen. Jacobson et n / . (1990) demonstrated that a combination of PEG-SOD and PEG-catalase provided dramatic protection against lung weight gain and alveolar-capillary permeability in the rabbit caused by hyperoxia. The second model of lung injury is that caused by a toxic substance. Martin et a / . (1986) used a-naphthylthiourea to cause canine lung injury and found SOD to attenuate the resulting increase in capillary permeability. Archer er (11. (1989) found liposome-entrapped SOD plus catalase. but not free enzymes, to protect isolated rat lungs perfused with xanthine plus xanthine oxidase. In models of stress, SOD has been shown to protect rat lungs against thermal skin injury (Till et a [ . , 1983) and radiation injury (Malaker and Das, 1988a,b). In a model of ischemia/reperfusion, Koyarna et al. (1987) demonstrated that reperfusion of an isolated clog lung lobe subjected to 6 hr of ischemia resulted in progressive injury, as assessed by increase in lung weight, which was markedly attenuated by SOD. Tsuji et al. (1989) exposed dog lungs to 1 hr of warm ischemia and found SOD to attenuate the decrease in the partial pressure of oxygen 2 hr after reperfusion. Models of pulmonary embolism have also been employed. Flick et a / . (1981) showed that SOD attenuates the increase in protein permeability of sheep lungs caused by air emboli. Dikshit ef al. (1989) induced pulmonary thromboembolism in mice by intravenous infusion of collagen and adrenaline, resulting in malondialdehyde release, which was ameliorated by SOD. Several sources of oxyradical production in the lung have been implicated. One important source seems to be xanthine oxidase. The presence of xanthine oxidase activity has been demonstrated in the lung of several animal species, including the dog and the rat (Parks and Granger, 1986). Cheronis et a / . (1987) have shown that the addition of tungsten to the diet of rats for 3 weeks prevented lung injury caused by hyperoxia. The slow conversion of xanthine dehydrogenase to xanthine oxidase in the lungs (Engerson et ul., 1987) raised some questions about the magnitude of damage contributed by this source of free radicals. However, such conversion might not necessarily be required since the initial xanthine oxidase content itself is high in some species and may contribute to oxidative

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stress. Another source of free radicals is the mitochondrion. Lung mitochondria have been shown to increase their hydrogen peroxide production dramatically as the oxygen tension rises (Turrens er al., 1982). Lung mitochondria, however, generate less hydrogen peroxide than liver mitochondria under the same conditions (Boveris and Chance, 1973), which may be an adaptive mechanism to the high oxygen tensions normally present in the lung. A third potential source of free radicals in the lung is the neutrophil. A number of reports support the involvement of neutrophils in the pathogenesis of hyperoxia-induced acute edematous lung injury (Fox et a/., 1981a,b; Tate and Repine, 1983). Neutrophils have been shown to accumulate along damaged endothelial cells in lungs from rats exposed to hyperoxia (Fox e t a / . , 1981a; Barry and Crapo, 1985). Furthermore, neutrophils were shown to make potent toxins, damage cultured lung endothelial cells and cause acute edematous injury when added to the perfusate of isolated lungs (Shasby et a / . , 1982). The mechanism of neutrophil-mediated lung injury has been suggested to involve a synergistic action of both lysosomal proteases and oxygen-derived free radicals (Ward et a l . , 1986; Repine et a / . , 1987). Some oxygen derivatives such as hydrogen peroxide have been shown to potentiate the effects of proteases, resulting in a modification of protein substrates such that they become much more susceptible to proteolysis (Fligiel et a / . , 1984).

F. Kidney Three models of kidney damage have been employed in the literature. First, we will consider drug-induced nephrotoxicity. McGinness er af. ( 1977) found that daily subcutaneous injections of SOD decreased the nephrotoxicity produced by cisplatin in female rats. Diamond et d.(1986) found SOD to ameliorate glomerular morphologic changes associated with nephrotoxicity from the aminonucleoside of puromycin. Kaur et a / . ( 1989) induced renal brush border membrane damage with xanthine plus xanthine oxidase and found that while lower SOD doses tended to exacerbate the damage, higher doses were protective. Paller (1985) found no protection with SOD against mercuric chloride-induced acute renal failure in rats. Models of nephritis have generally been amenable to SOD treatment. Adachi et a/. (1986) showed that SOD reduces malondialdehyde release from rat kidneys following the injection of nephrotoxic serum. Birtwistle er al. (1989) induced glomerulonephritis in rats using sheep anti-glomerular basement membrane antibody, resulting in proteinuria, which was attenuated by SOD. Sato et d.(1989) used a rat model of lupus nephritis and found SOD to decrease proteinuria and ameliorate the associated histolog-


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ical changes. Matsumoto et al. (1990) found SOD to suppress renal scarring following bacterial pyelonephritis. Webb er al. (1985) failed to demonstrate protection with SOD against a rat model of nephrotoxic nephritis. The last model is ischemia/reperfusion to the kidney. Hansson et al. ( 1983)demonstrated improved reperfusion following ischemia of the rabbit kidney when SOD was used. Paller et (11. (1984) exposed rat kidneys to 60 min of ischemia and found SOD to improve the recovery of plasma creatinine, inulin clearance, and renal blood flow after reperfusion. Koyama et al. (1985) isolated pig kidneys, stored them for 24 hr at 4"C, and transplanted them to other pigs and found SOD to improve the recovery of creatine clearance in a dose-dependent manner. Ouriel er al. (1985) subjected canine kidneys to 60 min of ischemia and found SOD to decrease renal edema and renovascular resistance and preserve glomerular filtration rate and urine flow following reperfusion. Baker et ul. (1983, Schneider et al. (1987), Wolgast et al. (1988), Rosati et al., (1988), and Vicens et al. (1990) all reported protective effects when using SOD in different protocols of rat kidney ischemiaireperfusion. Winchell and Halasz ( 1989), however, failed to demonstrate any functional improvement with SOD against renal ischemia/reperfusion in the rabbit. Evidence for the involvement of xanthine oxidase in free radical production in the kidney comes from the protective effects of the xanthine oxidase inhibitor allopurinol in dogs (Chatterjee and Berne, 1976), rabbits (Hansson e f al., 19821, and rats (Paller et f i l . , 1984; Bayati et al., 1985). The involvement of neutrophils in ischemic renal injury is inconclusive. Hellberg et al. (1988) reported that neutrophil depletion with anti-neutrophil serum (ANS) caused a modest increase in the immediate reperfusion glomerular filtration rate in the rat. Klausner et al. (1989) reported that ANS-treated rats developed less azotemia 24 hr after ischemia than did nonneutropenic controls. In contrast, Paller (1989) and Thornton er af. (1989) failed to show any protection from either ANS or Mo-1 antibody against ischemia-reperfusion in rabbit and rat kidneys. The protection against oxygen-induced tissue damage is of relevance to the kidneys, especially during transplantation, when ischemia is followed by reperfusion (Parks et al., 1983).

V. The Pharmacodynamics and Pharmacokinetics of the Superoxide Dismutases

There are several possible explanations as to why SOD failed in some animal models. For example, SOD may not have access to critical compartments in the organ, or superoxide may simply not contribute to tissue

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necrosis. Much of our knowledge about the pharmacological properties of SOD has been obtained in the heart. The heart is peculiar in that it has continuous capillaries that selectively sieve macromolecules based on their effective molecular size (Parker and Perry, 1984; Parker er al., 1981; Taylor and Granger, 1984). The charge-selective nature of continuous capillaries, however, is unknown. The discussion of the pharmacodynamic and pharmacokinetic properties of SOD will concentrate on the myocardium, where much of the controversy exists. Other organs presumably follow the same pattern. SOD has peculiar dose-response characteristics. Several studies showed that the amount of protection SOD confers to the myocardium is dose dependent. Riva et al. (1987) and Bernier et al. (1989) showed that protection against reperfusion arrhythmias in the rat is lost at higher doses of SOD. We have carried out dose-response studies using both rabbit and rat hearts. Using isolated rat hearts (Omar et al., 1991) subjected to 40 min of hypoxia and 10 min of reoxygenation, lower Cu,Zn-SOD doses (2.3,7, and 20 mglliter) resulted in a lower creatine kinase release, while a higher (50 mg/liter) Cu,Zn-SOD dose resulted in enzyme release that was not significantly different from controls. Using isolated rabbit hearts (Omar er al., 1991) subjected to 60 min of hypoxia and 60 min of reoxygenation, lower Cu,Zn-SOD doses (0.5, I , and 5 mg/liter) resulted in a lower lactate dehydrogenase release, while a higher (50 mg/liter) Cu,Zn-SOD dose resulted in enzyme release that was not significantly different from controls. Finally, we examined the dose-response effect of SOD on infarct size in the rabbit heart in vivo (Omar er al., 1991) and found that while lower SOD doses provided modest protection against infarct size, a high (50 mg/kg) dose caused an increase in infarct size. A 50 mglkg dose of SOD injected as an intravenous bolus would produce a theoretical initial plasma concentration of over 1400 mg/liter, which is much higher than any of the concentrations attempted in vitro. The rapid clearance rate, however, coupled with a slow rate of equilibration between intravascular and interstitial spaces, results in a pharmacologically complex and rapidly changing scenario. A11 three dose-response curves are replotted in Fig. 1 to demonstrate their bell-shaped characteristics. It is remarkable to note the consistent loss of protection at higher doses of SOD in all three models, even though the models differ considerably with respect to species (rat versus rabbit), end point measured (infarct size. versus enzyme release), source of the SOD used (yeast in the rat and human recombinant in the rabbit) and its schedule of administration (continuous versus a single bolus). Our data could have predicted the outcome of some reported negative studies in the literature using the isolated rat (Menasche et al., 1986) and rabbit hearts ( C . L. Myers et al., 1985) and the rabbit heart in vivo (Miura et al., 1989) points 1 , 2, and 3 on Fig. 1).

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Superoxide Disrnufase

0.1

1

10

100

Superoxide disrnutase (mg/l or rng/kg)

Fig. 1 Dose-response curves for SOD in three models of myocardial damage. The upper curve represents creatine phosphokinase release from isolated rat hearts subjected to 40 min of hypoxia and 10 min of reoxygenation. Optimal protection is at about 10 mg/liter of yeast Cu,Zn-SOD. The middle curve represents lactate dehydrogenase release from isolated rabbit hearts subjected to I hr of hypoxia and 1 hr of reoxygenation. Optimal protection is at about 4 mgiliter of human recombinant Cu,Zn-SOD. The lower curve represents infarct size in rabbit hearts subjected to 45 min of coronary artery ligation iti uiuo, followed by 3 hr of reperfusion. Optimal protection is at about 7 mgikg of human recombinant Cu,Zn-SOD. The open symbols labeled 1 , 2, and 3 represent values calculated from the literature, from studies using identical models, which reported negative findings: (1) Menasche er a / . (1986); (2) C. L. Myers et al. (1985); (3) Miura et al. ( 1989).

Pretreatment and posttreatment with SOD may be required for maximum efficacy. Cu,Zn-SOD has a relatively short half-life in the plasma of 6-10 min (McCord and Wong, 1979). Moreover, it has a net negative charge at physiologic pH. Both of these factors could hinder sufficient delivery of the molecule to sites of oxygen radical generation. SOD does not stay long enough in the circulation to equilibrate adequately with the tissues. Furthermore, its negative charge is not favorable for tissue equilibration since endothelial cells, basement membrane, and interstitial matrix carry negative charges in most tissues, which would retard its plasmainterstitial transport. Therefore, a long equilibration time may be required

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before adequate Cu,Zn-SOD could reach a site where it may exert some protective effect. We examined the effect of preischemic equilibration of the isolated rabbit heart with SOD on its tolerance to ischemia-reperfusion (Omar and McCord, 1991). After either 15 or 50 min of equilibration the hearts were subjected to 1 hr of ischemia followed by reperfusion. Fluid weeping from the ventricular surface was collected and assumed to represent cardiac lymph. Only 15 min of preischemic equilibration with the positively charged human recombinant Mn-SOD was sufficient to load the lymph and protect function. This period, however, was insufficient for either the negatively charged human recombinant Cu,Zn-SOD or the large molecular size and negatively charged PEG-SOD to equilibrate with the lymph. This effect was reflected by the lack of protection by both of these forms of SOD. However, when the equilibration period was raised to 50 min, a sufficient amount of time for the Cu,Zn-SOD to fully equilibrate with the interstitium, Cu,Zn-SOD did show good protection. PEG-SOD showed very poor equilibration even after 50 min, and did not provide protection. Figure 2 illustrates the strong correlation between SOD activity in the lymph and recovery of developed tension for the various SOD treatments and preischemic equilibration times. This study, in addition to pointing out the importance of pretreatment with Cu,Zn-SOD, also gives insight into the charge selectivity of the myocardial continuous capillaries. Mn-SOD had the peculiar ability of being large enough (about twice the molecular radius of Cu,Zn-SOD) to remain in the plasma with a half-life of about 7 hr (Baret er al., 19841, while at the same time being positive enough to equilibrate rapidly in the lymph and exert marked protection to the myocardium. The schedule of administering SOD is apparently very important. Many investigators have overlooked the fact that free radical production is a long-term process that starts with ischemia itself (especially purrial ischemia), and not just at reperfusion. The fact that SOD was shown to be effective during ischemia alone supports this idea (Manning et al., 1984). Moreover, Ferrari et al. (1985) and Curello er ul. (1985) showed that ischemia alone resulted in approximately 50% reduction in mitochondria1 SOD in the rabbit heart and, therefore, suggested the presence of oxidative stress during ischemia. Przyklenk and Kloner (1987) showed that SOD, given in combination with catalase, managed to reduce the infarct size in dogs subjected to 6 hr of coronary artery ligation by 46%, when SOD was given as a continuous infusion throughout ischemia. However, when SOD was discontinued and the hearts were reperfused for 30-48 hr the protective effect of SOD was reduced to 7%, which was not statistically significant. Jolly et af. (1984) showed that SOD, in combination with catalase,

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100

]

75 50 25

0

10

20

30

40

50

60

Equilibration time ( minutes ) Fig. 2 Myocardial protection correlates with extent of interstitial equilibration of SOD. The lines represent the time course of the appearance of SOD in the interstitial relative to vascular compartments. The bars show recovery of developed tension in hearts after I hr of ischemia and 1 hr of reperfusion, after 15 or 50 min of equilibration with the indicated type of SOD. (a) hr-Mn-SOD; (b) hr-Cu.7,n-SOD; ( c ) hr-PEG-SOD.

given as a continuous infusion 15 min before a 90-min coronary artery occlusion in dogs and ending 15 rnin after reperfusion, was able to reduce the infarct size by 53%. However, when they administered SOD for 1 hr only, starting 40 min after reperfusion, they found no significant effect. Engler and Gilpin (1989) have recently reviewed several studies of the effect of SOD on myocardial infarct size. They have identified some important variables as possibly causing the discrepant findings among SOD trials in the literature. These variables included collateral flow in dogs, the use of either tetrazolium or histology in assessing infarct size, the length of ischemic period, and the duration of reperfusion. To these variables we can add the length of treatment with SOD prior to reperfusion. Dog hearts enjoy an ample supply of collateral flow, and lymphatic equilibration during regional ischemia is. therefore, not unlikely. It is interesting to note that four out of the five positive dog studies that Engler and Gilpin reviewed administered SOD at least 60 min prior to reperfusion (Werns et

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al., 1985, 1988; Chambers et al., 1985; Jolly et a/., 1984). On the other hand, all of the five negative dog studies that they reported gave SOD only 25 min or less before reperfusion (Uraizee et ul., 1987; Gallagher et ul., 1986; Nejima et al., 1989; Pate1 ef ul., 1988; Richard et al., 1988). There is no doubt as to the importance of the schedule of administering SOD. In our studies in uitro, SOD was present in the perfusate throughout. Hence, we do not know when the drug can be withdrawn. Recent studies with PEG-SOD (Tamura et al., 1988; Chi et al., 1989; Ohkubo e f al., 1990) suggest that prolonged posttreatment is indeed a requirement. PEG-SOD has a plasma half-life in excess of 1 day. However, Ooiwa et al. (1989) saw no limitation of infarct size in a rabbit model with PEG-SOD given at 1000 U/kg [the same dose as that of Tamura e f ul. (1988)l when histology was used to size the infarcts. Tanaka et al. (1989) also failed to find protection with PEG-SOD in dog hearts using histology. PEG-SOD probably fails to reduce infarct size as a result of its inability to enter the interstitial space. Even though PEG-SOD may not salvage myocardium, posttreatment may still be a requirement for a positive outcome, especially if neutrophils are the source of injurious free radicals. Note: None of the heart studies discussed above maintained any significant posttreatment. Moreover, PEG-SOD has shown more success in other organs using different models, especially where the injury is primarily endothelial rather than parenchymal.

VI. Chemical Modification of Superoxide Dismutases Since the discovery of SOD (McCord and Fridovich, 1969), the involvement of 0; has been documented in many pathological states discussed above, including inflammation (Petrone et al., 1980; Salin and McCord, 1975), ischemia-reperfusion (Granger e f al., 1981; McCord, 1985), and oxygen toxicity (Crapo and McCord, 1976: White et al., 1989). In the first reported use of SOD to prevent reperfusion injury in uivo (to feline intestine) (Granger et ul., I981), we found that Cu,Zn-SOD was minimally effective unless the animal’s kidneys were ligated to prevent rapid clearance. Others confirmed the failure of native cytosolic Cu,Zn-SOD to protect against oxygen toxicity in uiuo, as a result of its poor pharmacokinetic and pharmacodynamic properties, although liposomeencapsulated enzymes were effective (Turrens et ul., 1984). To deal with this problem of adverse pharmacological properties of SOD, the enzyme has been chemically modified in a variety of ways in attempts to enhance its efficacy. These modifications have focused almost entirely on the Cu,Zn-SOD, perhaps because of its ready availability and remarkable


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stability. The modifications have been aimed at changing the effective size, charge, or lipophilicity of the molecule. Shortly after Babior found that activated neutrophils produce superoxide as a bactericidal weapon (Babior et ul., 1973), we found that the production of the radical was the major factor responsible for the “suicide” of activated phagocytes (Salin and McCord, 1975). The addition of native bovine Cu,Zn-SOD to the medium in which activated neutrophils were suspended could virtually eliminate their premature demise, but curiously high concentrations of SOD were required to effect this protection. The human Mn-SOD and the porcine Cu,Zn-SOD, which have higher isoelectric points than the bovine enzyme, were able to protect the neutrophils at much lower concentrations. Reasoning that external cell surfaces are negatively charged and that the native bovine SOD is also negatively charged at neutral pH, we hypothesized that the SOD might be effectively excluded from the shell of solvent surrounding the surface of the neutrophil. We hypothesized further that we might attain nonspecific binding of SOD if a positively charged enzyme could be produced from the bovine Cu,Zn-SOD by chemical modification. Polylysyl-SOD was prepared by incubating purified bovine liver SOD with I-ethyl-3(3dimethylamino-propy1)-carbodiimide and polylysine (average M,.2000) (McCord and Salin, 1977). This charge-modified SOD was assessed in a model of superoxide-dependent cytotoxicity: prevention of the radicalmediated “suicide” of phagocytosing neutrophils (Salin and McCord, 1975). The positively charged polylysyl-SOD was able to protect the neutrophils at one-tenth the concentration of the native bovine SOD. Ironically, a similar motif was found to have been used by Mother Nature in the design of ECSOD. When the deduced amino acid sequence was published (Hjalmarsson et ul., 1987), the C terminus was seen to be a highly positively charged “tail” that was suggested to permit binding to heparin, which was in turn bound to the surface of the endothelial cells. Subsequent studies proved this to be the case (Karlsson and Marklund, 1987; Adachi and Marklund, 1989). In viuo, most of the ECSOD is bound to cell surfaces, and can be displaced into the plasma by the administration of a large dose of heparin. Another promising avenue for the modification of SOD appears to be alterations in lipophilicity . N-Hydroxysuccinimide esters of a variety of fatty acids with different chain lengths were reacted with human Cu,ZnSOD, covalently attaching up to 6 mol of fatty acidlmol of enzyme by reaction with E-amino groups of lysyl residues (Y. Ando er ul., 1988). These amphiphathic derivatives bind to plasma membranes of erythrocytes and neutrophils, and to corneal surfaces, and can dismute superoxide at these membrane surfaces. In a model of corneal inflammation,

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acylated SOD was protective whereas native SOD was not (E. Ando et al., 1990). Similarly, the two cysteinyl residues of the human Cu,Zn-SOD have been covalently modified with a large, hydrophobic organic anion, a4-{[6-(N-maleimido)hexanoyloxymethyl]cumyl}half-butyl-esterifiedpoly(styrene-co-maleic acid) (lnoue et al., 1989). The resulting derivative, called SM-SOD, binds reversibly to serum albumin, producing a circulating half-life of 6 hr. Furthermore, the SM-SOD was found to bind to cell membranes, particularly when the pH was decreased. The authors suggest that the propensity to accumulate at sites of low pH may render SM-SOD 1989b). well suited to treat ischemic heart disease (Watanabe et d., Following intravenous injection in the rat, native Cu,Zn-SOD has a half-life of only 6 min (McCord and Wong, 1979).If the SOD is derivatized by covalently linking it to Ficoll (a branched polymer with an average molecular weight of 70,000),to dextran (a linear polymer with an average molecular weight of 70,000),or to polyethylene glycol (PEG, a linear polymer with an average molecular weight of 1900),the derivatives show dramatically increased circulating lifetime of up to 35 hr (McCord er a / . , 1979).The Ficoll derivative was assessed for antiinflammatory activity and showed a dramatic increase in efficacy in uiuo by two models, the reverse passive Arthus reaction and carrageenan-induced foot edema (Petrone et al., 1980).These results suggest a profound effect of circulating half-life on therapeutic efficacy in uiuo, at least in models of inflammation. By far, the greatest number of studies dealing with covalently modified SODS have utilized the attachment of polyethylene glycol. PEG-proteins not only show increased circulating half-lives, but also show decreased immunogenicity (Abuchowski er a / . , 1977).PEG-SOD has been found effective in reducing cotton twine-induced granuloma formation (Pyatak el af., 1980), in suppressing carrageenan-induced foot edema (Veronese et af., 1983)and pleurisy (Conforti et al., 1987).in preventing streptozotocininduced hyperglycemia (Asplund et al., 1984),in preventing free radicalinduced microvascular permeability changes (Ley and Arfors, 1982), in preserving function of renal allografts (Bennett et al., 1987),and in protecting rats against pulmonary oxygen toxicity (White et a/., 1989). It should be kept in mind that not all PEG-SOD preparations are equivalent pharmacokinetically, and few studies have provided full characterization of the particular product used. The number of polymers attached per mole of SOD, as well as the average polymer length, has been varied, producing PEG-SODS with clearance half-times ranging from about 1.5 to >25 hr (Boccu et a / . , 1982). While PEG modification may dramatically improve plasma half-life, other pharmacokinetic properties are also changed, such as the rate of equilibration between vascular and interstitial spaces, as discussed above. Whether this becomes an asset or a liability


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may depend on the nature of the specific pathology under treatment. Thus, when the plasma half-life of Cu,Zn-SOD is increased more than 100-fold, huge increases in efficacy may be produced in certain models of inflammation, despite the fact that the modified enzyme may be significantly impaired in gaining access to the extravascular spaces. In models of reperfusion injury the burst of free radical production may be of relatively short duration. If SOD were used in the clinical treatment of myocardial infarction (coupled, e.g., with infusion of streptokinase) a fast-acting form of the enzyme would be more desirable than a long-acting form. In other words, the improvement of one pharmacokinetic property at the expense of another may or may not be advantageous, depending on the application.

VII. Genetic Modification of Superoxide Dismutases Even though all three described vertebrate SOD isoenzymes catalyze the same reaction, they have different cellular and subcellular distributions. Presumably, cellular loci of active superoxide radical generation are well supplied with SOD activity. The proposal (McCord, 1987) that free radicals are generated during periods of ischemia and reperfusion and during inflammation has attained wide acceptance. During these periods, exterior surfaces of cell membranes are exposed to high levels of superoxide radical due to its liberation into extracellular fluids by activated phagocytes. Unfortunately, the extracellular spaces of the vertebrate animals contain much lower concentrations of SOD than are found intracellularly (Marklund, 1984b). Most pharmacologic efforts have concentrated on utilizing the cytosolic Cu,Zn-SOD as an exogenously administered protective agent after exposure to various oxidative insults, with generally positive results (McCord, 1988). The initial laboratory successes prompted optimism in the usage of native Cu,Zn-SOD in clinical trials for the treatment of ischemia/reperfusion injury following a heart attack (Werns et al., 1989), after organ transplantation, or as an antiinflammatory agent (Emerit et al., 1989). However, extrapolation from animal models to humans has proven difficult. The limiting factors have been the undesirable pharmacological properties of the Cu,Zn-SOD molecule, as discussed above: a short (approximately 6 min) plasma half-life with rapid clearance by the kidney (McCord and Wong, 1979), a net negative charge that prevents close contact with cell surfaces (McCord and Salin, 1977), or its equilibration between vascular and interstitial spaces (Omar and McCord, 1991). Much current research therefore focuses on the development of genetic variants of SOD with improved pharmacologic properties, more

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suitable for therapeutic purposes. This section will discuss some of these modifications. Most studies published to date have employed the commercially available bovine, yeast, or human Cu,Zn-SODS. Adverse immunological reactions are often a problem when using xenogeneic enzymes as therapeutic agents, although the Cu,Zn-SODS are remarkably nonimmunogenic. Authentic human SODs (especially the Mn- and ECSODs) are therefore desirable for therapeutic purposes. Furthermore, any attempt at modifying the sequence of a particular SOD isoenzyme gene requires detailed knowledge about the wild-type sequence. Fortunately, the cDNA sequences for all three human SOD isoenzymes have already been reported (Beck e t a / . , 1987; Hallewell eta/., 1985; Iljalmarsson et a/., 1987). In addition, expression in yeast or bacterial cells has permitted the production of relatively large amounts of the human enzymes (for a review, see Touati, 1988). The Cu,Zn-SOD was the first of the SODs to be described and has been the most widely scrutinized. The protein-coding region of the human Cu,Zn-SOD cDNA sequence is approximately 84% homologous to the same region of the mouse Cu,Zn-SOD cDNA sequences (Ho and Crapo, 1987). In uiuo, the N-terminal methionine is removed and an acetylated alanine remains to produce the 153-amino acid negatively charged mature SOD (Hallewell et a / . , 1985). Hallewell et al. (1985) placed the human Cu,Zn-SOD gene under the control of the tac bacterial promoter and expressed the enzyme in E . coli. The recombinant human protein accounted for 5% of the total recovered protein. Hartman e f a / . (1986) also used a thermoinducible promoter to achieve expression of human SOD in bacteria. The expressed recombinant enzymes were not acetylated in either system, but they had full activity, demonstrating that acetylation of the N-terminal amino acid is not necessary for function. In contrast, expression in yeast cells resulted in an acetylated peptide (Hallewell et al., 1987). The eukaryotic Mn-SOD is synthesized in the cytoplasm and transported into the mitochondria1 matrix where it resides (Weisiger and Fndovich, 1973). There is no homology to the Cu,Zn-SOD sequences, but a high degree of homology to the bacterial Mn- and Fe-SODS (Steinman and Hill, 1973). A short stretch of amino acids preceding the N-terminal residue of the mature protein is a leader sequence that is presumably responsible for targeting and importation into the mitochondrion (Heckl, 1988; Ho and Crapo, 1987; Wispe el al., 1989). Extracellular SOD (ECSOD) was first described as a distinct human SOD isoenzyme by Marklund (1982; Marklund et af., 1982). ECSOD is found in a number of tissues but at concentrations that are lower than the other two intracellular SOD isoenzymes (Marklund, 1984a). Still, 90-99%

Superoxide Dbrnutase

I37

of the ECSOD in mammals is found in the extracellular space of the tissues (Marklund, 1984b). A distinguishing characteristic of ECSODs is their ability to bind to heparin-Sepharose columns (Karlsson and Marklund, 1987). Three molecular forms of the enzyme are distinguished based on this ability: ECSOD-A, with very little affinity; ECSOD-B, with intermediate affinity; and ECSOD-C, with relatively high heparin affinity. ECSOD-A and -B are mainly circulating forms in v i m (Karlsson and Marklund, 1988).The suggestion that ECSOD-C might be associated with endothelial cell surfaces through heparan sulfate proteoglycans was confirmed when an intravenous administration of heparin increased plasma ECSOD-C levels (Karlsson and Marklund, 1987). Cloning and sequencing (Hjalmarsson et al., 1987) of the ECSOD cDNA sequence provided an insight as to the mechanism of the cellular association: the presence of a 26-amino acid positively charged carboxy-terminal “tail” that is probably responsible for its affinity for the heparan sulfate proteoglycans of cell surfaces. In fact, almost all of the ECSOD-C was found to be complexed with cell surfaces (Marklund, 1990a,b). In addition to the basic tail, the ECSOD amino acid sequence as deduced from its cDNA sequence contains a leader sequence of 18 amino acids (Hjalmarsson et al., 1987). This sequence is homologous to known signal peptide sequences, implying that ECSOD is secreted after synthesis. The central 98-amino acid portion of the mature ECSOD is approximately 50% homologous with the region of the Cu,Zn-SOD isoenzyme involved in the active site and metal binding. The goal of protein engineering is to understand the contribution of particular amino acid residues to the stability and function of a protein. The process of natural selection designates which amino acids will be retained in a protein sequence after a mutation has occurred, depending on how much of an advantage the mutant protein will have over its predecessor. Site-directed mutagenesis allows us the luxury of manipulating amino acid residues to suit a defined purpose without having to wait on the leisurely time of the evolutionary scale. In this light, the amino acid residues of Cu,Zn-SOD involved in the active site, in dimer-dimer interactions, and i n substrate positioning have been identified and dissected (Getzoff rt al., 1983; Forman and Fridovich, 1973; Parge et al., 1986; Tainer rt al., 1982). By comparing the amino acid sequences of the Cu,Zn-SODS from mouse and man, two regions were identified as being the most variable: between residues 19 and 36 and between residues 88 and 105 (Getzoff et nl., 1989). The structure of these regions is predominantly hairpin loops exposed to and interacting with the solvent (Getzoff et al., 1989). Mutations in these regions would be better accommodated than mutations elsewhere in the structure. Indeed, when

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the Cu,Zn-SOD sequences from 15 different species were compared, 15 out of 23 invariant amino acid residues were located in the active sites (Getzoff et ul., 1989). For example, Arg-143 in human SOD resides in the active site cavity immediately adjacent to the copper ion (Bertini et al., 1988). This site attracts the superoxide anion and its contribution to catalytic activity is by electrostatic “docking” of the anion (Getzoff et al., 1983; Getzoff and Tainer, 1986). Thus, mutations that affect the charge of this residue will also affect catalytic activity of the enzyme. When Arg-143 was replaced by lysine, isoleucine, or glutamic or aspartic acid and the variants expressed in yeast, the specific activities of the mutant enzymes correlated with the polarity of the substituting amino acid: 43 and 11% of wild type for Lys-143 and Ile-143, respectively, and 2-4% of wild type for Glu-143 and Asp-143 (Beyer et al., 1987; Banci et al., 1988; Hallewell et al., 1989). These data suggest that not arginine per se, but rather a positive charge, is essential for the catalytic process. The decreasing affinity of the mutant enzymes for the inhibitor azide paralleled the decrease in activity as the positive charge of the substituting amino acid residue decreased (Banci et al., 1988). This suggested that the affinity of superoxide for the active site cavity was decreased as positive charge was lost. Substitution of Arg-143 also affected the rate at which the Cu2+ was removed by EDTA treatment (Banci et al., 1988). Removal of metals from the mutants was much slower than from the wild-type enzyme. Copper depletion required about 4 days for Glu-143,8 days for Lys-143, and about 20 days for Ile-143, whereas the wild type released copper completely in less than 2 days. In a series of related studies, the cloned gene of the bovine Cu,Zn-SOD was manipulated such that a cysteine at position4 was replaced by an alanine and the mutant protein expressed in yeast (McRee et al., 1990). At 21°C the mutant enzyme was fully active, suggesting that the mutation did not affect the folding and function of the enzyme. While the T , (melting temperature) of the mutant enzyme was decreased when compared to wild type, at 70°C the rate of thermal inactivation was only half as fast for the mutant. The authors speculated that preserving the integrity and function of an enzyme during thermal denaturation is more critical than increasing its T,: thermal inactivation due to unfolding is usually reversible and may occur at temperatures below the T,. On the other hand, a greater tendency toward irreversible denaturation can make proteins more difficult to purify and less useful as drugs. Active site-directed mutants also exhibited altered thermal denaturation profiles: the mutant Ile-143 proved more resistant than the wild type to the thermal treatment during the purification procedure (Beyer et al., 1988). In a series of very elegant experiments, Hallewell et a[. (1989)joined two


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human SOD genes in the same translational reading frame. The “designer” gene encoded two identical subunits of Cu.Zn-SOD covalently joined in a tail-to-head fashion or separated by a 19-amino acid spacer taken from the hinge region of IgA1. These constructs were efficiently expressed in both bacterial and yeast cells. The authors’ premise was that because of the very strong interactions between the SOD subunits, the two SOD subunits covalently joined with a hinge region would fold through intramolecular interactions to produce a single-chain enzyme that would be catalytically active. In addition, the covalently joined SOD protein constructs would provide a hydrophobic dimer surface that should allow for intermolecular interactions to produce catalytically active multimeric forms of the enzyme. When tested in uiuo, these multimers had half-lives of approximately 145 min (half-life of wild-type Cu,Zn-SOD is 6 min). Increasing the molecular weight of the SOD variants showed a linear relationship with clearance time by the kidneys until a plateau was reached. After this point, further increases in molecular weight had no effect on clearance time. It is clear from these experiments that long-life (hours) in plasma is independent of the kidneys and that characteristics of SOD other than molecular weight should be manipulated to increase its steady state levels in plasma. Only with a long-lived SOD can plasma concentrations of the enzyme be controlled for maximum efficiency. An interesting alternative would be to engineer SODS with increased affinity for endothelial cell surfaces. lnoue et ul. (1990a) addressed this possibility by constructing a fusion gene that encoded the human Cu,ZnSOD followed by a 26-amino acid C-terminal heparin-binding peptide similar to the heparin-binding domain of ECSOD. While native Cu,ZnSOD is unable to bind to vascular endothelial cells, the hybrid SOD bound to the cellular surfaces. The binding was inhibited by heparin, suggesting that the association occurred through the 26-amino acid tail. The hybrid peptide protected rat tissues against carrageenan-induced inflammation and cold-induced brain edema. It is obvious from these experiments that genetically engineered SODS can create the best combination of characteristics that determine stability, plasma clearance rate, and charge.

VIII. Down Syndrome and the Concept of ”Oxidant-Antioxidant Balance” When it was realized that biological systems are capable of producing free radicals and other potent oxidants, the natural tendency of conventional wisdom was to view the oxidants as “bad” and the antioxidants as

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“good.” Things are rarely that simple. There is a growing recognition that a balance between oxidants and antioxidants is a more realistic depiction of the relationship. There were many early clues, most of which were ignored or otherwise dismissed due to their circumstantial nature. Perhaps the first clue was the remarkable constancy of SOD activity across virtually all aerobic organisms (McCord et al., 1971). Very early in the history of SOD it was recognized that the protein might have therapeutic efficacy in certain pathological situations (Huber et af., 19681, bolstering the concept that “more is better.” Yet, if this were the case, why was the activity distributed within such narrow limits? If more were truly better, surely some organism would have discovered the advantage of boosting its SOD production-but this had not occurred. In 1973 it was discovered that activated phagocytes put radical production to a constructive use: the killing of bacteria (Babior et af.,1973). That might explain the advantage of having relatively little SOD in the extracellular fluids, where phagocytes roam. The human condition known as Down syndrome, or trisomy 21, has also provided some provocative clues regarding the concept of balance between oxidants and antioxidants. The human cytosolic Cu,Zn-SOD gene is located on chromosome 21 (Tan ef ul., 1973), and persons with trisomy 21 exhibit a gene-dosage effect (Sinet et af.,1974);i.e., their cells contain 50% more than the normal amount of Cu,Zn-SOD due to the presence of a third copy of the gene. Their platelets, however, contain one-third less Mn-SOD (Sinet et al., 1975). This suggests a regulatory mechanism that attempts to control SOD concentration within the cell, or conversely, to buffer the superoxide concentration at a low, but nonzero value. Down syndrome patients display a variety of metabolic and physiological aberrations, including abnormal neuromuscular junctions in the tongue (Yarom et a / ., 1987), evidence of increased lipid peroxidation in brain homogenates (Brooksbank and Balazs, 1984), and decreased uptake of serotonin by platelets (Schickler et a/., 1989). Because of the large number of genes present on the extra chromosome (actually only segment 21q22 is believed responsible for the syndrome), there was no reason to attribute any of these abnormalities to the extra SOD-until the development of models for specific gene-dosage effects. With the advent of molecular biology, it has become possible to manipulate levels of gene expression. Bacteria can be induced to produce huge quantities of recombinant SOD, accounting for > 10% of their total cellular protein (Hartman et al., 1986). Cultured mammalian cells (Elroy-Stein et al., 1986) and even intact transgenic rodents (Epstein et af., 1987) can be induced to produce up to six times the normal amount of SOD. These overproducing cells are not the “supercells” some expected they would

I4 1


be; rather, they exhibit some interesting and unexpected deficits and frailties. By so doing, they have shed considerable light on the pathological mechanisms of Down syndrome, and suggest that certain of the symptoms of the syndrome are, in fact, a reflection of the gene dosage of SOD per se. The tendency for lipid peroxidation seen in tissues from Down syndrome (Brooksbank and Balazs, 1984) is also seen in transfected human HeLa cells and mouse L-cells, which overexpress the Cu,Zn-SOD by about sixfold (Elroy-Stein et al., 1986). The diminished serotonin uptake by platelets seen in Down syndrome is also seen in platelets from transgenic mice with increased Cu,Zn-SOD activity (Schickler er a l . , 1989). Finally, and most amazingly, the abnormal neuromuscular junctions seen in the tongue in Down syndrome is reproduced in mice transgenic for the human Cu,Zn-SOD (Avraham er al., 1988). These observations make a strong case for the importance of the concept of balance between oxidants and antioxidants. The mechanism whereby too much SOD may become toxic to cells is not at all clear. At least one superoxide-utilizing enzyme, indoleamine-2,3dioxygenase, has been described (Taniguchi et al., 1977). This enzyme, and possibly others like it, are in competition with SOD for the available superoxide. Interestingly, the indoleamine-2,3-dioxygenasedegrades serotonin, a neurotransmitter, and dimethyltryptamine, a normal metabolite that is hallucinogenic. Michelson and co-workers surveyed a large variety of populations (old, young, rural, urban, sick, healthy, etc.), finding significantly higher levels of erythrocyte SOD in a population of mentally ill adult patients (Michelson et a / . . 1977) and among infants with develop1977). mental psychoses (Glose et d., Some believe that more SOD results in more formation of H202, the product of the dismutation. This can be true only under special conditions, namely, if much of the intracellular superoxide were serving to reduce another species: 02 + X - . O ? + X

Excess SOD would, in this case, force half the 0; into Hz02 production. However, if nearly all the superoxide were undergoing spontaneous dismutation, excess SOD would serve only to lower the steady state concentration of OT, but would have no effect on the rate of H202 formation (McCord and Fridovich, 1969). If, on the other hand, much of the intracellular superoxide were serving to oxidize another species: 0,

+ H X + H + + H202 + X

then excess SOD would force half the 0; into O2 production, decreasing

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the rate of H202production. Hence, in the absence of data showing that the first condition holds. the argument that excess SOD causes increased cellular production of H z 0 2is not a convincing one. We have recently suggested a mechanism whereby the low concentrations of superoxide (or its conjugate acid, the hydroperoxyl radical, HO;!.) produced by normal metabolism and buffered by normal concentrations of SOD may serve a useful role to the cell by scavenging the lipid peroxyl radicals (LOO.) that propagate lipid peroxidation (Omar ct al., 1991). The only way a radical may be eliminated, after all, is by an annihilation reaction with another radical. The superoxide radical is constantly supplied by normal metabolism and is, as radicals go, a mild-mannered and relatively nonreactive free radical. These qualities would seem to make it an ideal candidate for use in the annihilation of other more noxious radicals. In this scheme .OH represents any free radical capable of abstracting a hydrogen atom from an unsaturated lipid, LH, to initiate lipid peroxidation:

+ LH + H20 + L. (initiation) L. + 0 2 + LOO. LOO. + LH + LOOH + L. (propagation) LOO. + HO?. + LOOH + 0 2 (termination) .OH

Alternatively, if the lipid peroxidation is iron dependent, ferrous iron may cause the reductive lysis of the oxygen-oxygen bond in a preexisting lipid hydroperoxide molecule, giving rise to a lipid alkoxyl radical (LO.) that may then serve as an initiating radical in the scheme above. If this lipid alkoxyl radical were scavenged by 0, , then an entire chain of reactions would be prevented: Fe2+ + LOOH + Fe'+ LO. + HOz. -+ LOH +

+ LO. + OH- (preinitiation) 0 2

(termination)

Therefore, overscavenging of superoxide by increased amounts of SOD would eliminate important termination steps of lipid peroxidation, thereby amplifying cellular damage.

IX. Concluding Remarks In summary, it is clear that after two decades of intensive investigation into the biological roles of superoxide and the superoxide dismutases, we are far from a complete understanding. SOD has proven to be an invaluable tool in these investigations, and the tool is being honed to provide greater discrimination by delineation and alteration of its pharmacokinetic proper-


I43

ties. This knowledge may also contribute to the eventual success of SOD as a human therapeutic agent. It is clear that the balance between oxidants and antioxidants (or more specifically between superoxide and SOD) is indeed a sensitive and delicate one. It may be seriously upset by the pathological production of excess superoxide. Ironically, it may be upset in the other direction by the therapeutic overdosing of SOD, or by the genetic overproduction of SOD.

Acknowledgments This work was supported in part by a Glaxo Cardiovascular Discovery Grant and by National Research Service Award H L 07085-16 from the National Institutes of Health.

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Superoxide dismutase and Parkinson's disease.

catalase mimetics on human breast cancer cells.

Superoxide dismutase activity and superoxide dismutase-1 gene methylation in normal and tumoral human breast tissues.

Reconstitution of iron-superoxide dismutase.

Polarographic determination of superoxide dismutase.

zinc-superoxide dismutase transgenic mice.

Superoxide dismutase: "positive" spectrophotometric assays.

Superoxide dismutase and pulmonary ozone toxicity.

Active subunits from superoxide dismutase.

Evolutionary relationships in superoxide dismutase.

Manganese superoxide dismutase and oxidative stress modulation.

Singlet oxygen and superoxide dismutase (cuprein).

Superoxide-dismutase therapy in hyperuricaemic syndromes.

Isoelectric focusing of superoxide dismutase isoenzymes.

Radioprotection of mice by superoxide dismutase.

Expression of Mn-superoxide dismutase in carcinogenesis.

Nucleotide sequence of Coxiella burnetii superoxide dismutase.

Metal sites of copper-zinc superoxide dismutase.

Lamellar superoxide dismutase of isolated chloroplasts.

Superoxide dismutase: a photochemical augmentation assay.

Amphiphilic pentaazamacrocyclic manganese superoxide dismutase mimetics.

Superoxide dismutase of mammalian nervous system.

Superoxide dismutase activity in copper-deficient swine.

Effect of aging on pulmonary superoxide dismutase.