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Reoxygenation injury and antioxidant protection: A tale of two paradoxes
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 283, No. 2, December, pp. 223-226, 1990
INVITED PAPER Reoxygenation
Injury and Antioxidant
John M. C. Gutteridge’
and Barry
Protection:
A Tale of Two Paradoxes
Halliwell
Molecular Toxicology, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, Oklahoma 73104; and Pulmonary Medicine Division, UCD Professional Building, 4301 X Street, Sacramento, California 95817 Received April 30, 1990, and in revised form August 3, 1990
Under certain circumstances, added antioxidants can protect tissues against reoxygenation injury after ischemia. Yet reperfusing blood carries many antioxidants with it. The implications of this “antioxidant paradox” are discussed. o 1990 Academic PWS, IIN.
THE
OXYGEN
PARADOX
Oxygen deprivation is damaging to human tissues and the only treatment is rapid tissue reoxygenation by restoring blood flow as soon as possible. If ischemia is too prolonged, irreversible damage takes place, cells die and necrosis eventually occurs. There is no intervention therapy that will restore life to dead cells. The term “reperfusion injury” was first used by Hearse et al. in 1973 (1) and refers to an increase in damage observed when ischemia is corrected by restoring oxygen supply to an organ, the so-called “oxygen paradox.” For example, the heart can respond to reperfusion by showing several dysfunctions, such as arrhythmias, stunning, and infarction (211), depending on the time and extent of the initial ischemia. The molecular mechanisms responsible for the additional damage observed when oxygen is restored to ischemit tissue are still not completely understood, and several injury mechanisms may be operative. However, a great step forward was made when the involvement of oxygenderived species in reoxygenation injury was realized. Guarnieri et al. (4) observed oxidative damage to lipids and decreases in tissue antioxidants during the reoxygenation of hypoxic rat hearts. Parks, Granger, McCord, and their colleagues (5,6) proposed that the superoxide radical (0,) is a key intermediate in reperfusion damage to in1 Permanent address: National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG UK. 0003-9S61/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
testinal (5) and myocardial (6) systems. Their seminal proposal that proteolytic cleavage of xanthine dehydrogenase to xanthine oxidase, which then acts on one of its substrates (xanthine or hypoxanthine) to generate 0, and HzOz in the reperfused tissue has led to rapid advances in our knowledge (5, 6). However, it is now clear that many other mechanisms besides xanthine oxidase can contribute to the increased production of oxygen-derived species upon reperfusion, depending on the model used and the experimental conditions employed. These additional sources of reactive oxygen species include accumulation and activation of neutrophils in the reperfused tissue, increased “leakage” of electrons to oxygen from disrupted mitochondrial electron transport chains, increases in prostaglandin synthesis, oxidation of catecholamines, platelet activation, and increased 0, production by the vascular endothelium (7-l 1). Indeed, doubt exists as to whether xanthine oxidase makes any contribution at all to reoxygenation injury in human myocardium (11-13). Superoxide is produced in most, if not all, aerobic cells and the enzyme superoxide dismutase (SOD)’ is present intracellularly to remove it (14). Failure to remove 0, leads to deleterious consequences (14, 15). Dismutation of 0; gives hydrogen peroxide (HsO,), and two distinct intracellular enzymes, catalase and glutathione peroxidase (selenium-containing), exist to remove it (16). SOD and H,O,-removing enzymes protect the cell against reduced intermediates of oxygen produced during normal aerobic metabolism, but they seem unable to cope when production of 0; and H,O, is excessive (14, 16, 17). The mechanisms of cell damage by increased generation of reactive oxygen species are probably multifactorial (17-20) but considerable evidence suggests that at least some of the damage involves transition metal ions (such as iron), that convert 0, and H202 into aggressive oxidants such as hydroxyl radical (‘OH) by what is essentially Fenton-type 2 Abbreviation
used: SOD, superoxide
dismutase. 223
Inc. reserved.
224
GUTTERIDGE
chemistry (20-25). Thus it is usually proposed that excess generation of 0; and HzOz in reoxygenated tissues leads to formation of ‘OH (3-10) although the question of where the necessary iron ions come from has only recently been raised (26, 27). Evidence that Fenton-type chemistry is involved in reperfusion damage has usually come from the use of scavengers and of iron ion chelators. The most widely used chelator is desferrioxamine, which inhibits iron ion-dependent ‘OH generation under most experimental conditions (28, 29). However, desferrioxamine does many other things as well and it must never be assumed that it is exerting a protective effect by iron ion chelation (29). Attempts to detect release of iron ions using the bleomycin method (30) have so-far proved negative in ischemia-reperfusion systems (Gutteridge, Chevion, Bolli, and Halliwell, unpublished results). However, ischemia has been shown to increase the “availability” of iron in kidney (31, 32) and in dogs subjected to hemorrhagic shock (33). Further, a role for iron in reoxygenation damage is strongly suggested by the results of several other experiments [reviewed in (3, 29)], including the observation that reperfusion damage is considerably greater in iron-overloaded animals (34). Iron chelators other than desferrioxamine have also been reported to be protective (35). Implication of ‘OH, or some other species derived from 0; and HzOz, in reoxygenation injury has been thought necessary because of the observations that 0, and H202 are generally poorly reactive in aqueous solution and none of their described direct damaging effects (36) seem particularly threatening to human tissues. For example, cells subjected to oxidative stress show rapid DNA fragmentation, yet neither Opnor HzOz reacts with DNA (23,37). FROM THE OXYGEN PARADOX THE ANTIOXIDANT PARADOX
TO
During the 1980s there has been an explosion of research into reperfusion injury, mostly using isolated hearts or open-chest animals, but with a few human studies. In no model has the complete mechanism of reoxygenation injury been elucidated. However, a variety of antioxidants has been used to implicate O;, Hz02, and possibly ‘OH, (2-12). For example, SOD and catalase have been extensively explored as protective agents when added to reperfusates in many types of experiment. In some models significant protection is observed while in others the enzymes are without effect. The authors’ general impression is that SOD is often very protective against myocardial arrhythmias, and sometimes protective against stunning, whereas its ability to diminish infarct size is not clearly established (3, 38-40). Protection by antioxidants in at least one model system only occurs when the antioxidant is added preischemia or at the start of reperfusion. Addition of antioxidants after reperfusion is under way (even after only 60 s of reperfusion) gives no protective effect.
AND
HALLIWELL
These studies have been carried out using desferrioxamine (R. Bolli, personal communication) and mercaptopropionylglycine (41) in an open-chest dog model of myocardial stunning injury after reperfusion. Thus, with some satisfaction, scientists have been able to explain partially the “oxygen paradox.” Unfortunately, an antioxidant paradox has appeared. To the authors’ minds, an important question is “Why should antioxidants added extracellularly protect reperfused tissues against damage?” Myocardial cells themselves contain SOD, glutathione peroxidase, and catalase, so why should these same enzymes added extracellularly sometimes protect? Why should desferrioxamine and mercaptopropionylglytine protect reperfused dog heart when added at the start of reoxygenation and not 60 s later, since they are very unlikely to enter cells in that short time (41)? The logical explanation is that the damaging oxygen-derived species being scavenged are generated extracellularly. Here then is the paradox. When blood reenters an ischemic tissue, it carries with it a wide range of antioxidants. Thus erythrocytes possess substantial SOD, glutathione peroxidase, and catalase activities as well as an ion channel through which 0, can pass (42). Indeed, the ability of erythrocytes to protect against oxidative damage has been shown in several systems [e.g., Refs. (43, 44)]. Blood plasma has considerable antioxidant activity: it can inhibit metal iondependent lipid peroxidation and conversion of 0; and H202 into reactive species such as ‘OH (45). The highaffinity iron ion-binding protein of plasma is transferrin: iron bound to transferrins will not stimulate free radical reactions (45-47). The production of reactive oxygen species upon reoxygenation of ischemic myocardium has been amply demonstrated by spin-trapping experiments, although the identity of the trapped radicals is not always clear (4852). If production of these species is extracellular, where do they come from? If protection by desferrioxamine and other iron chelators is due to suppression of iron-iondependent ‘OH generation, where does the extracellular iron come from? Why does transferrin in the reperfusing blood not bind this iron and protect the tissue? It has been proposed (26) that the 0; involved in reperfusion injury is generated by an ischemia-induced derangement ofphysiological endothelial mechanisms (52) for producing 0;. It was proposed that endothelial cells can generate 0, because 0, can further interact with and inactivate endothelium-derived relaxing factor, widely thought to be identical to nitric oxide [reviewed in (53, 54)]. Thus ischemia might somehow not only increase 0, formation (52) but also cause iron ion liberation (26), converting the 0; into damaging species such as ‘OH that are reactive enough to immediately attack the tissue (26). It is possible that the binding of this iron to transferrin is slower than its binding to desferrioxamine, accounting for the failure of plasma transferrin to protect and for the protection observed when desferrioxamine is added at the start of
REOXYGENATION
INJURY
AND
ANTIOXIDANT
225
PROTECTION
ISCHAEMA STIMULATES: Oxidase : Kanthine Dehydrogenase---Xanthine reducing equivalents Idisruption of electron transport chain pro&glandin cascade catecholamines neutrophils activated Biochemical changes platelets facilitate oxidant formation
Cellular SOD CATALASE GSH(Se)pASE
I
L
RED CELL AEPEFlFUSlON STIMULATES
1
Transferrin kictoferrin Albumin Haemopexin Haptoglobins Caeruloplasmin 1,
t
I V ASCULAR
&+NOlo=t&JOt lFlOSBlNDlNG INACTWATING
ENDOTHELIUM
1 AND IRON POTENTLAL
NO PROTECTlON
SOD, CATALASE, XOD INHIBITORS SPIN TRAPS DESFERAIOXAMINE.
FIG. 1. The antioxidant protection.
paradox.
Despite the presence of antioxidants
reperfusion. Indeed, some iron ion complexes found in vivo are slow donors of iron to transferrin [discussed in (55)]. As we have already pointed out, however, a source for the iron has not yet been identified and attempts to detect its formation in myocardial systems have so far proved negative. Another important aspect of the NO/O; interaction has been raised by recent work (56,57). These two species react quickly (rate constant of about 4 X lo7 M-l s-l at pH 7.4) to form peroxynitrite, ONOO-, as the initial product (56). Peroxynitrite then decays rapidly, and it has been suggested that it gives rise to ‘OH [reviewed in (56)]. Consistent with this, Beckman et al. (57) have shown that peroxynitrite decomposition generates a strong oxidizing agent that is able to degrade deoxyribose and dimethylsulfoxide [‘OH attacks both of these molecules, but so do some other oxidants (58, 59)]. Desferrioxamine could also be oxidized by systems containing peroxynitrite (57). If production of 0;and of NO increases when ischemic tissues are reperfused the resulting peroxynitrite could injure cell membranes directly and/or by forming ‘OH in a reaction independent of metal ions (56,57,60). Desferrioxamine might then protect not by binding iron ions but by reacting preferentially with peroxynitrite (57). Peroxynitrite formation might also contribute to the cytotoxic actions of neutrophils and macrophages, which produce both NO and 0;. However, it must not be forgotten that NO can itself be toxic to cells (53, 54, 64). It is especially interesting to note that NO can induce release
in the cells and in the reperfusing
blood, added antioxidants
can offer
of iron from non-heme-iron proteins (61), perhaps providing a link between the O;/NO system and subsequent Fenton chemistry. It has been observed in some model systems [e.g., the myocardial “stunning” model used by Bolli et al. (62)] that the combination of SOD and catalase protects much better than either enzyme alone, even though either enzyme alone can protect almost completely against superoxide-driven Fenton chemistry in vitro (63). How can this be explained? In the model used by Bolli et al. the injury that leads to stunning occurs within the first few seconds of reperfusion (41). Perhaps there are two mechanisms of injury. 0; could do damage by reacting with NO: catalase would not be expected to protect against this (although it is possible that proteins at high concentration might be able to protect by direct reaction with peroxynitrite, thus emphasizing the need for appropriate “controls” with inactivated protein when studying the effects of antioxidant enzymes). SOD would prevent peroxynitrite formation by removing 0;. However, the increased Hz02 generation in the presence of SOD might produce extra damage by a different mechanism, perhaps involving Fenton chemistry. We predict that in the 1990s more and more attention will be directed to the molecular biochemistry taking place at the endothelial and myocyte surfaces during the first few seconds of reperfusion (Fig. 1). ACKNOWLEDGMENTS We are grateful to Drs. Bruce Freeman, Manfred Saran, Christa Michel, Wolf Bors, Joseph Beckman, and Roberto Bolli for their helpful
226
GUTTERIDGE
AND
comments and permission to quote work in press. J.M.C.G. was a 1988 Greenberg scholar. B.H. thanks the British Heart Foundation for research support.
REFERENCES 1. Hearse, D. J., Humphrey, S. M., and Chain, E. B. (1973) J. Mol. Cell. Cardiol. 5, 395-407. 2. Darley-Usmar, V. M., Stone, D., and Smith, D. R. (1989) Free Radical Res. Commun. 7, 247-254. 3. Bolli, R. (1988) J. Amer. Cob!. Cardiol. 12, 239-249. 4. Guarnieri, C., Flamigni, F., and Caldarera, C. M. (1980) J. Mol. Cell. Cardiol. 12,797-808. 5. Granger, D. N., Rutili, G., and McCord, J. M. (1981) GastroenteroZogy 89,22-29. 6. McCord, J. M. (1985) N. Engl. J. Med. 7. Flaherty, J. T., and Weisfeldt, 5,409-419.
312,159-163.
M. L. (1988) Free Radical Biol. Med.
8. Lucchesi, B. R., Werns, S. W., and Fantone, Cell. Cardiol. 21,1241-1251.
J. C. (1989) J. Mol.
9. Simpson, P. J., and Lucchesi, B. R. (1987) J. Lab. Clin. Med. 13-30. 10. Marklund,
110,
S. L. (1988) J. Mol. Cell. Cardiol. 20 (Suppl. 2), 23-30.
11. Kehrer, J. P. (1989) Free Radical Res. Commun. 5, 305-314. 12. Grum, C. M., Gallagher, K. P., Kirsch, M. M., and Shlafer, M. (1989) J. Mol. Cell. Cardiol. 21, 263-267. 13. Eddy, L. J., Stewart, J. R., Jones, H. P., Engerson, T. D., McCord, J. M., and Downey, J. M. (1987) Amer. J. Physior 263, H709-H711. 14. Fridovich, I. (1983) Annu. Rev. Pharmacol. Toxicol. 23, 239-257. 15. Touati,
D. (1989) Free Radical Res. Commun. 8, l-9.
16. Chance, B., Sies, H., and Boveris, A. (1979) Physiol. Rev. 59,527605. 17. Halliwell, B., and Gutteridge, J. M. C. (1989) Free RadicaZs in Biology and Medicine, second ed., Clarendon Press, Oxford. 18. Dypbukt, J. M., Thor, H., and Nicotera, Commun. 8,347-354.
P. (1990) Free Radical Res.
19. Cantoni, O., Scotili, P., Cattabeni, F., Bellomo, G., Pou, S., Cohen, M. and Cerutti, P. (1989) Eur. J. Biochem. 182,209-212. 20. Halliwell, B. (1987) FASEB J. 1, 358-364. 21. Halliwell, B., and Gutteridge, J. M. C. (1990) in Methods in Enzymology (Packer, L., and Glazer, A. N., Eds.), Vol. 186, pp. 1-91, Academic Press, San Diego. 22. Kyle, M. E., Nakae, D., Sakaida, I., Miccadei, S., Farber, J. L. (1988) J. Biol. Chem. 263,3784-3789. 23. Schraufstatter, I. Hyslop, P. A., Jackson, J. H., and Cochrane, C. G. (1988) J. Clin. Znuest. 82, 1040-1050. 24. Imlay, J. A., and Linn, S. (1988) Science 240,
1302-1309.
25. Larramendy, M., Mello-Filho, A. C., Leme Martins, neghini, R. (1987) Mutat. Res. 178, 57-63.
E. A., and Me-
26. Halliwell, B. (1989) Free Radical Res. Commun. 5, 315-218. 27. Puppo, A., and Halliwell, B. (1988) Free Radical Res. Commun. 4, 415-422. 28. Gutteridge, J. M. C., Richmond, R., and Halhwell, B. (1979) B&hem. J. 184,469-472. 29. Halliwell, B. (1989) Free Radical Biol. Med. 7, 645-651. 30. Gutteridge, 113-142.
J. M. C., and Halliwell,
B. (1987) Life Chem. Rep. 4,
HALLIWELL
31. Gower, J., Healing, G., and Green, C. (1989) Free Radical Res. Commum 5,291-299. 32. Paller, S., and Hedlund, B. E. (1988) Kidney Znt. 34,474-480. 33. Saran, S., Sharma, G., Malhotra, R., Sanan, D. P., Jain, P., and Vadhera, P. (1989) Free Radical Res. Commun. 6, 29-38. 34. Van der Kraaij, A. M. M., Mostert, L. J., van Eijk, H. G., and Koster, J. F. (1988) Circulation 78,442-449. 35. Van der Kraaij, A. M. M., van Eijk, H. G., and Koster, J. F. (1989) Circulation 80. 158-164. 36. Fridovich, I. (1986) Arch. Biochem. Biophys. 247, l-11. 37. Aruoma, 0. I., Halliwell, B., and Dizdaroglu, M. (1989) J. Biol. Chem. 264, 13,024-13,028. 38. Englar, R., and Gilpin, E. (1989) Circulation 79, 1137-1142. 39. Cohen, M. V. (1989) Ann. Znt. Med. 111,918-931. 40. Downey, J. M. (1990) Annu. Reu. Physiol. 52,487-504. 41. Bolli, R., Jeroudi, M. O., Patel, B. S., Aruoma, 0. I., Halliwell, B., Lai, E. K., and McCay, P. B. (1989) Circ. Res. 65, 607-622. 42. Lynch, R. E., and Fridovich, I. (1978) J. Biol. Chem. 263, 46974699. 43. Toth, K. M., Clifford, D. P., Berger, E. M., White, C. W., and Repine, J. E. (1984) J. Clin Znuest. 74,292-295. 44. Tsan, M. F., and White, J. E. (1988) Proc. Sot. Exp. Biol. Med. 188, 323-327. 45. Halliwell, B., and Gutteridge, J. M. C. (1990) Arch. B&hem. Biophys. 280, l-8. 46. Gutteridge, J. M. C., Paterson, S. K., Segal, A. W., and Halliwell, B. (1981) Biochem. J. 199,259-261. 47. Aruoma, 0. I., and Halliwell, B. (1987) Biochem. J. 241, 273-278. 48. Bolli, R., and McCay, P. B. (1990) Free Radical Res. Commun. 9, 169-180. 49. Shuter, S. L., Davies, M. J., Garlick, P. B., Hearse, D. J., and Slater, T. F. (1990) Free Radical Res. Commun. 9, 223-232. 50. Blasig, I. E., Ebert, E., and Lowe, H. (1986) St&a Biophysics 116, 35-42. 51. Garlick, P. B., Davies, M. J., Hearse, D. J., and Slater, T. F. (1987) Circ. Res. 61, 757-760. 52. Arroyo, C. M., Carmichael, A. J., Bouscarel, B., Liang, J. H., and Weglicki, W. B. (1990) Free Radical Res. Commun. 9, 287-296. 53. Marletta, M. A. (1989) Trends Biochem Sci. 14,488-492. 54. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1989) Biochem. Pharmacol. 38,1709-1715. 55. Aruoma, 0. I., Bomford, A., Polson, R. J., and Halliwell, B. (1988) Blood 72,1416-1419. 56. Saran, M., Michel, C., and Bars, W (1990) Free Radical Res. Commun.
10,221-226. 57. Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A., and Freeman, B. A. (1990) Proc. Natl. Acad. Sci. USA 87, 1620-1624. 58. Puppo, A., and Halliwell, B. (1989) Free Radical Res. Commun. 5, 277-281. 59. Puppo, A., and Halliwell, B. (1988) B&hem. J. 249, 185-190. 60. Beckman, J. S. (1990) Nature (London) 345, 27-28. 61. Hibbs, J. B., Jr., Taintor, R. R., Vavrin, Z., and Rachlin, E. M. (1988) Biochem. Biophys. Res. Commun. 157,87-94. 62. Bolli, R., Jeroudi, M. O., Patel, B. S., Du Bose, C. M., Lai, E. K., Roberts, R., and McCay, P. B. (1988) Proc. Natl. Acad Sci. USA 86,4695-4699. 63. Halliwell, B. (1978) FEBS Z&t. 92, 321-326. 64. Liew, F. Y., Millot, S., Parkinson, C., Palmer, R. M. J., and Moncada, S. (1990) J. Zmmunol. 144,4794-4797.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 283, No. 2, December, pp. 223-226, 1990
INVITED PAPER Reoxygenation
Injury and Antioxidant
John M. C. Gutteridge’
and Barry
Protection:
A Tale of Two Paradoxes
Halliwell
Molecular Toxicology, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, Oklahoma 73104; and Pulmonary Medicine Division, UCD Professional Building, 4301 X Street, Sacramento, California 95817 Received April 30, 1990, and in revised form August 3, 1990
Under certain circumstances, added antioxidants can protect tissues against reoxygenation injury after ischemia. Yet reperfusing blood carries many antioxidants with it. The implications of this “antioxidant paradox” are discussed. o 1990 Academic PWS, IIN.
THE
OXYGEN
PARADOX
Oxygen deprivation is damaging to human tissues and the only treatment is rapid tissue reoxygenation by restoring blood flow as soon as possible. If ischemia is too prolonged, irreversible damage takes place, cells die and necrosis eventually occurs. There is no intervention therapy that will restore life to dead cells. The term “reperfusion injury” was first used by Hearse et al. in 1973 (1) and refers to an increase in damage observed when ischemia is corrected by restoring oxygen supply to an organ, the so-called “oxygen paradox.” For example, the heart can respond to reperfusion by showing several dysfunctions, such as arrhythmias, stunning, and infarction (211), depending on the time and extent of the initial ischemia. The molecular mechanisms responsible for the additional damage observed when oxygen is restored to ischemit tissue are still not completely understood, and several injury mechanisms may be operative. However, a great step forward was made when the involvement of oxygenderived species in reoxygenation injury was realized. Guarnieri et al. (4) observed oxidative damage to lipids and decreases in tissue antioxidants during the reoxygenation of hypoxic rat hearts. Parks, Granger, McCord, and their colleagues (5,6) proposed that the superoxide radical (0,) is a key intermediate in reperfusion damage to in1 Permanent address: National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG UK. 0003-9S61/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
testinal (5) and myocardial (6) systems. Their seminal proposal that proteolytic cleavage of xanthine dehydrogenase to xanthine oxidase, which then acts on one of its substrates (xanthine or hypoxanthine) to generate 0, and HzOz in the reperfused tissue has led to rapid advances in our knowledge (5, 6). However, it is now clear that many other mechanisms besides xanthine oxidase can contribute to the increased production of oxygen-derived species upon reperfusion, depending on the model used and the experimental conditions employed. These additional sources of reactive oxygen species include accumulation and activation of neutrophils in the reperfused tissue, increased “leakage” of electrons to oxygen from disrupted mitochondrial electron transport chains, increases in prostaglandin synthesis, oxidation of catecholamines, platelet activation, and increased 0, production by the vascular endothelium (7-l 1). Indeed, doubt exists as to whether xanthine oxidase makes any contribution at all to reoxygenation injury in human myocardium (11-13). Superoxide is produced in most, if not all, aerobic cells and the enzyme superoxide dismutase (SOD)’ is present intracellularly to remove it (14). Failure to remove 0, leads to deleterious consequences (14, 15). Dismutation of 0; gives hydrogen peroxide (HsO,), and two distinct intracellular enzymes, catalase and glutathione peroxidase (selenium-containing), exist to remove it (16). SOD and H,O,-removing enzymes protect the cell against reduced intermediates of oxygen produced during normal aerobic metabolism, but they seem unable to cope when production of 0; and H,O, is excessive (14, 16, 17). The mechanisms of cell damage by increased generation of reactive oxygen species are probably multifactorial (17-20) but considerable evidence suggests that at least some of the damage involves transition metal ions (such as iron), that convert 0, and H202 into aggressive oxidants such as hydroxyl radical (‘OH) by what is essentially Fenton-type 2 Abbreviation
used: SOD, superoxide
dismutase. 223
Inc. reserved.
224
GUTTERIDGE
chemistry (20-25). Thus it is usually proposed that excess generation of 0; and HzOz in reoxygenated tissues leads to formation of ‘OH (3-10) although the question of where the necessary iron ions come from has only recently been raised (26, 27). Evidence that Fenton-type chemistry is involved in reperfusion damage has usually come from the use of scavengers and of iron ion chelators. The most widely used chelator is desferrioxamine, which inhibits iron ion-dependent ‘OH generation under most experimental conditions (28, 29). However, desferrioxamine does many other things as well and it must never be assumed that it is exerting a protective effect by iron ion chelation (29). Attempts to detect release of iron ions using the bleomycin method (30) have so-far proved negative in ischemia-reperfusion systems (Gutteridge, Chevion, Bolli, and Halliwell, unpublished results). However, ischemia has been shown to increase the “availability” of iron in kidney (31, 32) and in dogs subjected to hemorrhagic shock (33). Further, a role for iron in reoxygenation damage is strongly suggested by the results of several other experiments [reviewed in (3, 29)], including the observation that reperfusion damage is considerably greater in iron-overloaded animals (34). Iron chelators other than desferrioxamine have also been reported to be protective (35). Implication of ‘OH, or some other species derived from 0; and HzOz, in reoxygenation injury has been thought necessary because of the observations that 0, and H202 are generally poorly reactive in aqueous solution and none of their described direct damaging effects (36) seem particularly threatening to human tissues. For example, cells subjected to oxidative stress show rapid DNA fragmentation, yet neither Opnor HzOz reacts with DNA (23,37). FROM THE OXYGEN PARADOX THE ANTIOXIDANT PARADOX
TO
During the 1980s there has been an explosion of research into reperfusion injury, mostly using isolated hearts or open-chest animals, but with a few human studies. In no model has the complete mechanism of reoxygenation injury been elucidated. However, a variety of antioxidants has been used to implicate O;, Hz02, and possibly ‘OH, (2-12). For example, SOD and catalase have been extensively explored as protective agents when added to reperfusates in many types of experiment. In some models significant protection is observed while in others the enzymes are without effect. The authors’ general impression is that SOD is often very protective against myocardial arrhythmias, and sometimes protective against stunning, whereas its ability to diminish infarct size is not clearly established (3, 38-40). Protection by antioxidants in at least one model system only occurs when the antioxidant is added preischemia or at the start of reperfusion. Addition of antioxidants after reperfusion is under way (even after only 60 s of reperfusion) gives no protective effect.
AND
HALLIWELL
These studies have been carried out using desferrioxamine (R. Bolli, personal communication) and mercaptopropionylglycine (41) in an open-chest dog model of myocardial stunning injury after reperfusion. Thus, with some satisfaction, scientists have been able to explain partially the “oxygen paradox.” Unfortunately, an antioxidant paradox has appeared. To the authors’ minds, an important question is “Why should antioxidants added extracellularly protect reperfused tissues against damage?” Myocardial cells themselves contain SOD, glutathione peroxidase, and catalase, so why should these same enzymes added extracellularly sometimes protect? Why should desferrioxamine and mercaptopropionylglytine protect reperfused dog heart when added at the start of reoxygenation and not 60 s later, since they are very unlikely to enter cells in that short time (41)? The logical explanation is that the damaging oxygen-derived species being scavenged are generated extracellularly. Here then is the paradox. When blood reenters an ischemic tissue, it carries with it a wide range of antioxidants. Thus erythrocytes possess substantial SOD, glutathione peroxidase, and catalase activities as well as an ion channel through which 0, can pass (42). Indeed, the ability of erythrocytes to protect against oxidative damage has been shown in several systems [e.g., Refs. (43, 44)]. Blood plasma has considerable antioxidant activity: it can inhibit metal iondependent lipid peroxidation and conversion of 0; and H202 into reactive species such as ‘OH (45). The highaffinity iron ion-binding protein of plasma is transferrin: iron bound to transferrins will not stimulate free radical reactions (45-47). The production of reactive oxygen species upon reoxygenation of ischemic myocardium has been amply demonstrated by spin-trapping experiments, although the identity of the trapped radicals is not always clear (4852). If production of these species is extracellular, where do they come from? If protection by desferrioxamine and other iron chelators is due to suppression of iron-iondependent ‘OH generation, where does the extracellular iron come from? Why does transferrin in the reperfusing blood not bind this iron and protect the tissue? It has been proposed (26) that the 0; involved in reperfusion injury is generated by an ischemia-induced derangement ofphysiological endothelial mechanisms (52) for producing 0;. It was proposed that endothelial cells can generate 0, because 0, can further interact with and inactivate endothelium-derived relaxing factor, widely thought to be identical to nitric oxide [reviewed in (53, 54)]. Thus ischemia might somehow not only increase 0, formation (52) but also cause iron ion liberation (26), converting the 0; into damaging species such as ‘OH that are reactive enough to immediately attack the tissue (26). It is possible that the binding of this iron to transferrin is slower than its binding to desferrioxamine, accounting for the failure of plasma transferrin to protect and for the protection observed when desferrioxamine is added at the start of
REOXYGENATION
INJURY
AND
ANTIOXIDANT
225
PROTECTION
ISCHAEMA STIMULATES: Oxidase : Kanthine Dehydrogenase---Xanthine reducing equivalents Idisruption of electron transport chain pro&glandin cascade catecholamines neutrophils activated Biochemical changes platelets facilitate oxidant formation
Cellular SOD CATALASE GSH(Se)pASE
I
L
RED CELL AEPEFlFUSlON STIMULATES
1
Transferrin kictoferrin Albumin Haemopexin Haptoglobins Caeruloplasmin 1,
t
I V ASCULAR
&+NOlo=t&JOt lFlOSBlNDlNG INACTWATING
ENDOTHELIUM
1 AND IRON POTENTLAL
NO PROTECTlON
SOD, CATALASE, XOD INHIBITORS SPIN TRAPS DESFERAIOXAMINE.
FIG. 1. The antioxidant protection.
paradox.
Despite the presence of antioxidants
reperfusion. Indeed, some iron ion complexes found in vivo are slow donors of iron to transferrin [discussed in (55)]. As we have already pointed out, however, a source for the iron has not yet been identified and attempts to detect its formation in myocardial systems have so far proved negative. Another important aspect of the NO/O; interaction has been raised by recent work (56,57). These two species react quickly (rate constant of about 4 X lo7 M-l s-l at pH 7.4) to form peroxynitrite, ONOO-, as the initial product (56). Peroxynitrite then decays rapidly, and it has been suggested that it gives rise to ‘OH [reviewed in (56)]. Consistent with this, Beckman et al. (57) have shown that peroxynitrite decomposition generates a strong oxidizing agent that is able to degrade deoxyribose and dimethylsulfoxide [‘OH attacks both of these molecules, but so do some other oxidants (58, 59)]. Desferrioxamine could also be oxidized by systems containing peroxynitrite (57). If production of 0;and of NO increases when ischemic tissues are reperfused the resulting peroxynitrite could injure cell membranes directly and/or by forming ‘OH in a reaction independent of metal ions (56,57,60). Desferrioxamine might then protect not by binding iron ions but by reacting preferentially with peroxynitrite (57). Peroxynitrite formation might also contribute to the cytotoxic actions of neutrophils and macrophages, which produce both NO and 0;. However, it must not be forgotten that NO can itself be toxic to cells (53, 54, 64). It is especially interesting to note that NO can induce release
in the cells and in the reperfusing
blood, added antioxidants
can offer
of iron from non-heme-iron proteins (61), perhaps providing a link between the O;/NO system and subsequent Fenton chemistry. It has been observed in some model systems [e.g., the myocardial “stunning” model used by Bolli et al. (62)] that the combination of SOD and catalase protects much better than either enzyme alone, even though either enzyme alone can protect almost completely against superoxide-driven Fenton chemistry in vitro (63). How can this be explained? In the model used by Bolli et al. the injury that leads to stunning occurs within the first few seconds of reperfusion (41). Perhaps there are two mechanisms of injury. 0; could do damage by reacting with NO: catalase would not be expected to protect against this (although it is possible that proteins at high concentration might be able to protect by direct reaction with peroxynitrite, thus emphasizing the need for appropriate “controls” with inactivated protein when studying the effects of antioxidant enzymes). SOD would prevent peroxynitrite formation by removing 0;. However, the increased Hz02 generation in the presence of SOD might produce extra damage by a different mechanism, perhaps involving Fenton chemistry. We predict that in the 1990s more and more attention will be directed to the molecular biochemistry taking place at the endothelial and myocyte surfaces during the first few seconds of reperfusion (Fig. 1). ACKNOWLEDGMENTS We are grateful to Drs. Bruce Freeman, Manfred Saran, Christa Michel, Wolf Bors, Joseph Beckman, and Roberto Bolli for their helpful
226
GUTTERIDGE
AND
comments and permission to quote work in press. J.M.C.G. was a 1988 Greenberg scholar. B.H. thanks the British Heart Foundation for research support.
REFERENCES 1. Hearse, D. J., Humphrey, S. M., and Chain, E. B. (1973) J. Mol. Cell. Cardiol. 5, 395-407. 2. Darley-Usmar, V. M., Stone, D., and Smith, D. R. (1989) Free Radical Res. Commun. 7, 247-254. 3. Bolli, R. (1988) J. Amer. Cob!. Cardiol. 12, 239-249. 4. Guarnieri, C., Flamigni, F., and Caldarera, C. M. (1980) J. Mol. Cell. Cardiol. 12,797-808. 5. Granger, D. N., Rutili, G., and McCord, J. M. (1981) GastroenteroZogy 89,22-29. 6. McCord, J. M. (1985) N. Engl. J. Med. 7. Flaherty, J. T., and Weisfeldt, 5,409-419.
312,159-163.
M. L. (1988) Free Radical Biol. Med.
8. Lucchesi, B. R., Werns, S. W., and Fantone, Cell. Cardiol. 21,1241-1251.
J. C. (1989) J. Mol.
9. Simpson, P. J., and Lucchesi, B. R. (1987) J. Lab. Clin. Med. 13-30. 10. Marklund,
110,
S. L. (1988) J. Mol. Cell. Cardiol. 20 (Suppl. 2), 23-30.
11. Kehrer, J. P. (1989) Free Radical Res. Commun. 5, 305-314. 12. Grum, C. M., Gallagher, K. P., Kirsch, M. M., and Shlafer, M. (1989) J. Mol. Cell. Cardiol. 21, 263-267. 13. Eddy, L. J., Stewart, J. R., Jones, H. P., Engerson, T. D., McCord, J. M., and Downey, J. M. (1987) Amer. J. Physior 263, H709-H711. 14. Fridovich, I. (1983) Annu. Rev. Pharmacol. Toxicol. 23, 239-257. 15. Touati,
D. (1989) Free Radical Res. Commun. 8, l-9.
16. Chance, B., Sies, H., and Boveris, A. (1979) Physiol. Rev. 59,527605. 17. Halliwell, B., and Gutteridge, J. M. C. (1989) Free RadicaZs in Biology and Medicine, second ed., Clarendon Press, Oxford. 18. Dypbukt, J. M., Thor, H., and Nicotera, Commun. 8,347-354.
P. (1990) Free Radical Res.
19. Cantoni, O., Scotili, P., Cattabeni, F., Bellomo, G., Pou, S., Cohen, M. and Cerutti, P. (1989) Eur. J. Biochem. 182,209-212. 20. Halliwell, B. (1987) FASEB J. 1, 358-364. 21. Halliwell, B., and Gutteridge, J. M. C. (1990) in Methods in Enzymology (Packer, L., and Glazer, A. N., Eds.), Vol. 186, pp. 1-91, Academic Press, San Diego. 22. Kyle, M. E., Nakae, D., Sakaida, I., Miccadei, S., Farber, J. L. (1988) J. Biol. Chem. 263,3784-3789. 23. Schraufstatter, I. Hyslop, P. A., Jackson, J. H., and Cochrane, C. G. (1988) J. Clin. Znuest. 82, 1040-1050. 24. Imlay, J. A., and Linn, S. (1988) Science 240,
1302-1309.
25. Larramendy, M., Mello-Filho, A. C., Leme Martins, neghini, R. (1987) Mutat. Res. 178, 57-63.
E. A., and Me-
26. Halliwell, B. (1989) Free Radical Res. Commun. 5, 315-218. 27. Puppo, A., and Halliwell, B. (1988) Free Radical Res. Commun. 4, 415-422. 28. Gutteridge, J. M. C., Richmond, R., and Halhwell, B. (1979) B&hem. J. 184,469-472. 29. Halliwell, B. (1989) Free Radical Biol. Med. 7, 645-651. 30. Gutteridge, 113-142.
J. M. C., and Halliwell,
B. (1987) Life Chem. Rep. 4,
HALLIWELL
31. Gower, J., Healing, G., and Green, C. (1989) Free Radical Res. Commum 5,291-299. 32. Paller, S., and Hedlund, B. E. (1988) Kidney Znt. 34,474-480. 33. Saran, S., Sharma, G., Malhotra, R., Sanan, D. P., Jain, P., and Vadhera, P. (1989) Free Radical Res. Commun. 6, 29-38. 34. Van der Kraaij, A. M. M., Mostert, L. J., van Eijk, H. G., and Koster, J. F. (1988) Circulation 78,442-449. 35. Van der Kraaij, A. M. M., van Eijk, H. G., and Koster, J. F. (1989) Circulation 80. 158-164. 36. Fridovich, I. (1986) Arch. Biochem. Biophys. 247, l-11. 37. Aruoma, 0. I., Halliwell, B., and Dizdaroglu, M. (1989) J. Biol. Chem. 264, 13,024-13,028. 38. Englar, R., and Gilpin, E. (1989) Circulation 79, 1137-1142. 39. Cohen, M. V. (1989) Ann. Znt. Med. 111,918-931. 40. Downey, J. M. (1990) Annu. Reu. Physiol. 52,487-504. 41. Bolli, R., Jeroudi, M. O., Patel, B. S., Aruoma, 0. I., Halliwell, B., Lai, E. K., and McCay, P. B. (1989) Circ. Res. 65, 607-622. 42. Lynch, R. E., and Fridovich, I. (1978) J. Biol. Chem. 263, 46974699. 43. Toth, K. M., Clifford, D. P., Berger, E. M., White, C. W., and Repine, J. E. (1984) J. Clin Znuest. 74,292-295. 44. Tsan, M. F., and White, J. E. (1988) Proc. Sot. Exp. Biol. Med. 188, 323-327. 45. Halliwell, B., and Gutteridge, J. M. C. (1990) Arch. B&hem. Biophys. 280, l-8. 46. Gutteridge, J. M. C., Paterson, S. K., Segal, A. W., and Halliwell, B. (1981) Biochem. J. 199,259-261. 47. Aruoma, 0. I., and Halliwell, B. (1987) Biochem. J. 241, 273-278. 48. Bolli, R., and McCay, P. B. (1990) Free Radical Res. Commun. 9, 169-180. 49. Shuter, S. L., Davies, M. J., Garlick, P. B., Hearse, D. J., and Slater, T. F. (1990) Free Radical Res. Commun. 9, 223-232. 50. Blasig, I. E., Ebert, E., and Lowe, H. (1986) St&a Biophysics 116, 35-42. 51. Garlick, P. B., Davies, M. J., Hearse, D. J., and Slater, T. F. (1987) Circ. Res. 61, 757-760. 52. Arroyo, C. M., Carmichael, A. J., Bouscarel, B., Liang, J. H., and Weglicki, W. B. (1990) Free Radical Res. Commun. 9, 287-296. 53. Marletta, M. A. (1989) Trends Biochem Sci. 14,488-492. 54. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1989) Biochem. Pharmacol. 38,1709-1715. 55. Aruoma, 0. I., Bomford, A., Polson, R. J., and Halliwell, B. (1988) Blood 72,1416-1419. 56. Saran, M., Michel, C., and Bars, W (1990) Free Radical Res. Commun.
10,221-226. 57. Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A., and Freeman, B. A. (1990) Proc. Natl. Acad. Sci. USA 87, 1620-1624. 58. Puppo, A., and Halliwell, B. (1989) Free Radical Res. Commun. 5, 277-281. 59. Puppo, A., and Halliwell, B. (1988) B&hem. J. 249, 185-190. 60. Beckman, J. S. (1990) Nature (London) 345, 27-28. 61. Hibbs, J. B., Jr., Taintor, R. R., Vavrin, Z., and Rachlin, E. M. (1988) Biochem. Biophys. Res. Commun. 157,87-94. 62. Bolli, R., Jeroudi, M. O., Patel, B. S., Du Bose, C. M., Lai, E. K., Roberts, R., and McCay, P. B. (1988) Proc. Natl. Acad Sci. USA 86,4695-4699. 63. Halliwell, B. (1978) FEBS Z&t. 92, 321-326. 64. Liew, F. Y., Millot, S., Parkinson, C., Palmer, R. M. J., and Moncada, S. (1990) J. Zmmunol. 144,4794-4797.