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Molecular aspects of myocardial ischemia/reperfusion injury and the protective effects of allopurinol Ko, Robert K. M. 1990

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MOLECULAR ASPECTS OF MYOCARDIAL ISCHEMIA/REPERFUSION INJURY AND THE PROTECTIVE EFFECTS OF ALLOPURINOL By ROBERT K.M. KO B . S c , The C h i n e s e U n i v e r s i t y o f Hong Kong, 1984 M . S c , The C h i n e s e U n i v e r s i t y o f Hong Kong, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department o f Pharmacology & T h e r a p e u t i c s , F a c u l t y o f M e d i c i n e ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1990 © R o b e r t K.M. Ko, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of P h a r m a c o l o g y & T h e r a p e u t i c s The University of British Columbia Vancouver, Canada Date J u l y 26, 1990  DE-6 (2/88) ABSTRACT A growing body of evidence has now accumulated supporting the involvement of oxygen-derived free r a d i c a l s in the development of myocardial ischemia/reperfusion (I/R) injury. We have, therefore, undertaken the present study to examine (1) I/R-related a l t e r a t i o n s i n myocardial antioxidant capacity i n pentobarbital anesthetized open-chest rabbits subjected to l e f t circumflex coronary artery l i g a t i o n followed by reperfusion; (2) the protective effects of pretreatrnent with a l l o p u r i n o l or the 21-aminosteroid U74006F; (3) a l t e r n a t i v e mechanisms to xanthine oxidase i n h i b i t i o n for a l l o p u r i n o l protection against I/R injury; and (4) the e f f e c t of a l l o p u r i n o l treatment on the antioxidant capacity of erythrocytes i n pigs used in a heart-lung transplantation study. In the rabbit myocardium, a marked impairment in myocardial antioxidant capacity developed in association with the onset of i r r e v e r s i b l e injury, as r e f l e c t e d in the enhancement in glutathione (GSH) depletion and formation of t h i o b a r b i t u r i c acid-reactive substances (TBARS) following in  v i t r o incubation of tissue homogenate with t e r t -butylhydroperoxide (TBHP). During the course of post-ischemic reperfusion, the protracted time-course of a l t e r a t i o n s in antioxidant capacity dissociated them from the early burst of r a d i c a l formation known to occur at the onset of post-ischemic reperfusion of the myocardium. When the time-dependent changes i n functional indices of antioxidant status (TBHP-induced GSH depletion and formation of TBARS) were analysed i n r e l a t i o n to a c t i v i t i e s of antioxidant enzymes, evidence suggestive of functionally relevant impairments in Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and glutathione reductase (GRD) a c t i v i t i e s was found. These r e s u l t s and our demonstration of s i g n i f i c a n t decreases in the a c t i v i t y of GSH-dependent antioxidant enzymes under a c i d o t i c conditions suggest that a transient impairment in the functioning of antioxidant enzymes may be involved in t r i g g e r i n g i r r e v e r s i b l e myocardial I/R injury. Repetitive b r i e f episodes of I/R produced a progressive decrease i n myocardial ATP l e v e l s , which was not associated with any detectable changes in myocardial antioxidant capacity. Ischemic preconditioning produced by b r i e f episodes of I/R did not a f f e c t the severity of subsequently induced I/R injury. These r e s u l t s suggest that b r i e f episodes of myocardial ischemia do not produce oxidative t i s s u e damage and the ischemia-induced depletion in myocardial ATP l e v e l i s at least p a r t i a l l y dissociable from the I/R-related impairment in tissu e antioxidant capacity. Isolated Langendorff-perfused rabbit hearts subjected to I/R did not show any changes i n antioxidant capacity. However, when inta c t hearts were subjected to ischemia in  vivo and a subsequent reperfusion in v i t r o , an impairment in myocardial antioxidant capacity became apparent. These iv r e s u l t s suggest that blood elements, possibly activated neutrophils, may be a c r u c i a l factor involved i n the development of I/R-induced oxidant injury. Chronic a l l o p u r i n o l pretreatment (1 mg/ml i n drinking water or approximately 75 mg/kg/day) for 7 days p r i o r to ischemia provided s i g n i f i c a n t protection against I/R-induced a l t e r a t i o n s i n myocardial antioxidant capacity, but not the decrease i n tissue ATP l e v e l s . This chronic a l l o p u r i n o l regimen was found to enhance myocardial GRD a c t i v i t y in non-ischemic t i s s u e . In addition, both a l l o p u r i n o l and oxypurinol i n h i b i t e d the t r a n s i t i o n metal ion-catalysed ascorbate oxidation and l i p i d peroxidation in v i t r o , l i k e l y as a consequence of t h e i r metal chelating properties. S i m i l a r l y , myoglobin-TBHP-catalysed oxidation of u r i c acid and l i p i d peroxidation were also suppressed by a l l o p u r i n o l . A l l these suggest that a l l o p u r i n o l may favorably a l t e r myocardial antioxidant capacity d i r e c t l y by v i r t u e of i t s t r a n s i t i o n metal chelating properties and i t s antioxidant actions i n myoglobin-mediated oxidative processes. The acute administration of 21-aminosteroid U74006F (3 mg/kg, i.v) under conditions comparable to those known to protect against trauma-induced damage in the central nervous system f a i l e d to reduce manifestations of oxidative injury in rabbit hearts subjected to ischemia and reperfusion. Although reactive oxy-radicals have been implicated in both types of tissue damage, the observed difference in s u s c e p t i b i l i t y to protection by t h i s s t e r o i d a l antioxidant s u g g e s t s t h a t t h e m o l e c u l a r mechanisms i n v o l v e d a r e not i d e n t i c a l . I n t h e h e a r t - l u n g t r a n s p l a n t a t i o n s t u d y , e r y t h r o c y t e s from a l l o p u r i n o l - t r e a t e d p i g s ( g i v e n r e p e a t e d l y a t an o r a l dose o f 50 mg/kg) showed a time/dose-dependent i n c r e a s e i n a n t i o x i d a n t c a p a c i t y as r e f l e c t e d i n t h e d e c r e a s e i n m a l o n d i a l d e h y d e p r o d u c t i o n f o l l o w i n g i n v i t r o o x i d a t i v e c h a l l e n g e . The e x t e n t o f r e d c e l l p r o t e c t i o n i n b o t h donor and r e c i p i e n t a n i m a l s c o r r e l a t e d s i g n i f i c a n t l y w i t h t h e f u n c t i o n a l v i a b i l i t y o f t h e t r a n s p l a n t e d l u n g t i s s u e , as a s s e s s e d by t i s s u e w a t e r c o n t e n t . These r e s u l t s s u g g e s t t h a t t h e measurement o f e r y t h r o c y t e a n t i o x i d a n t c a p a c i t y may p r o v i d e an u s e f u l assessment o f g e n e r a l i z e d a l t e r a t i o n s i n t i s s u e a n t i o x i d a n t s t a t u s produced by p h a r m a c o l o g i c a l i n t e r v e n t i o n s . v i TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v i LIST OF TABLES x i i i LIST OF FIGURES x i v LIST OF ABBREVIATIONS x v i i ACKNOWLEDGEMENTS x i x DEDICATION xx 1. INTRODUCTION 1 1.1 Free Radicals i n B i o l o g i c a l Systems 1 1.1.1 C e l l u l a r D e f e n s e M e c h a n i s m s A g a i n s t F r e e R a d i c a l s 2 1.1.2 A n t i o x i d a n t E n z y m e s 3 1.1.3 N o n - E n z y m a t i c A n t i o x i d a n t s 4 1.1.4 E x t r a c e l l u l a r A n t i o x i d a n t S y s t e m 5 1.1.5 A n t i o x i d a n t I n t e r a c t i o n s 6 1.1.6 C y t o t o x i c E f f e c t s o f H y d r o x y 1 R a d i c a l O t h e r R e a c t i v e O x y g e n - D e r i v e d S p e c i e s . . . 7 1.2 Myocardial Ischemia/Reperfusion Injury 9 1.2.1 R o l e o f R e a c t i v e O x y g e n - D e r i v e d R a d i c a l s i n M y o c a r d i a l I s c h e m i a / R e p e r f u s i o n I n j u r y 10 1.2.2 C y t o t o x i c E f f e c t s o f O x y - R a d i c a l s on t h e M y o c a r d i u m 12 1.2.3 S o u r c e s o f O x y - R a d i c a l s i n t h e I s c h e m i c M y o c a r d i u m 13 1.2.3.1 X a n t h i n e O x i d a s e 13 1.2.3.2 A c t i v a t e d N e u t r o p h i l s 14 v i i 1.2.3.3 D i s r u p t i o n o f t h e M i t o c h o n d r i a l E l e c t r o n T r a n s p o r t S y s t e m 15 1.2.3.4 M e t a b o l i s m o f A r a c h i d o n i c A c i d 16 1.2.3.5 O x i d a t i o n o f C a t e c h o l a m i n e s 16 1.2.3.6 T r a n s i t i o n M e t a l C a t a l y s e d O x y - R a d i c a l P r o d u c t i o n 17 1.2.4 P o s s i b l e S t r a t e g i e s i n t h e P r e v e n t i o n o f O x y - R a d i c a l - M e d i a t e d M y o c a r d i a l I s c h e m i a / R e p e r f u s i o n I n j u r y 17 1.3 Rationale and Objectives of the Study 19 1.3.1 C l i n i c a l S i g n i f i c a n c e o f I s c h e m i a / R e p e r f u s i o n I n j u r y 19 1.3.2 P r o t e c t i v e E f f e c t s o f A l l o p u r i n o l A g a i n s t I s c h e m i a / R e p e r f u s i o n I n j u r y 21 1.3.3 E x p e r i m e n t a l A p p r o a c h e s 2 3 1.3.3.1 M o l e c u l a r A s p e c t s o f M y o c a r d i a l I s c h e m i a / R e p e r f u s i o n I n j u r y a n d t h e P r o t e c t i v e E f f e c t s o f A l l o p u r i n o l 2 3 1.3.3.2 I s c h e m i a a n d R e p e r f u s i o n i n a S w i n e M o d e l o f H e a r t - L u n g T r a n s p l a n t a t i o n a n d t h e E f f e c t s o f A l l o p u r i n o l P r e t r e a t m e n t 27 1.3.3.4 M e t a l C h e l a t i n g a n d o t h e r A n t i o x i d a n t P r o p e r t i e s o f A l l o p u r i n o l 29 2. MATERIALS AND METHODS 3 2 2.1 Surgical Procedures 3 2 2.1.1 A n i m a l C a r e 3 2 2.1.2 E x p e r i m e n t a l M o d e l o f I s c h e m i a / R e p e r f u s i o n I n j u r y 32 2.1.2.1 P e n t o b a r b i t a l A n e s t h e t i z e d O p e n - C h e s t R a b b i t 3 2 2.1.2.2 I n d u c t i o n o f R e g i o n a l I s c h e m i a . . 3 3 v i i i 2.1.2.3 I d e n t i f i c a t i o n of the Occluded Zone 3 3 2.1.2.4 Sham-Operated Animals 3 3 2.1.3 I s o l a t e d L a n g e n d o r f f - P e r f u s e d Rabbit Heart 3 4 2.1.3.1 I n d u c t i o n o f Regiona l Ischemia i n the I s o l a t e d Rabbit Heart .... 34 2.1.3.2 Sham-Control f o r I s o l a t e d Rabbit Heart 3 4 2.1.3.3 I n d u c t i o n of Regiona l Ischemia .. 3 5 In V i v o Followed by In V i t r o R e p e r f u s i o n 2.1.4 Heart-Lung T r a n s p l a n t a t i o n i n Pigs 3 5 2.2 Drug Treatment Protocols 37 2.2.1 Chronic A l l o p u r i n o l Treatment i n Rab b i t s 37 2.2.2 Acute A l l o p u r i n o l and Oxypurinol Treatment i n Rabbits 37 2.2.3 Acute U74006F Treatment i n Rabbits 37 2.3 Biochemical and Chemical Analyses 2.3.1 T i s s u e S u s c e p t i b i l i t y t o In V i t r o Peroxide Challenge 38 2.3.1.1 P r e p a r a t i o n of T i s s u e Homo-genates 38 2.3.1.2 S u s c e p t i b i l i t y of T i s s u e t o TBHP-Induced D e p l e t i o n of GSH ... 38 2.3.1.3 S u s c e p t i b i l i t y of T i s s u e s t o L i p i d P e r o x i d a t i o n 39 2.3.2 E r y t h r o c y t e S u s c e p t i b i l i t y t o In V i t r o Peroxide Challenge 4 0 2.3.2.1 P r e p a r a t i o n of Packed E r y t h r o c y t e s 4 0 2.3.2.2 S u s c e p t i b i l i t y of E r y t h r o c y t e s t o TBHP-Induced L i p i d P e r o x i d a t i o n 4 0 i x 2.3.2.3 Hemoglobin Assay 41 2.3.3 Tissue Antioxidant Enzyme A c t i v i t i e s . . . . 41 2.3.3.1 Preparation of Cytosolic Fractions 42 2.3.3.2 Catalase 42 2.3.3.3 Cu,Zn-Superoxide Dismutase 43 2.3.3.4 Glutathione Peroxidase 4 3 2.3.3.5 Glutathione Reductase 4 4 2.3.3.6 Hemoglobin Assay 4 5 2.3.3.7 Correction for Enzyme A c t i v i t y Contributed by Blood 4 5 2.3.4 Erythrocyte Antioxidant Enzyme A c t i v i t i e s 45 2.3.4.1 Preparation of Hemolysates 45 2.3.4.2 Catalase 46 2.3.4.3 Cu,Zn-Superoxide Dismutase 46 2.3.4.4 Glutathione Peroxidase 4 6 2.3.4.5 Glutathione Reductase 4 6 2.3.4.6 Hemoglobin Assay 4 7 2.3.5 Mitochondrial ATPase A c t i v i t y 47 2.3.5.1 Is o l a t i o n of Mitochondria 47 2.3.5.2 Mitochondrial Azide-Sensitive ATPase A c t i v i t y 4 8 2.3.6 Tissue ATP 49 2.3.7 Preparation of Erythrocyte Membranes . . . . 50 2.3.8 F e r r i c Chloride-Induced Oxidation of Erythrocyte Membrane Lipids 51 2.3.9 Cupric Chloride-TBHP-Induced Oxidation of Erythrocyte Membrane Lipids 52 2.3.10 Myoglobin-TBHP-Induced Oxidation of Erythrocyte Membrane Lipids 5 3 X 2.3.11 Transition Metal Ion-Catalysed Oxidation of Ascorbic Acid 53 2.3.12 UV Absorption Spectroscopy of Ascorbate/Allopurinol/Copper Ion 54 2.3.13 Myoglobin-TBHP-Catalysed Oxidation of U r i c Acid 55 2.3.14 S t a t i s t i c a l Analyses 56 3. RESULTS 3.1 S u s c e p t i b i l i t y of Tissues to Peroxide Challenge 57 3.2 Altered Antioxidant Capacity i n Ischemic/ Reperfused Myocardial Tissues 62 3.3 Alter a t i o n s i n Myocardial Antioxidant Capacity i n Rabbits Subjected to Increasing Periods of Ischemia 75 3.3.1 E f f e c t s of A l l o p u r i n o l Pretreatrnent 80 3.3.2 Ef f e c t s of Acute A l l o p u r i n o l and Oxypurinol Pretreatrnent 8 0 3.3.3 Ef f e c t s of Chronic A l l o p u r i n o l Treatment on the A c t i v i t y of Myocardial Antioxidant Enzymes 87 3.3.4 Eff e c t s of Acute U74006F Treatment on Ischemia/Reperfusion-Induced Alte r a t i o n s in Rabbit Myocardium 87 3.4 Time-Course of Alterations i n Myocardial Antioxidant Capacity During Post-Ischemic Reperfusion 91 3.4.1 Alterations in the A c t i v i t i e s of Antioxidant Enzymes i n Ischemic/ Reperfused Myocardial Tissues 100 3.4.2 pH Dependence of Antioxidant Enzyme A c t i v i t i e s 101 3.5 B r i e f Episodes of Ischemia 104 3.5.1 Ef f e c t s of Cumulative Brief Episodes of Ischemia on Myocardial Antioxidant Capacity and ATP Levels 104 x i 3.5.2 Ef f e c t s of Ischemic Preconditioning on I/R-related Myocardial Alterations ... 109 3.6 I/R-Induced Alterations i n Antioxidant Capacity i n Isolated Langendorff-Perfused Hearts 112 3.7 Heart-Lung Transplantation 115 3.7.1 Ef f e c t s of A l l o p u r i n o l Pretreatment on the Antioxidant Capacity of Pig Erythrocytes 115 3.8 Metal Chelating Properties of A l l o p u r i n o l .... 122 3.8.1 Ef f e c t s of A l l o p u r i n o l and Oxypurinol on Transition Metal Ion-Catalysed Oxidation of Ascorbic Acid 123 3.8.2 Transition Metal Ion-Catalysed Oxidation of Erythrocyte Membrane Lipids 12 8 3.8.2.1 F e r r i c Chloride-Induced Oxidat-ion of Erythrocyte Membrane Lipids 129 3.8.2.2 Ef f e c t s of A l l o p u r i n o l and Oxypurinol on F e r r i c Chloride-Stimulated Oxidation of Erythrocyte Membrane Lipids .... 132 3.8.2.3 Ef f e c t s of A l l o p u r i n o l and Oxypurinol on Cupric Ion-TBHP-Induced Oxidation of Erythrocyte Membrane Lipids .... 137 3.8.3 UV Absorption Spectra of A l l o p u r i n o l / Ascorbic Acid/Copper Ion 140 3.9 Myoglobin-TBHP-Catalysed Oxidation of Uric Uric Acid and Erythrocyte Membrane Lipids .... 145 3.9.1 Ef f e c t s of A l l o p u r i n o l and Oxypurinol on Myoglobin-TBHP-Catalysed Oxidation of Uric Acid 145 3.9.2 Ef f e c t s of Al l o p u r i n o l on Myoglobin-TBHP-Induced Oxidation of Erythrocyte Membrane Lipids 148 4 DISCUSSION 154 4.1 Assessment of Tissue Antioxidant Capacity .... 154 x i i 4.2 A l t e r a t i o n s i n A n t i o x i d a n t C a p a c i t y i n Ischemic/Reperfused Rabbit Myocardium 156 4.3 Time-Course of A l t e r a t i o n s i n M y o c a r d i a l A n t i o x i d a n t C a p a c i t y and A n t i o x i d a n t Enzyme A c t i v i t i e s During Post-Ischemic R e p e r f u s i o n 159 4.4 The E f f e c t s of Cumulative B r i e f Episodes of Ischemia on M y o c a r d i a l A n t i o x i d a n t C a p a c i t y and ATP L e v e l s 162 4.5 I/R-Induced A l t e r a t i o n s i n My o c a r d i a l A n t i o x i d a n t C a p a c i t y i n I s o l a t e d L a n g e n d o r f f - P e r f u s e d Rabbit Hearts 172 4.6 The E f f e c t s of A l l o p u r i n o l Pretreatrnent on My o c a r d i a l I/R I n j u r y 166 4.7 The E f f e c t s of Acute U74006F Treatment on I/R-Induced A l t e r a t i o n s i n Rabbit Myocardium 169 4.8 The E f f e c t s of A l l o p u r i n o l Pretreatrnent on the A n t i o x i d a n t C a p a c i t y of E r y t h r o c y t e s and the F u n c t i o n a l v i a b i l i t y of T r a n s p l a n t e d Lung T i s s u e 172 4.9 I n h i b i t o r y E f f e c t s of A l l o p u r i n o l and Oxy p u r i n o l on T r a n s i t i o n Metal Ion-C a t a l y s e d Ascorbate O x i d a t i o n and L i p i d P e r o x i d a t i o n 177 4.10 The E f f e c t s of A l l o p u r i n o l on Myoglobin-TBHP-Catalysed U r i c A c i d O x i d a t i o n and L i p i d P e r o x i d a t i o n 184 4.11 Summary and Co n c l u s i o n s 187 5. REFERENCES 193 xi i i Table LIST OF TABLES Page I E f f e c t s of c h r o n i c a l l o p u r i n o l treatment on 88 myoc a r d i a l a n t i o x i d a n t c a p a c i t y . II E f f e c t s of a c u t e l y a d m i n i s t e r e d a l l o p u r i n o l 89 or o x y p u r i n o l (50 mg/kg, i . v . ) on I/R i n j u r y . III E f f e c t s of c h r o n i c a l l o p u r i n o l treatment on the 90 a c t i v i t y of myocardial a n t i o x i d a n t enzymes. IV E f f e c t s of U74006F pretreatment on I/R-induced 92 a l t e r a t i o n s i n r a b b i t myocardium. V Time-course of p o s t - i s c h e m i c r e p e r f u s i o n i n j u r y 102 i n r a b b i t myocardium: e f f e c t s on a n t i o x i d a n t enzymes. VI C o r r e l a t i o n s between the a c t i v i t i e s of 103 a n t i o x i d a n t enzymes and a n t i o x i d a n t c a p a c i t y of i s c h e m i c / r e p e r f u s e d r a b b i t myocardium. VII E f f e c t s of a c i d o t i c pH on a n t i o x i d a n t 105 enzyme a c t i v i t i e s . VIII E f f e c t s of b r i e f cumulative episodes of 107 ischemia on myocardial a n t i o x i d a n t c a p a c i t y and ATP l e v e l s . IX E f f e c t s of b r i e f cumulative episodes of 108 ischemia on myocardial a n t i o x i d a n t enzyme a c t i v i t i e s . X E f f e c t s of ischemic p r e c o n d i t i o n i n g on I/R- 110 induced myocardial a l t e r a t i o n s . XI E f f e c t s of ischemic p r e c o n d i t i o n i n g on 111 myocardial a n t i o x i d a n t enzyme a c t i v i t i e s . XII I/R-induced a l t e r a t i o n s i n a n t i o x i d a n t 114 c a p a c i t y i n i n t a c t or i s o l a t e d Langendorff-p e r f u s e d r a b b i t h e a r t s . XIII I n t e r - a n i m a l v a r i a t i o n i n a l l o p u r i n o l - 119 induced p r o t e c t i o n a g a i n s t l i p i d p e r o x i d a t i o n i n p i g e r y t h r o c y t e s . XIV Absorbance changes of the a l l o p u r i n o l - 144 a s c o r b a t e - c u p r i c c h l o r i d e mixture: e f f e c t s of EDTA. xiv Fig. LIST OF FIGURES Page 1 TBHP-induced d e p l e t i o n of GSH i n t i s s u e 59 homogenates prepared from non-ischemic t i s s u e s from r a b b i t . 2 TBHP-induced formation o f TBARS i n t i s s u e 61 homogenates prepared from non-ischemic t i s s u e s from r a b b i t . 3 C a t a l a s e a c t i v i t i e s i n r a b b i t t i s s u e s . 64 4 Cu,Zn-SOD a c t i v i t i e s i n r a b b i t t i s s u e s . 66 5 G l u t a t h i o n e p e r o x i d a s e a c t i v i t i e s i n r a b b i t 68 t i s s u e s . 6 G l u t a t h i o n e reductase a c t i v i t i e s i n r a b b i t 70 t i s s u e s . 7 TBHP-induced d e p l e t i o n of GSH i n myocardial 72 t i s s u e homogenates f o l l o w i n g a 40 min p e r i o d of ischemia w i t h or without subsequent r e p e r f u s i o n f o r 60 min i n r a b b i t s . 8 TBHP-induced TBARS formation i n myocardial 74 t i s s u e homogenates f o l l o w i n g a 40 min p e r i o d of ischemia with or without subsequent r e p e r f u s i o n f o r 60 min i n r a b b i t s . 9 Changes i n s u s c e p t i b i l i t y of myocardial t i s s u e 77 to TBHP-induced d e p l e t i o n of GSH a f t e r v a r y i n g p e r i o d s of coronary a r t e r y l i g a t i o n w i t h or without 60 min of r e p e r f u s i o n i n r a b b i t s . 10 Changes i n s u s c e p t i b i l i t y of myocardial t i s s u e 79 to TBHP-induced formation of TBARS a f t e r v a r y i n g p e r i o d s of coronary a r t e r y l i g a t i o n with or without 60 min of r e p e r f u s i o n i n r a b b i t s . 11 E f f e c t of c h r o n i c a l l o p u r i n o l treatment on the 82 s u s c e p t i b i l i t y of myocardial t i s s u e to TBHP-induced d e p l e t i o n of GSH a f t e r a 30 min p e r i o d of coronary a r t e r y l i g a t i o n f o l l o w e d by 60 min of r e p e r f u s i o n i n r a b b i t s . XV 12 E f f e c t of c h r o n i c a l l o p u r i n o l treatment on the 84 s u s c e p t i b i l i t y of myocardial t i s s u e t o TBHP-induced formation of TBARS a f t e r a 3 0 min p e r i o d of coronary a r t e r y l i g a t i o n f o l l o w e d by 60 min of r e p e r f u s i o n i n r a b b i t s . 13 E f f e c t of c h r o n i c a l l o p u r i n o l treatment on the 86 t i s s u e ATP l e v e l a f t e r a 30 min p e r i o d of coronary a r t e r y l i g a t i o n f o l l o w e d by 60 min of r e p e r f u s i o n i n r a b b i t s 14 Time-course of a l t e r a t i o n s i n myocardial GSH 95 l e v e l s d u r i n g the course of p o s t - i s c h e m i c r e p e r f u s i o n i n r a b b i t s . 15 Time-course of a l t e r a t i o n s i n GSH d e p l e t i o n o f 97 m y o c a r d i a l t i s s u e d u r i n g the course of p o s t -ischemic r e p e r f u s i o n i n r a b b i t s . 16 Time-course of a l t e r a t i o n s i n formation of 99 TBARS i n myocardial t i s s u e d u r i n g the course of p o s t - i s c h e m i c r e p e r f u s i o n i n r a b b i t s . 17 E f f e c t of a l l o p u r i n o l treatment on 117 s u s c e p t i b i l i t y of p i g e r y t h r o c y t e s t o l i p i d p e r o x i d a t i o n . 18 C o r r e l a t i o n between e r y t h r o c y t e MDA and lung 121 water (LW) l e v e l s . 19 C u p r i c i o n - c a t a l y s e d o x i d a t i o n of ascorbate: 125 e f f e c t s of a l l o p u r i n o l . 20 C u p r i c and f e r r i c i o n - c a t a l y s e d o x i d a t i o n of 127 a s c o r b a t e i n the presence of EDTA. 21 F e r r i c i o n - induced formation of TBARS i n 131 e r y t h r o c y t e membranes 22 E f f e c t s of GSH and a s c o r b i c a c i d on the time- 134 course of f e r r i c i o n - induced formation of e r y t h r o c y t e membranes. 23 F e r r i c i o n - induced formation of TBARS i n 136 e r y t h r o c y t e membranes: e f f e c t s of a l l o p u r i n o l and o x y p u r i n o l . 24 C u p r i c ion-TBHP - induced formation of 139 TBARS i n e r y t h r o c y t e membranes: e f f e c t s of a l l o p u r i n o l and o x y p u r i n o l . A x v i 25 UV absorption spectrum of the a l l o p u r i n o l - 142 ascorbate-cupric chloride mixture: e f f e c t s of EDTA. 2 6 Myoglobin-TBHP - catalysed oxidation of u r i c 147 acid: e f f e c t s of a l l o p u r i n o l and oxypurinol. 27 Time-course of myoglobin-TBHP - induced 150 peroxidation of erythrocyte membrane l i p i d s . 28 Myoglobin-TBHP-induced formation of 152 TBARS i n erythrocyte membranes: e f f e c t of a l l o p u r i n o l . LIST OP ABBREVIATIONS ALP a l l o p u r i n o l ANS l - a m i n o - 2 - n a p h t h o l - 4 - s u l f o n i c a c i d ASC a s c o r b i c a c i d ATP a d e n o s i n e t r i p h o s p h a t e ATPase a d e n o s i n e t r i p h o s p h a t a s e BHT b u t y l a t e d h y d r o x y t o l u e n e CAT c a t a l a s e DTNB 5 , 5 ' - d i t h i o - b i s - ( 2 - n i t r o b e n z o i c a c i d ) EDTA e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d EGTA e t h y l e n e g l y c o l - b i s - ( b e t a - a m i n o e t h y l e t h e r ) N , N ' - t e t r a a c e t i c a c i d GPX g l u t a t h i o n e p e r o x i d a s e GRD g l u t a t h i o n e r e d u c t a s e GSH g l u t a t h i o n e , reduced form GSSG g l u t a t h i o n e d i s u l f i d e HO* h y d r o x y l r a d i c a l I/R i s c h e m i a / r e p e r f u s i o n ISC/NON i s c h e m i c / n o n - r e p e r f u s e d ISC/REP i s c h e m i c / r e p e r f u s e d LW l u n g w a t e r MDA m a l o n d i a l d e h y d e x v i i i NADPH n i c o t i n a m i d e adenine d i n u c t e o t i d e phosphate, reduced form. 0 2*~ s u p e r o x i d e a n i o n r a d i c a l OXYP o x y p u r i n o l Pi i n o r g a n i c phosphate pC>2 a r t e r i a l p a r t i a l p r e s s u r e o f oxygen PRECON p r e c o n d i t i o n i n g RBC r e d b l o o d c e l l s SHAM-CON sham-operated c o n t r o l SOD s u p e r o x i d e d i s m u t a s e TBA t h i o b a r b i t u r i c a c i d TBARS t h i o b a r b i t u r i c a c i d - r e a c t i v e s u b s t a n c e s TBHP t e r t - b u t y l h y d r o p e r o x i d e TCA t r i c h l o r o a c e t i c a c i d T r i s t r i s [ h y d r o x y m e t h y l ] a m i n o m e t h a n e URC u r i c a c i d A C K N O W L E D G E M E N T S I would l i k e to express my sincere gratitude to my supervisor Dr. David V. Godin for his continuous encouragement and guidance. Much thank i s due to my colleague Ms. Maureen E. Garnett whose profound technical knowledge and valuable suggestions have been very h e l p f u l . Special gratitude i s also expressed to Mrs. Janet Garnett for her warm caring. I am indebted to Drs. Sidney Katz, Catherine C.Y. Pang, Michael J.A. Walker and Richard A. Wall for t h e i r p a r t i c i p a t i o n i n my thesis committee. I am g r a t e f u l to the faculty and s t a f f members of Department of Pharmacology & Therapeutics for allowing me to use t h e i r f a c i l i t i e s . I also express my gratitude to Drs. A.K. Qayumi and W.R.E. Jamieson at Vancouver General Hospital for giving me opportunity to collaborate with them i n the heart-lung transplantation study. F i n a l l y , the f i n a n c i a l support of the Canadian Commonwealth Scholarships & Fellowships Plan for my study in Canada i s greatly appreciated. X X A l l dedicated to my parents and my sweet heart Tammy for t h e i r passion, patience and unlimited support 1 1. INTRODUCTION 1.1 Free Radicals i n B i o l o g i c a l Systems A free r a d i c a l i s a molecule with an odd (unpaired) electron i n i t s outer o r b i t ; t h i s unpaired electron makes the molecule unstable and highly reactive, e s p e c i a l l y towards b i o l o g i c a l molecules such as l i p i d s , proteins and DNA. Free r a d i c a l s can be produced i n b i o l o g i c a l systems and may be important i n a vari e t y of c e l l u l a r processes such as d i f f e r e n t i a t i o n [1,2], aging [2,3], mutagenesis [4], carcinogenesis [3] and phagocyte-dependent inflammation [5]. In addition, oxygen-derived free r a d i c a l s have been also implicated i n the pathogenesis of many diseases including ischemia/reperfusion (I/R) injury in brain [3], myocardium [6-8], l i v e r [9], kidney [10], inte s t i n e [9], pancreas [3,9], s k e l e t a l muscle [9] and skin [3], oxygen t o x i c i t y of lung [11,12] and ret i n a [3], and rheumatoid a r t h r i t i s [13]. Furthermore, the toxic e f f e c t s of some xenobiotics and chemical pollutants, such as paraquat, carbon te t r a c h l o r i d e and c i g a r e t t e smoke, have been attributed to free r a d i c a l -mediated tissu e damage [14,15]. On the other hand, the free r a d i c a l generating properties of some xenobiotics, such as chloramphenicol [16], t e t r a c y c l i n e [16] and doxorubicin [17], have been exploited for t h e i r therapeutic use as a n t i -microbial and a n t i - p r o l i f e r a t i v e agents. 2 1.1.1 Cellular Defense Mechanisms Against Free Radicals Under normal p h y s i o l o g i c a l c o n d i t i o n s , t h e oxygen m o l e c u l e undergoes t e t r a v a l e n t r e d u c t i o n i n a e r o b i c c e l l s v i a t h e m i t o c h o n d r i a l cytochrome o x i d a s e pathway w i t h r e s u l t i n g f o r m a t i o n o f wa t e r [ 1 8 ] , D u r i n g t h i s normal c o u r s e o f m e t a b o l i s m , s e v e r a l r e a c t i v e oxygen i n t e r m e d i a t e s a r e formed. The f i r s t e l e c t r o n t r a n s f e r t o oxygen produces a s u p e r o x i d e a n i o n r a d i c a l (02*~) w h i c h , i n t u r n , g e n e r a t e s hydrogen p e r o x i d e (H2O2) f o l l o w i n g t h e a c c e p t a n c e o f t h e second e l e c t r o n . W i t h t h e t h i r d e l e c t r o n t r a n s f e r , a h y d r o x y l r a d i c a l (HO*) i s formed. F i n a l l y , a c c e p t a n c e of t h e f o u r t h e l e c t r o n g e n e r a t e s a H2O m o l e c u l e . I n a d d i t i o n , u n i v a l e n t r e d u c t i o n o f O2 a l s o o c c u r s i n t h e m i t o c h o n d r i o n ; f o r i n s t a n c e , t h e semiquinone r a d i c a l p r o d u c e d from u b i q u i n o n e d u r i n g e l e c t r o n t r a n s p o r t can g i v e r i s e t o a s u p e r o x i d e a n i o n r a d i c a l [ 1 9 ] . I n t h e i n t a c t m i t o c h o n d r i o n , most o f t h e p a r t i a l l y r e d u c e d r e a c t i v e oxygen s p e c i e s a r e t i g h t l y bound t o t h e e n z y m a t i c s i t e s i n v o l v e d i n t h e i r g e n e r a t i o n . To t h e e x t e n t t h a t s m a l l amounts o f r e a c t i v e oxygen s p e c i e s l e a k i n t o t h e c y t o s o l , endogenous p r o t e c t i v e mechanisms, w h i c h c o n s i s t o f b o t h e n z y m a t i c and non-e n z y m a t i c components, a r e c a p a b l e of i n a c t i v a t i n g them, t h e r e b y p r o t e c t i n g t h e c e l l from r a d i c a l - m e d i a t e d o x i d a t i v e damage t o membranes and a l t e r a t i o n s i n s u b c e l l u l a r o r g a n e l l e s t r u c t u r a l and f u n c t i o n a l i n t e g r i t y [ 2 0 ] . 3 1.1.2 Antioxidant Enzymes S u p e r o x i d e a n i o n r a d i c a l ( O 2 * - ) i s c a t a l y t i c a l l y c o n v e r t e d by s u p e r o x i d e d i s m u t a s e (SOD) i n t o m o l e c u l a r oxygen and H2O2 [ 2 1 ] . Two forms o f SOD have been d e s c r i b e d - a m i t o c h o n d r i a l enzyme (Mn-SOD) w h i c h i s c y a n i d e i n s e n s i t i v e and a c y t o s o l i c enzyme (Cu,Zn-SOD) wh i c h i s h i g h l y s e n s i t i v e t o i n h i b i t i o n by c y a n i d e [ 2 1 ] . H2°2' a l t h o u g h not a f r e e r a d i c a l , may i t s e l f o r i n t h e presence o f ®2~' < j i v e r i s e t o HO* and s i n g l e t oxygen v i a t h e t r a n s i t i o n m e t a l i o n - c a t a l y s e d Fenton o r Haber-Weiss r e a c t i o n , r e s p e c t i v e l y [ 2 2 ] . The d e c o m p o s i t i o n o f H2O2 i s c a t a l y s e d by c a t a l a s e (CAT) whose a c t i o n , when c o u p l e d w i t h t h a t o f SOD, can p r e v e n t t h e g e n e r a t i o n o f h y d r o x y l r a d i c a l s . O r g a n i c h y d r o p e r o x i d e s a r i s i n g from h y d r o x y l r a d i c a l - m e d i a t e d p e r o x i d a t i o n r e a c t i o n s a r e d e t o x i f i e d by t h e g l u t a t h i o n e p e r o x i d a s e (GPX) [ 2 3 ] . Two forms o f GPX e x i s t o f w h i c h t h e s e l e n i u m - c o n t a i n i n g enzyme (Se-GPX) a c t s on a v a r i e t y o f s u b s t r a t e s , i n c l u d i n g hydrogen p e r o x i d e and o r g a n i c h y d r o p e r o x i d e s [ 2 4 ] , w h i l e t h e no n - s e l e n i u m -dependent GPX does not u t i l i z e H2O2 and b e l o n g s t o a f a m i l y o f g l u t a t h i o n e t r a n s f e r a s e enzymes [ 2 5 ] . The c a t a l y t i c d e c o m p o s i t i o n o f h y d r o p e r o x i d e s by GPX i s a s s o c i a t e d w i t h t h e s i m u l t a n e o u s o x i d a t i o n of g l u t a t h i o n e (GSH) t o i t s d i s u l f i d e form (GSSG), w h i c h i s r e g e n e r a t e d by g l u t a t h i o n e r e d u c t a s e (GRD) u s i n g NADPH d e r i v e d m a i n l y from t h e hexose monophosphate shunt enzyme g l u c o s e - 6 - p h o s p h a t e dehydrogenase as a r e d u c i n g agent [ 2 6 ] . The a f o r e m e n t i o n e d a n t i o x i d a n t 4 enzymes are widely d i s t r i b u t e d i n c e l l s of tissues, e s p e c i a l l y those with high rates of aerobic metabolism, with highest a c t i v i t i e s often being found i n l i v e r c e l l s [26,27]. 1.1.3 Non-Enzymatic Antioxidants In addition to the c r i t i c a l r o le of antioxidant enzymes i n preventing t i s s u e oxidative damage, endogenous molecules, such as a-tocopherol, /3-carotene, ascorbic acid, u r i c acid and GSH or protein t h i o l s etc., are also important i n the d e t o x i f i c a t i o n of oxy-radicals. Alpha-tocopherol (Vitamin E) , a major l i p i d - s o l u b l e antioxidant present i n b i o l o g i c a l membranes, protects against peroxidation of membrane l i p i d s [28]; i t can scavenge a v a r i e t y of reactive oxy-radicals, including O2* ~ [29], HO* and peroxyl r a d i c a l s [30], as well as s i n g l e t oxygen [31]. Beta-carotene, a major carotenoid precursor of Vitamin A, i s also present i n c e l l u l a r membranes [32], and l i k e l y has antioxidant functions s i m i l a r to those of a-tocopherol. Thus, l i p o p h i l i c antioxidants, which are i n t e r c a l a t e d i n b i o l o g i c a l membranes, are capable of terminating p o t e n t i a l l y deleterious free r a d i c a l chain reactions occurring i n the l i p i d b i l a y e r s . In aqueous domains, ascorbic acid (Vitamin C) reacts d i r e c t l y with O2" ~ [33], HO* [34] and s i n g l e t oxygen [35], while u r i c acid, a product derived from purine metabolism, not only has an antioxidant p r o f i l e s i m i l a r to that of ascorbic acid, but also reduces oxidants produced from the reaction of peroxide with hemoproteins and inactivates c a t a l y t i c a l l y active 5 t r a n s i t i o n m e t a l i o n s by c h e l a t i o n [ 3 6 ] . I n a d d i t i o n t o s e r v i n g as a r e d u c t a n t i n t h e GSH-dependent p e r o x i d a s e system, GSH a l s o r e a c t s d i r e c t l y w i t h a wide v a r i e t y o f f r e e r a d i c a l s p e c i e s , i n c l u d i n g c a r b o n - c e n t r e d , p e r o x y l , p h e n o x y l and semiquinone r a d i c a l s [ 3 7 , 3 8 ] , Presumably, t h i o l groups of p r o t e i n m o l e c u l e s i n t e r a c t w i t h f r e e r a d i c a l s p e c i e s i n a s i m i l a r manner [ 3 9 ] . 1.1.4 E x t r a c e l l u l a r A n t i o x i d a n t System I n e x t r a c e l l u l a r f l u i d s , a n t i o x i d a n t enzyme a c t i v i t y i s v e r y low [ 4 0 , 4 1 ] . However, t h e i r o n - b i n d i n g p r o t e i n t r a n s f e r r i n [42] i s p r e s e n t i n such a q u a n t i t y t h a t t h e amount o f r e a c t i v e i r o n a v a i l a b l e i n e x t r a c e l l u l a r f l u i d s f o r c a t a l y s i n g t h e g e n e r a t i o n o f HO* from O2 * ~ and/or ^2^2 i s u s u a l l y n e g l i g i b l e . Moreover, t h e p r o t e i n c e r u l o p l a s m i n , i n a d d i t i o n t o b i n d i n g copper i o n s , can c a t a l y s e t h e c o n v e r s i o n o f f e r r o u s t o f e r r i c i o n [ 4 3 ] , t h e l a t t e r b e i n g u n r e a c t i v e towards H2O2. I n a d d i t i o n , a l b u m i n , a major c o n s t i t u e n t o f t h e plasma, i s c a p a b l e o f b i n d i n g f r e e copper i o n s r e l e a s e d i n t o t h e plasma [ 1 3 ] . T h e r e f o r e , i n c o n t r a s t t o t h e i n t r a c e l l u l a r e n z y m a t i c systems, e x t r a c e l l u l a r a n t i o x i d a n t p r o t e c t i o n i s l a r g e l y d i r e c t e d towards p r e v e n t i n g t h e g e n e r a t i o n o f h y d r o x y l r a d i c a l s r a t h e r t h a n d e t o x i f y i n g ^2 * ~ a n c * ^2^2* When t h e t o t a l a n t i o x i d a n t c a p a c i t y o f plasma i s a s s e s s e d i n terms o f a b i l i t y t o i n a c t i v a t e p e r o x y l r a d i c a l s , c o n t r i b u t i o n s by v a r i o u s components t o t h e t o t a l a n t i o x i d a n t a c t i v i t y o f plasma a r e 6 as follows: a-tocopherol 5%, ascorbic acid 15%, u r i c acid 25% and protein t h i o l s 50% [39]. The time-course of changes i n concentrations of plasma antioxidants caused by peroxyl r a d i c a l s indicates that the f i r s t l i n e of defense against r a d i c a l attack i s provided by plasma t h i o l s , u r i c acid (the second vulnerable antioxidant i n plasma) being spared during the i n i t i a l stages of the reaction [39]. Moreover, ascorbic acid seems to play a p i v o t a l r o l e i n protecting plasma l i p i d s from peroxidation i n i t i a t e d e i t h e r by aqueous peroxyl r a d i c a l s or by activated polymorphonuclear leukocytes; the depletion of plasma ascorbic acid i s followed by formation of l i p i d peroxidation products [44]. 1.1.5 Antioxidant Interactions In enzymatic antioxidant systems, a d e l i c a t e balance in the a c t i v i t i e s of these key enzymes must be maintained i n order to cope with oxidative challenges. Antioxidant enzymes are protected from free radical-induced i n a c t i v a t i o n i n a co-operative manner; for instance, CAT can prevent the H202~induced i n a c t i v a t i o n of SOD [45], and r e c i p r o c a l l y , SOD protects CAT and GPX from O2*~ - mediated i n a c t i v a t i o n [46]. Moreover, the continuous functioning of GPX requires a sustained supply of GSH regenerated from GSSG v i a the GRD-catalysed reaction, which, i n turn, u t i l i z e s hexose monophosphate shunt-derived NADPH as reductant, as mentioned previously. With regard to non-enzymatic antioxidant systems, a-tocopherol can protect /5-carotene from 7 oxidation [47]. On the other hand, a-tocopherol can be continuously regenerated at the expense of ascorbic acid with r e s u l t i n g preservation of l i p i d - s o l u b l e antioxidants [48]. In the presence of t r a n s i t i o n metals, ascorbic acid can produce free r a d i c a l s v i a oxidation reactions [49]; however, u r i c acid protects t h i s ascorbic acid oxidation by v i r t u e of i t s metal chelating properties [50,51]. Since superoxide r a d i c a l s can a r i s e from reaction of GSH with free r a d i c a l s through the intermediacy of t h i y l (GS') r a d i c a l [52], the antioxidant e f f e c t of GSH per se requires the concomitant action of SOD [53]. In addition, a-tocopherol and GPX are believed to act s y n e r g i s t i c a l l y in the prevention of l i p i d peroxidation [28]. Furthermore, the redox c y c l i n g of myoglobin induced by GSH or ascorbic acid i s thought to be an important antioxidant mechanism in protecting muscles against oxidative injury [54,55]. Given the inter-dependence and integration of the entire antioxidant system, an impairment i n any one component may upset the whole defense mechanism for protecting against free r a d i c a l attack, leading to i r r e v e r s i b l e c e l l u l a r damage. 1.1.6 Cytotoxic E f f e c t s of Hydroxyl Radical and Other  Reactive Oxygen - Derived Species Under conditions that antioxidant processes become impaired and/or overwhelmed by increased production of free r a d i c a l s , oxidative damage w i l l r e s u l t from free r a d i c a l -8 m e d i a t e d r e a c t i o n s w h i c h a r e m a i n l y t r i g g e r e d by HO*. T h i s h i g h l y r e a c t i v e s p e c i e s can cause t h e p e r o x i d a t i o n o f membrane l i p i d s by i n i t i a t i n g f r e e r a d i c a l c h a i n r e a c t i o n s , w i t h r e s u l t i n g d i s r u p t i o n o f c e l l u l a r o r s u b c e l l u l a r s t r u c t u r a l and f u n c t i o n a l i n t e g r i t y [ 5 6 , 5 7 ] . The l i p i d p e r o x i d a t i o n r e a c t i o n b e g i n s w i t h HO* a b s t r a c t i o n o f a hydrogen atom from t h e l i p i d m o l e c u l e , w h i c h t h e n becomes a l i p i d f r e e r a d i c a l . RH + HO* > R* + H 20 F o l l o w i n g t h e r e s t r u c t u r i n g o f i n t r a m o l e c u l a r u n s a t u r a t e d l i n k a g e s , t h e l i p i d r a d i c a l r e a c t s w i t h 0 2 t o form a l i p i d h y d r o p e r o x y l r a d i c a l . R* + 0 2 > R-0-0* The p e r o x y l r a d i c a l can r e a c t w i t h o t h e r l i p i d m o l e c u l e s i n t h e membrane i n t h e c h a i n p r o p a g a t i n g phase of t h e r e a c t i o n . R-0-0* + RH > R-OOH + R* The l i p i d h y d r o p e r o x i d e formed can undergo a v a r i e t y of r e a c t i o n s t o y i e l d breakdown p r o d u c t s , such as a l k a n e s (eg. e t h a n e and p e n t a n e ) , m a l o n d i a l d e h y d e and a l d e h y d e s . The p r e s e n c e o f t r a n s i t i o n m e t a l s can enhance t h e r e a c t i o n c a s c a d e by c a t a l y s i n g t h e d e g r a d a t i o n o f l i p i d h y d r o p e r o x i d e s [ 5 8 ] . On t h e o t h e r hand, t h e c h a i n r e a c t i o n w i l l be t e r m i n a t e d i f two o f t h e s e f r e e r a d i c a l m o l e c u l e s combine w i t h each o t h e r o r i f t h e y a r e i n a c t i v a t e d by c e l l u l a r a n t i o x i d a n t s , such as a - t o c o p h e r o l and ^ - c a r o t e n e [ 2 8 , 5 9 ] . However, i f t h e l i p i d b i l a y e r o f c e l l membranes i s p e r o x i d i z e d t o t h e e x t e n t t h a t t h e c e l l becomes i n c a p a b l e of 9 m a i n t a i n i n g e l e c t r o l y t e and volume h o m e o s t a s i s , c e l l d e a t h w i l l ensue. P r o t e i n and n u c l e i c a c i d m o l e c u l e s a r e a l s o o x i d a t i o n s e n s i t i v e [ 6 0 , 6 1 ] . F r e e r a d i c a l - i n d u c e d damage t o t h i o l g r o u p - c o n t a i n i n g enzymes and o t h e r p r o t e i n s can c u l m i n a t e i n i n a c t i v a t i o n , c r o s s - l i n k i n g , o r d e n a t u r a t i o n . N u c l e i c a c i d m o l e c u l e s can undergo m o d i f i c a t i o n o r even s c i s s i o n i n t h e p r e s e n c e o f HO* and t h e r e s u l t i n g damage can g i v e r i s e t o c a r c i n o g e n e s i s o r m u t a g e n e s i s . O x i d a t i v e damage t o c a r b o h y d r a t e s can a l t e r any o f t h e i r c e l l u l a r r e c e p t o r f u n c t i o n s , i n c l u d i n g t h o s e m e d i a t i n g hormonal and n e u r o t r a n s m i t t e r a c t i o n s . F u r t h e r m o r e , a l d e h y d e s such as m a l o n d i a l d e h y d e and h y d r o x y n o n e n a l r e s u l t i n g f r om f r e e r a d i c a l - i n d u c e d d e g r a d a t i o n o f p o l y u n s a t u r a t e d f a t t y a c i d s can cause c r o s s - l i n k i n g i n l i p i d s , p r o t e i n s and n u c l e i c a c i d s , l e a d i n g t o marked f u n c t i o n a l i m p a i r m e n t . 1.2 Myocardial Ischemia/Reperfusion Injury C o r o n a r y r e p e r f u s i o n a c c o m p l i s h e d by t h r o m b o l y t i c o r a n g i o p l a s t i c p r o c e d u r e s has become a s t a n d a r d t r e a t m e n t f o r p a t i e n t s s u f f e r i n g from a c u t e m y o c a r d i a l i n f a r c t i o n [ 6 2 , 6 3 ] . An e x t e n s i v e c l i n i c a l t r i a l w h i c h examined i n t r a v e n o u s s t r e p t o k i n a s e t r e a t m e n t showed t h a t e a r l y r e p e r f u s i o n , e s p e c i a l l y w i t h i n 3 hours f o l l o w i n g t h e o n s e t o f m y o c a r d i a l i n f a r c t i o n , r e d u c e d t h e i n c i d e n c e o f sudden d e a t h and improved l o n g e v i t y [ 6 4 ] . W h i l e t h e b e n e f i c i a l e f f e c t s o f e a r l y c o r o n a r y r e p e r f u s i o n a r e u n d i s p u t a b l e , t h e r e a r e some d e l e t e r i o u s consequences o f r e p e r f u s i o n , i n c l u d i n g 10 i n t r a m y o c a r d i a l hemorrhage, a r r h y t h m i a s , t h e " n o - r e f l o w " phenomenon, p r o l o n g e d f u n c t i o n a l i m p a i r m e n t and m y o c a r d i a l c e l l n e c r o s i s [ 6 5 ] . As a r e s u l t , an i n c r e a s e d m o r t a l i t y i n p a t i e n t s on t h e f i r s t day f o l l o w i n g s t r e p t o k i n a s e t r e a t m e n t was o b s e r v e d [ 6 6 ] . Moreover, l a r g e h e m o r r h a g i c i n f a r c t s have been n o t e d i n p a t i e n t s d y i n g a f t e r t h e c o r o n a r y bypass s u r g e r y [ 6 7 ] , D u r i n g t h e p a s t few y e a r s , a g r o w i n g body o f e v i d e n c e has a c c u m u l a t e d s u g g e s t i n g t h a t r e p e r f u s i o n may i n d e e d have an i n j u r i o u s component, termed " r e p e r f u s i o n i n j u r y " , w h i c h a c c o u n t s , a t l e a s t i n p a r t , f o r t h e a d v e r s e s e q u e l a e f o l l o w i n g p o s t - i s c h e m i c r e p e r f u s i o n o f t h e myocardium. I n t h i s r e g a r d , o x y g e n - d e r i v e d f r e e r a d i c a l s have been i m p l i c a t e d i n t h e p a t h o g e n e s i s o f m y o c a r d i a l i s c h e m i a / r e p e r f u s i o n (I/R) i n j u r y i n v a r i o u s e x p e r i m e n t a l and c l i n i c a l s e t t i n g s [ 6 8 , 6 9 ] . 1.2.1 Role of Reactive Oxygen-Derived Radicals i n Myocardial  Ischemia/Reperfusion Injury The i n v o l v e m e n t o f endogenous o x y - r a d i c a l s i n t h e development o f m y o c a r d i a l I/R i n j u r y was e a r l y s u g g e s t e d by t h e p r o t e c t i v e e f f e c t s o f a n t i - f r e e r a d i c a l i n t e r v e n t i o n s i n v a r i o u s e x p e r i m e n t a l s e t t i n g s . Many i n v i t r o s t u d i e s u s i n g i s o l a t e d h e a r t s p r e p a r e d from s e v e r a l s p e c i e s ( i n c l u d i n g r a t s , r a b b i t s and p i g s ) showed t h a t o x y - r a d i c a l s c a v e n g e r s and i r o n c h e l a t o r s , when a d m i n i s t e r e d a t t h e t i m e o f r e f l o w , c o u l d b l u n t t h e m y o c a r d i a l i n j u r y and d y s f u n c t i o n i n d u c e d by h y p o x i a w i t h subsequent r e o x y g e n a t i o n [ 7 0 - 7 2 ] . I n s t u d i e s 11 u s i n g i n v i v o s e t t i n g s o f m y o c a r d i a l i s c h e m i a and r e p e r f u s i o n , t h e a d m i n i s t r a t i o n o f o x y - r a d i c a l s c a v e n g e r s d u r i n g t h e p e r i o d o f r e p e r f u s i o n c a u s e d a r e d u c t i o n i n m y o c a r d i a l i n f a r c t s i z e when compared w i t h u n t r e a t e d a n i m a l s [ 7 3 , 7 4 ] . Moreover, o x y - r a d i c a l s c a v e n g e r s added t o p r e s e r v a t i v e s o l u t i o n s c o u l d r e d uce r e p e r f u s i o n i n j u r y i n h e a r t s f o l l o w i n g t r a n s p l a n t a t i o n [ 7 5 - 7 7 ] . R e c e n t l y , e a r l i e r d i f f i c u l t i e s i n v o l v e d i n t h e d i r e c t ' d e t e c t i o n and c h a r a c t e r i z a t i o n o f o x y - r a d i c a l s i n t h e s e t t i n g o f m y o c a r d i a l i s c h e m i a and r e p e r f u s i o n have been overcome by u s i n g e l e c t r o n s p i n resonance measurements w i t h s p i n t r a p s s u ch as DMPO ( 5 , 5 - d i m e t h y l - l - p y r o l i n e - N - o x i d e ) [ 7 8 ] . S e v e r a l i n v i t r o s t u d i e s w i t h i s o l a t e d h e a r t s s u b j e c t e d t o g l o b a l i s c h e m i a and r e p e r f u s i o n have d e s c r i b e d a b u r s t o f o x y - r a d i c a l f o r m a t i o n w i t h i n t h e f i r s t m i n u t e s o f r e p e r f u s i o n [ 7 8 - 8 0 ] , Carbon- and o x y g e n - c e n t e r e d r a d i c a l s i n m y o c a r d i a l t i s s u e s h a r v e s t e d from c a n i n e h e a r t s s u b j e c t e d t o r e g i o n a l i s c h e m i a were a l s o o b s e r v e d [ 8 1 ] . S i m i l a r l y , B o l l i e t . a l . [82] r e p o r t e d an a b r u p t i n c r e a s e i n r a d i c a l c o n c e n t r a t i o n i n t h e c o r o n a r y venous e f f l u e n t i m m e d i a t e l y a f t e r t h e o n s e t o f r e p e r f u s i o n i n dogs s u b j e c t e d t o 15 m i n u t e s o f c o r o n a r y o c c l u s i o n . Taken t o g e t h e r , a l l t h e f o r e g o i n g s t u d i e s p r o v i d e b o t h i n d i r e c t and d i r e c t e v i d e n c e o f o x y - r a d i c a l p r o d u c t i o n i n t h e s e t t i n g o f m y o c a r d i a l i s c h e m i a and r e p e r f u s i o n . 12 1 . 2 . 2 Cytotoxic E f f e c t s of Oxy-Radicals on the Myocardium Direct cytotoxic e f f e c t s of oxy-radicals on the myocardium have been demonstrated i n studies involving the exposure of in v i t r o preparations to exogenously generated oxy-radicals. Oxy-radicals generated from a mixture of purine plus xanthine oxidase caused functional impairment of iso l a t e d p a p i l l a r y muscles [83,84]. S i m i l a r l y , the exposure of i s o l a t e d r a t hearts to O 2"~ generated by infusion of hypoxanthine and xanthine oxidase reduced l e f t v e n t r i c u l a r developed pressure, depleted high energy phosphate l e v e l s , and caused c e l l u l a r edema [85]. Moreover, exposure of the myocardium to HO* generated by infusion of xanthine and xanthine oxidase i n the presence of iron-loaded t r a n s f e r r i n caused severe u l t r a s t r u c t u r a l damage to myocardial c e l l s , including swelling and disruption of mitochondria, blebbing of the sacrolemma and breaks within the sarcolemmal membrane [86]. In i n vivo preparations, intracoronary infusion of xanthine oxidase and purine plus iron-loaded t r a n s f e r r i n to anesthetized dogs resulted i n l e f t v e n t r i c u l a r wall-motion abnormalities i n the perfused region of the heart [87]. High concentrations of xanthine oxidase/purine/transferrin solution administered into the a o r t i c root through the ca r o t i d artery produced myocardial contraction bands and i n t e r s t i t i a l edema in rat hearts [88]. A l l these functional, chemical and u l t r a s t r u c t u r a l a l t e r a t i o n s induced by exogenous oxy-radicals resembled those seen in 13 i s c h e m i c / r e p e r f u s e d h e a r t s and c o u l d be prevented by oxy-r a d i c a l scavengers such as SOD and CAT. Although the c a r d i o t o x i c e f f e c t s of o x y - r a d i c a l s are w e l l e s t a b l i s h e d , t h e r e i s s t i l l some c o n t r o v e r s y r e g a r d i n g which of the o x y - r a d i c a l s p e c i e s i s the major c u l p r i t . C u r r e n t o p i n i o n seems t o suggest t h a t o x y - r a d i c a l s p e c i e s , such as 02*~, H2O2 and HO*, may a l l c o n t r i b u t e t o the damage. 1.2.3 S o u r c e s o f O x y - R a d i c a l s i n t h e I s c h e m i c Myocardium 1.2.3.1 X a n t h i n e O x i d a s e One p o t e n t i a l source of r a d i c a l p r o d u c t i o n i n ischemia i s x a n t h i n e o x i d a s e , a p u r i n e - d e g r a d a t i o n enzyme prese n t mainly i n e n d o t h e l i a l c e l l s [89,90]. In i s c h e m i c t i s s u e s , d e g r a d a t i o n of hig h energy adenine n u c l e o t i d e s can l e a d t o the accumulation of hypoxanthine and xa n t h i n e , which are n a t u r a l s u b s t r a t e s f o r xanthine o x i d a s e . The d e p l e t i o n of c e l l u l a r energy s t o r e s caused by isc h e m i a can imp a i r i o n homeostasis, r e s u l t i n g , f o r example, i n an i n c r e a s e of c y t o s o l i c c a l c i u m and sodium c o n c e n t r a t i o n s [ 9 1 ] . The i n c r e a s e d i n t r a c e l l u l a r c a l c i u m a c t i v a t e s a p r o t e a s e [89] which presumably c o n v e r t s xanthine dehydrogenase, a NAD +-dependent ( n o n - r a d i c a l producing) form of the enzyme t h a t accounts f o r about 90% of the t o t a l a c t i v i t y i n non-ischemic t i s s u e , t o the oxygen-dependent ( r a d i c a l producing) xanthine o x i d a s e [92]. Subsequent r e p e r f u s i o n ( i e . reoxygenation) of the p r e v i o u s l y i s c h e m i c t i s s u e i n the presence of high 14 c o n c e n t r a t i o n s o f x a n t h i n e o x i d a s e and i t s s u b s t r a t e s can l e a d t o a marked i n c r e a s e i n t h e p r o d u c t i o n o f C>2*~ [ 9 3 ] , w i t h r e s u l t i n g c y t o t o x i c e f f e c t s m e d i a t e d by t h e t r a n s i t i o n m e t a l - c a t a l y s e d f o r m a t i o n o f HO" [ 9 4 ] . T h i s h y p o t h e s i s i s s t r o n g l y s u p p o r t e d by numerous s t u d i e s w h i c h have shown p r o t e c t i v e e f f e c t s o f a l l o p u r i n o l (a x a n t h i n e o x i d a s e i n h i b i t o r [95]) on I / R - r e l a t e d damage i n a v a r i e t y of t i s s u e s [ 9 0 ] . There i s no doubt t h a t o x y - r a d i c a l s can be g e n e r a t e d by t h e x a n t h i n e o x i d a s e pathway, but t h e i m p o r t a n c e o f t h i s pathway i n I/R i n j u r y may be s p e c i e s dependent. X a n t h i n e o x i d a s e i s p r e s e n t i n measurable q u a n t i t i e s i n c a n i n e and r a t h e a r t s , b u t has been found t o be u n d e t e c t a b l e i n r a b b i t and human myocardium [ 9 6 - 9 8 ] . 1.2.3.2 A c t i v a t e d N e u t r o p h i l s A c t i v a t e d n e u t r o p h i l s a r e a n o t h e r p o s s i b l e s o u r c e of o x y - r a d i c a l s [ 6 8 , 9 9 ] . I f m y o c a r d i a l i s c h e m i a produces an i n c r e a s e d f l u x o f C>2"~ , t h e 0 2 * ~ - s e n s i t i v e c h e m o t a c t i c f a c t o r p r e s e n t i n e x t r a c e l l u l a r f l u i d s w i l l be a c t i v a t e d [ 5 ] . The p r e s e n c e o f t h i s c h e m o t a c t i c f a c t o r would t h e n c a use n e u t r o p h i l s t o adhere t o t h e e n d o t h e l i u m , t o e x t r a v a s a t e , and e v e n t u a l l y e n t e r t h e i n t e r s t i c e s [ 5 ] . A c t i v a t e d n e u t r o p h i l s p o s s e s s a plasma membrane-associated NADPH-oxidase t h a t r e d u c e s O2 t o O 2 " - , w i t h c o n c o m i t a n t o x i d a t i o n o f c y t o s o l i c NADPH [ 1 0 0 ] , S i n c e t h e i n c r e a s e d p r o d u c t i o n o f c a n a t t r a c t and a c t i v a t e more n e u t r o p h i l s , a s e l f - p e r p e t u a t i n g c y c l e would ensue. I n 15 addition, myeloperoxidase (located within granules of the neutrophil) can, i n the presence of H2O2 (derived from O 2 ' - ) , lead to formation of hypochlorous acid (H0C1), a highly reactive substance capable of causing extensive c e l l u l a r damage [101]. In addition to the foregoing actions of c i r c u l a t i n g neutrophils, mast c e l l s and macrophages resident i n the heart may also play an important role i n the development of myocardial I/R injury. In t h i s regard,' a c t i v a t i o n of the resident cardiac leukocytes has been demonstrated i n is o l a t e d rat hearts subjected to hypoxic perfusion with neutrophil-free solution followed by reoxygenation [102]. 1.2.3.3 Disruption of the Mitochondrial Electron Transport  System Another potential source of oxy-radicals i s the production of O 2 ' - due to leakage of electrons from the electron transport system within mitochondria [69,103]. During ischemia, the i n t r a c e l l u l a r adenine nucleotide pool i s depleted by the increased energy demands and degradative processes, and electron c a r r i e r s i n the mitochondrial respiratory chain are converted to t h e i r reduced state. The exposure of O2 to these reduced ( i . e , unreoxidized) c a r r i e r s during reperfusion w i l l lead to the increased formation of O2"~. The impairment i n mitochondrial antioxidant capacity during ischemia [104] would enhance the s u s c e p t i b i l i t y of mitochondria to r a d i c a l attack. This self-induced 16 mitochondrial damage would progress, leading to greater and greater fluxes of reactive r a d i c a l species derived from molecular oxygen. 1.2.3.4 Metabolism of Arachidonic Acid During ischemia, the increase i n c y t o s o l i c calcium concentration can activate phospholipase A2 [105] which, in turn, releases arachidonic acid from plasma membranes of myocardial c e l l s [106]. Arachidonic acid produces b i o l o g i c a l l y active prostanoids, such as the vasoactive thromboxane A2 and prostacyclin, through the action of cyclooxygenases, which reside mainly in endothelial c e l l s . This cyclooxygenase pathway of arachidonic acid metabolism produces endoperoxides, which are reactive oxidants with the a b i l i t y to i n i t i a t e deleterious chain reactions [107]. In addition, arachidonic acid i s metabolized by lipoxygenases present i n leukocytes to produce leukotrienes and other metabolites that act as chemotaxins to a t t r a c t and activate more leukocytes [108]. 1.2.3.5 Oxidation of Catecholamines The oxidation of catecholamines may also be a source of oxy-radicals leading to damage in the ischemic/reperfused myocardium [109,110]. The oxidation of epinephrine by microsomal preparations produces O2 * ~ and other r a d i c a l intermediates [111,112], Similar oxidation of epinephrine can occur by other mechanisms involving i r o n -17 c o n t a i n i n g p r o t e i n s [113] and iron-heme p r o t e i n s w i t h H2O2 [ 1 1 4 ] . I n a d d i t i o n , t r a n s i t i o n m e t a l i o n s , such as Cu and 3+ Fe , c a t a l y s e t h e o x i d a t x o n o f e p i n e p h r i n e , w i t h t h e g e n e r a t i o n o f o x y - r a d i c a l s as b y p r o d u c t s [115,116]. Thus, o x i d a t i o n o f c a t e c h o l a m i n e s r e l e a s e d from t h e i s c h e m i c h e a r t s c o u l d c a use a b u r s t o f o x y - r a d i c a l p r o d u c t i o n a t t h e o n s e t o f r e p e r f u s i o n [ 1 1 7 ] . 1.2.3.6 Tr a n s i t i o n Metal Catalysed Oxy-Radical Production T r a n s i t i o n m e t a l i o n s , p a r t i c u l a r l y c e r t a i n a c t i v e forms o f i r o n , can c a t a l y s e t h e p r o d u c t i o n o f a d d i t i o n a l o x y - r a d i c a l s i n i s c h e m i c t i s s u e [ 1 3 ] . The i r o n - l o a d e d t r a n s f e r r i n appears t o s u p p o r t t h e c a t a l y s i s of t h e Haber-Weiss r e a c t i o n t o produce HO" [ 1 1 8 ] . A d d i t i o n a l c a t a l y t i c a l l y a c t i v e forms o f i r o n may be p r o d u c e d from t h e i n t e r a c t i o n o f h e moproteins w i t h p e r o x i d e s [ 1 1 9 ] . I n a d d i t i o n , ®2~ i n t e r a c t s w i t h f e r r i t i n ( i r o n s t o r a g e p r o t e i n ) t o r e l e a s e i r o n t h a t can c a t a l y s e t h e f o r m a t i o n o f r e a c t i v e o x y - r a d i c a l s [ 1 2 0 ] . 1.2.4 Possible Strategies i n the Prevention of Oxy-Radical -Mediated Myocardial Ischemia/Reperfusion Injury I t now seems q u i t e c e r t a i n t h a t o x y - r a d i c a l s p l a y an i m p o r t a n t r o l e i n t h e development of i r r e v e r s i b l e I/R i n j u r y i n t h e myocardium. Two main s t r a t e g i e s c o u l d , t h e r e f o r e , be a d o p t e d t o p r e v e n t t h i s c l i n i c a l l y r e l e v a n t form o f t i s s u e damage: (1) a d m i n i s t r a t i o n o f o x y - r a d i c a l s c a v e n g e r s o r (2) 18 r e d u c t i o n of o x y - r a d i c a l p r o d u c t i o n . P r e t r e a t m e n t s w i t h SOD and/or CAT have been shown t o p r o t e c t a g a i n s t m y o c a r d i a l I/R i n j u r y i n v a r i o u s e x p e r i m e n t a l s e t t i n g s [73,77,121], and t o p r e v e n t t h e f o r m a t i o n o f o x y - r a d i c a l s i n i s c h e m i c / r e p e r f u s e d myocardium [ 7 8 ] . The c o n j u g a t e d forms o f s u p e r o x i d e d i s m u t a s e (such as t h e p o l y e t h y l e n e g l y c o l c o n j u g a t e s ) , w h i c h have an e x t e n d e d h a l f - l i f e [ 1 2 2 ] , were more e f f e c t i v e t h a n t h e u n c o n j u g a t e d form i n t h e p r o t e c t i o n a g a i n s t I/R-i n d u c e d damage i n v i v o [122,123]. Moreover, e n d o t o x i n p r e t r e a t m e n t , w h i c h i n c r e a s e s endogenous m y o c a r d i a l c a t a l a s e a c t i v i t y , was found t o d e c r e a s e t h e e x t e n t o f I/R i n j u r y i n i s o l a t e d r a t h e a r t s [ 1 2 4 ] . I n a d d i t i o n , p r e t r e a t m e n t w i t h n on-enzymatic a n t i o x i d a n t s , such as d i h y d r o q u i n o l o n e d e r i v a t i v e s [ 1 2 5 ] , a - t o c o p h e r o l [ 1 2 6 ] , a s c o r b i c a c i d [127] and i t s l i p o p h i l i c d e r i v a t i v e 2 - o c t a d e c y l a s c o r b i c a c i d [ 1 2 8 ] , and t h e t h i o l - c o n t a i n i n g compound N - a c e t y l c y s t e i n e [ 1 2 9 ] , were a l l found c a p a b l e o f r e d u c i n g t h e s e v e r i t y of I/R i n j u r y . On t h e o t h e r hand, t h e r a p e u t i c i n t e r v e n t i o n s might a l s o be d i r e c t e d towards n e u t r o p h i l - r e l a t e d oxy-r a d i c a l f o r m a t i o n . I n t h i s r e g a r d , p r e t r e a t m e n t w i t h n e u t r o p e n i c a gents o r i n h i b i t o r s of n e u t r o p h i l a c t i v a t i o n was shown t o r e d u c e i n f a r c t s i z e f o l l o w i n g i s c h e m i a and r e p e r f u s i o n i n dogs [1 3 0 , 1 3 1 ] , A n o t h e r p o t e n t i a l t a r g e t f o r t h e r a p e u t i c i n t e r v e n t i o n i s t h e x a n t h i n e o x i d a s e - m e d i a t e d f o r m a t i o n o f o x y - r a d i c a l s . I n f a c t , t h e a d m i n i s t r a t i o n of a l l o p u r i n o l has been shown t o reduce t h e s i z e o f m y o c a r d i a l i n f a r c t s i n d u c e d by c o r o n a r y a r t e r y l i g a t i o n [132-135] and 19 r e p e r f u s i o n - i n d u c e d a r r h y t h m i a s [134,136]. The c a t a l y s i s o f o x y - r a d i c a l p r o d u c t i o n i n i s c h e m i c t i s s u e c o u l d a l s o be p r e v e n t e d by t h e t r e a t m e n t w i t h t r a n s i t i o n m e t a l i o n c h e l a t o r s . T h i s has been de m o n s t r a t e d i n t h e c a s e of d e f e r o x a m i n e w h i c h p r o t e c t e d a g a i n s t t i s s u e damage i n normal o r i r o n - l o a d e d r a t h e a r t s s u b j e c t e d t o a p e r i o d o f a n o x i a f o l l o w e d by r e o x y g e n a t i o n [137] and t h e o x y - r a d i c a l g e n e r a t i o n d u r i n g c a r d i o p u l m o n a r y bypass i n man [ 1 3 8 ] , I n a d d i t i o n , p r e t r e a t r n e n t w i t h t h e o r a l l y a c t i v e i r o n c h e l a t o r 1 , 2 - d i m e t h y - 3 - h y d r o x y - 4 - p y r i d o n e was a l s o a b l e t o p r o t e c t r a t h e a r t s from r e p e r f u s i o n i n j u r y [ 1 3 9 ] . 1.3 Rationale and Objectives of the Study 1.3.1 C l i n i c a l S i g n i f i c a n c e of Ischemia/Reperfusion Injury C a r d i o v a s c u l a r d i s e a s e remains t h e major cause o f d e a t h i n N o r t h A m e r i c a and Western Europe, and t h e m a j o r i t y of t h e s e d eaths a r e a t t r i b u t a b l e t o a t h e r o s c l e r o s i s - r e l a t e d i s c h e m i c h e a r t d i s e a s e [ 1 4 0 ] . I n a c u t e m y o c a r d i a l i n f a r c t i o n r e s u l t i n g from t h r o m b o s i s , e a r l y c o r o n a r y r e p e r f u s i o n t h r o u g h t h r o m b o l y s i s has been shown t o r e duce m o r t a l i t y [ 6 2 , 6 4 ] . W h i l e t h e r e i s no doubt about t h e need t o r e e s t a b l i s h p e r f u s i o n o f t h e i s c h e m i c myocardium, t h e r e a r e some p o t e n t i a l l y n e g a t i v e a s p e c t s o f r e p e r f u s i o n w h i c h may a c c o u n t f o r t h e h i g h i n c i d e n c e o f p o s t - i n f a r c t i o n m o r t a l i t y and a d v e r s e s e q u e l a e [141,142]. T i m e l y r e p e r f u s i o n o f i s c h e m i c myocardium can r e duce t h e amount o f t i s s u e n e c r o s i s a f t e r a c u t e c o r o n a r y a r t e r y o c c l u s i o n , but 20 r e p e r f u s i o n , w h i l e t e r m i n a t i n g t h e i s c h e m i a , m ight a l s o cause f u r t h e r damage t o i s c h e m i c t i s s u e s . Over t h e p a s t decade, o x y g e n - d e r i v e d f r e e r a d i c a l s g e n e r a t e d d u r i n g t h e r e p e r f u s i o n phase have been s u g g e s t e d t o p l a y an i m p o r t a n t r o l e i n t h e p a t h o g e n e s i s o f i r r e v e r s i b l e t i s s u e damage f o l l o w i n g i s c h e m i a and r e p e r f u s i o n . A g r o w i n g body o f e v i d e n c e has now emerged s u p p o r t i n g t h e i n v o l v e m e n t o f oxy-r a d i c a l s i n t h e development o f m y o c a r d i a l I/R i n j u r y [68, 143,144]. R e p e r f u s i o n o f t h e p r e v i o u s l y i s c h e m i c myocardium has become f e a s i b l e i n many c l i n i c a l s i t u a t i o n s . I n t e r v e n t i o n s such as t h e a d m i n i s t r a t i o n o f p l a s m i n o g e n a c t i v a t o r o r s t r e p t o k i n a s e , and per c u t a n e o u s t r a n s l u m i n a l a n g i o p l a s t y have been shown t o s u c c e s s f u l l y r e e s t a b l i s h c o r o n a r y f l o w i n p a t i e n t s d u r i n g m y o c a r d i a l i n f a r c t i o n [ 6 2 , 6 3 ] . I n a d d i t i o n , p a t i e n t s u n d e r g o i n g c a r d i o p u l m o n a r y bypass p r o c e d u r e s d u r i n g c a r d i a c s u r g e r y a r e a l s o exposed t o p e r i o d s o f i s c h e m i a and r e p e r f u s i o n , i n w h i c h i s c h e m i a i s i n d u c e d by a o r t i c c r o s s -c l a m p i n g w i t h r e p e r f u s i o n b e i n g i n i t i a t e d by r e l e a s i n g t h e c r o s s - c l a m p . S i m i l a r l y , i n o r t h o t o p i c h e a r t t r a n s p l a n t a t i o n , t h e t r a n s p l a n t e d h e a r t i s r e p e r f u s e d a f t e r a p r o l o n g e d p e r i o d o f s t o r a g e under c o n d i t i o n s o f h y p o t h e r m i c g l o b a l i s c h e m i a . P r o t e c t i o n a g a i n s t I/R i n j u r y has t h e r e f o r e become a c r u c i a l f a c t o r i n o p t i m i z i n g t h e e f f e c t i v e n e s s o f p o s t -i n f a r c t i o n m y o c a r d i a l s a l v a g e , t h r o m b o l y t i c o r r e v a s c u l a r i z a t i o n p r o c e d u r e s , c a r d i o p l e g i a and organ t r a n s p l a n t a t i o n [ 1 4 3 ] . 21 1.3.2 Protective E f f e c t s of A l l o p u r i n o l Against Ischemia/  Reperfusion Injury The c l i n i c a l l y relevant consequences of reperfusion injury have led to intensive investigations of possible therapeutic interventions aimed at reducing the extent of t i s s u e i n j u r y . The pretreatrnent with antioxidant enzymes, such as SOD and CAT, can protect against tiss u e damage i n a var i e t y of experimental models of I/R injury [73,122,145,146]. However, the li m i t e d c l i n i c a l usefulness of these macromolecular substances, which usually have a r e l a t i v e l y short i n vivo h a l f - l i f e , has provided a strong impetus to devise pharmacological interventions using compounds with more convenient pharmacokinetic properties. One of the more a c t i v e l y investigated agents i n t h i s regard i s a l l o p u r i n o l , which has been shown to reduce manifestations of ischemic injury i n a number of systems, including s k e l e t a l muscle [147], l i v e r [148], kidney [149], brain [150,151], i n t e s t i n e [152] and, most notably, the myocardium i n several species, including rat [153], dog [133,154], rabbit [155,156] and pig [157]. A l l o p u r i n o l , a xanthine oxidase i n h i b i t o r , was synthesized i n the late 1950s and used c l i n i c a l l y to i n h i b i t the oxidative degradation of mercaptopurine, an a n t i -p r o l i f e r a t i v e agent [95]. Moreover, i n h i b i t i o n of xanthine oxidase by a l l o p u r i n o l i s p a r t i c u l a r l y useful i n managing complications of hyperuricemia, including primary gout and urate stone formation i n the kidney [158], a l l o p u r i n o l being 22 a s s o c i a t e d w i t h an i n c i d e n c e o f a d v e r s e r e a c t i o n s o f about 3.5% a t normal t h e r a p e u t i c dose [ 1 5 9 ] . The a b i l i t y o f a l l o p u r i n o l t o p r o t e c t a g a i n s t I/R i n j u r y was i n i t i a l l y a t t r i b u t e d t o i n h i b i t i o n o f x a n t h i n e o x i d a s e whose a c t i o n on h y p o x a n t h i n e a r i s i n g from ATP d e g r a d a t i o n d u r i n g t h e c o u r s e of i s c h e m i a and r e p e r f u s i o n l e a d s t o t h e g e n e r a t i o n of s u p e r o x i d e a n i o n r a d i c a l s [ 8 9 ] . However, t h e f i n d i n g t h a t x a n t h i n e o x i d a s e a c t i v i t y i s v i r t u a l l y u n d e t e c t a b l e i n r a b b i t and p i g h e a r t s [96,98] has c a s t c o n s i d e r a b l e doubt on t h e p r i m a r y r o l e o f x a n t h i n e o x i d a s e i n h i b i t i o n i n a l l o p u r i n o l p r o t e c t i o n a g a i n s t I/R i n j u r y . T h i s i s f u r t h e r s t r e n g t h e n e d by t h e f i n d i n g t h a t x a n t h i n e o x i d a s e a c t i v i t y , when p r e s e n t , i s found almost e x c l u s i v e l y i n v a s c u l a r e n d o t h e l i a l c e l l s r a t h e r t h a n i n c a r d i a c myocytes [160,161]. A number of o t h e r p o s s i b l e mechanisms of a l l o p u r i n o l p r o t e c t i o n have been pr o p o s e d , i n c l u d i n g an i n c r e a s e d e f f i c i e n c y o f ATP s a l v a g e and r e s y n t h e s i s [162,163], f a c i l i t a t i o n o f m i t o c h o n d r i a l e l e c t r o n t r a n s f e r [164] and d i r e c t i n a c t i v a t i o n o f endogenously formed r e a c t i v e s p e c i e s , such as h y d r o x y l r a d i c a l s o r m y e l o p e r o x i d a s e - d e r i v e d h y p o c h l o r o u s a c i d [ 1 6 5 ] . 23 1.3.3 Experimental Approaches 1.3.3.1 Molecular Aspects of Myocardial Ischemia/Reperfusion  Injury and the Protective E f f e c t s of A l l o p u r i n o l As n o t e d i n p r e v i o u s s t u d i e s from our l a b o r a t o r y , c h r o n i c a l l o p u r i n o l t r e a t m e n t can p r o t e c t a g a i n s t I/R-i n d u c e d u l t r a s t r u c t u r a l and b i o c h e m i c a l a l t e r a t i o n s i n r a b b i t myocardium wh i c h c o n t a i n s u n d e t e c t a b l e x a n t h i n e o x i d a s e a c t i v i t y [155,156], We h y p o t h e s i z e d t h a t a l l o p u r i n o l p r e t r e a t r n e n t may d e c r e a s e t i s s u e s u s c e p t i b i l i t y t o o x i d a n t i n j u r y ( i . e . , i n c r e a s e t i s s u e a n t i o x i d a n t c a p a c i t y ) by m o d i f y i n g o t h e r p r o c e s s e s i n v o l v e d i n t h e g e n e r a t i o n o r s c a v e n g i n g of r e a c t i v e o x y - r a d i c a l s w h i c h a r e u n r e l a t e d t o x a n t h i n e o x i d a s e i n h i b i t i o n . As an approach t o i n v e s t i g a t i n g t h e t i m e - c o u r s e o f I/R-r e l a t e d a l t e r a t i o n s i n m y o c a r d i a l a n t i o x i d a n t s t a t u s and d e t e r m i n i n g whether i t c o i n c i d e s t e m p o r a l l y w i t h t h o s e o f u l t r a s t r u c t u r a l and b i o c h e m i c a l m a n i f e s t a t i o n s o f i r r e v e r s i b l e i n j u r y o b s e r v e d i n t h e p r e v i o u s s t u d i e s [155,156], p e n t o b a r b i t a l a n e s t h e t i z e d open-chest r a b b i t s were s u b j e c t e d t o i n c r e a s i n g p e r i o d s o f c o r o n a r y a r t e r y l i g a t i o n , w i t h o r w i t h o u t a subsequent 60 min o f r e p e r f u s i o n . A n t i o x i d a n t c a p a c i t y o f m y o c a r d i a l t i s s u e was a s s e s s e d i n terms o f s u s c e p t i b i l i t y t o i n v i t r o o x i d a t i v e c h a l l e n g e . The e f f e c t s o f p r e t r e a t r n e n t w i t h a l l o p u r i n o l ( p r e v i o u s l y found t o p r o t e c t a g a i n s t 1/R-induced u l t r a s t r u c t u r a l and b i o c h e m i c a l a l t e r a t i o n s i n r a b b i t myocardium) and U74006F (a n o v e l 2 1 - a m i n o s t e r o i d a n t i o x i d a n t 24 which has been shown to be markedly protective i n various experimental models of brain or spinal cord ischemic injury [166-168]) were examined with regard to t h e i r possible e f f e c t s on I/R-related a l t e r a t i o n s i n myocardial antioxidant capacity. One possible mechanism of a l l o p u r i n o l protection against myocardial I/R injury might involve a l t e r a t i o n s i n antioxidant enzyme a c t i v i t i e s . The e f f e c t s of chronic a l l o p u r i n o l treatment on the a c t i v i t y of myocardial antioxidant enzymes were, therefore, examined i n rabbits not subjected to myocardial ischemia and reperfusion. Findings regarding I/R-induced alt e r a t i o n s i n the a c t i v i t y of myocardial antioxidant enzymes from other investigators have been inconsistent. The a c t i v i t y of myocardial glutathione peroxidase has been reported to be decreased [169], increased [170] or unchanged [104,171,172] following ischemia and/or reperfusion. Peterson et. a l . [154] have demonstrated an i n i t i a l decrease i n catalase a c t i v i t y i n ischemic dog myocardium aft e r 30 min of reflow, with complete restoration of a c t i v i t y a f t e r 2 hr of reperfusion. In contrast, evidence that glutathione reductase a c t i v i t y i s t r a n s i e n t l y increased a f t e r ischemia suggests that the pattern of changes may vary with the p a r t i c u l a r enzyme studied [170]. Although a burst of r a d i c a l formation has been observed during the early phase of post-ischemic reperfusion i n the myocardium [78,80], i t has been shown i n dogs that the maximal degree of myocardial necrosis induced 25 by a short period of coronary occlusion i s not attained u n t i l several hours a f t e r the i n i t i a t i o n of reperfusion [130]. These suggest that secondary oxidative processes occurring during the prolonged period of post-ischemic reperfusion are also important i n the development of i r r e v e r s i b l e I/R injury. Other time-dependent changes i n myocardial antioxidant enzyme a c t i v i t i e s and l i p i d peroxidation during the course of ischemia and reperfusion have also been observed [173,174]. In the present investigation, we have, therefore, undertaken a time-course study of reperfusion-related a l t e r a t i o n s i n myocardial antioxidant capacity and antioxidant enzyme a c t i v i t i e s in rabbits subjected to a 40 min period of ischemia to address the following questions: (1) are there a l t e r a t i o n s in myocardial antioxidant capacity during the course of post-ischemic reperfusion ; (2) what i s the time-course involved in the foregoing changes i n myocardial antioxidant capacity ( i . e . , does i t correlate with the burst of oxy-radical formation occurring within the f i r s t few minutes of post-ischemic reperfusion [78,80]); and (3) i s there i n a c t i v a t i o n or possibly a transient impairment in the functioning of myocardial antioxidant enzymes during the course of post-ischemic reperfusion, which i s l i k e l y related to ischemia-associated acidosis [175,176] and might be c r u c i a l in t r i g g e r i n g myocardial I/R injury ? In the "stunned myocardium", some early changes, such as c o n t r a c t i l e impairment and ATP depletion, occurring a f t e r 26 a b r i e f period of ischemia are not r e a d i l y r e v e r s i b l e with reperfusion [177]. It has also been suggested that intermittent b r i e f periods of ischemia may exert a cumulative e f f e c t and cause the progressive development of myocardial necrosis and v e n t r i c u l a r dysfunction [178]. In contrast, findings reported by Lange et. a l . [179] and Swain et. a l . [180] have shown that myocardial a l t e r a t i o n s in coronary blood flow, regional function and ATP content were not cumulative a f t e r r e p e t i t i v e b r i e f episodes of ischemia. As an approach to investigating whether I/R-related a l t e r a t i o n s i n myocardial antioxidant capacity can be influenced by the p a r t i a l depletion of t i s s u e ATP l e v e l induced by ischemia, the e f f e c t s of b r i e f episodes of ischemia and reperfusion on myocardial antioxidant status in r e l a t i o n to t i s s u e ATP l e v e l s were examined i n pentobarbital anesthetized open-chest rabbits. In addition, the e f f e c t s of ischemic preconditioning (by r e p e t i t i v e b r i e f episodes of ischemia), which has been shown to reduce i n f a r c t size in pigs [181], were also investigated. As mentioned e a r l i e r , oxidants derived from activated neutrophils have been shown to be a c r u c i a l factor involved i n the development of I/R injury [182,183]. The role of blood elements, mainly neutrophils, in causing I/R-induced oxidative injury was investigated in i s o l a t e d Langendorff-perfused rabbit hearts subjected to coronary artery l i g a t i o n followed by reperfusion, and the I/R-related a l t e r a t i o n s in myocardial antioxidant capacity were examined. 27 A l l o p u r i n o l p r o t e c t i o n a g a i n s t I/R i n j u r y has been d e m o n s t r a t e d i n numerous e x p e r i m e n t a l models o f organ t r a n s p l a n t a t i o n , i n v o l v i n g k i d n e y [ 1 8 4 ] , l i v e r [185] and h e a r t [ 1 8 6 ] . The f a c t t h a t t h e p r o t e c t i v e a c t i o n s o f a l l o p u r i n o l a r e d e m o n s t r a b l e i n a v a r i e t y o f t i s s u e s , i n c l u d i n g k i d n e y [ 1 8 7 ] , l i v e r [188] and cerebrum [ 1 5 0 ] , s u g g e s t s i t s e f f e c t s on a n t i o x i d a n t s t a t u s may be g e n e r a l i z e d and w i d e s p r e a d . T h i s p o s s i b i l i t y has been e x p l o r e d by e x a m i n i n g t h e e f f e c t s o f c h r o n i c a l l o p u r i n o l t r e a t m e n t on t h e a n t i o x i d a n t c a p a c i t y o f r e d c e l l s , as r e f l e c t e d i n t h e i r s u s c e p t i b i l i t y t o i n v i t r o o x i d a t i v e c h a l l e n g e . These e x p e r i m e n t s were p e r f o r m e d i n c o n n e c t i o n w i t h a p a r a l l e l h e a r t - l u n g t r a n s p l a n t a t i o n i n v e s t i g a t i o n e x a m i n i n g t h e e f f e c t s o f p r e t r e a t i n g donor and r e c i p i e n t a n i m a l s w i t h a l l o p u r i n o l on t h e f u n c t i o n a l v i a b i l i t y o f t r a n s p l a n t e d h e a r t and l u n g t i s s u e s as d e s c r i b e d below. 1.3.3.2 Ischemia and Reperfusion Injury a Swine Model of Heart-Lung Transplantation and the E f f e c t s of A l l o - purinol Pretreatment R e p e r f u s i o n o f t r a n s p l a n t e d organs w h i c h have been s u b j e c t e d t o p e r i o d s o f c o l d and/or warm i s c h e m i a d u r i n g p r e - o p e r a t i v e s t o r a g e may cause d e l e t e r i o u s e f f e c t s , l e a d i n g t o p o s t - t r a n s p l a n t a t i o n o r gan f a i l u r e [ 9 ] . A l t h o u g h t h e e x a c t mechanism r e s p o n s i b l e f o r t h e i r r e v e r s i b l e damage t o t r a n s p l a n t e d organs has y e t t o be d e t e r m i n e d , t h e r e i s ample e v i d e n c e from s t u d i e s o f I/R i n j u r y i n v a r i o u s e x p e r i m e n t a l 28 and c l i n i c a l s e t t i n g s [74,189] t o s u g g e s t t h e i n v o l v e m e n t o f o x y - r a d i c a l s . H e a r t - l u n g t r a n s p l a n t a t i o n has been used s u c c e s s f u l l y i n t h e t r e a t m e n t o f e n d - s t a g e c a r d i o p u l m o n a r y d i s e a s e s i n c e 1981, w i t h hundreds o f p a t i e n t s now h a v i n g undergone s u c c e s s f u l h e a r t - l u n g t r a n s p l a n t a t i o n w o r l d w i d e . The e f f e c t i v e n e s s o f o r gan p r e s e r v a t i o n f o r t r a n s p l a n t a t i o n seems t o be a c r u c i a l f a c t o r i n d e t e r m i n i n g t h e f u n c t i o n a l v i a b i l i t y o f t r a n s p l a n t e d organs [190,191]. D e s p i t e t h e r e l a t i v e s u c c e s s o f t h i s most c o m p l i c a t e d s u r g i c a l p r o c e d u r e , c u r r e n t l y a v a i l a b l e methods f o r p r e s e r v a t i o n o f t h e l u n g from I/R i n j u r y f o l l o w i n g p r o l o n g e d p e r i o d s o f i s c h e m i a a r e f a r from o p t i m a l [192,193]. T h e r e f o r e , f o r s e v e r a l y e a r s , i t was c o n s i d e r e d n e c c e s s a r y t o have donor and r e c i p i e n t a t t h e same c e n t r e f o r performance o f t h e t r a n s p l a n t a t i o n p r o c e d u r e . A l t h o u g h s e v e r a l methods of p r e s e r v a t i o n have been e v a l u a t e d , none of them has been s u c c e s s f u l i n p r e v e n t i n g h i g h l y v u l n e r a b l e l u n g t i s s u e from I/R i n j u r y [ 1 92,193]. I n t h i s r e g a r d , a l l o p u r i n o l , whose e f f e c t i v e n e s s i n p r e s e r v i n g t r a n s p l a n t e d organs has been w e l l documented [184-186], may be e f f e c t i v e i n p r o t e c t i n g l u n g t i s s u e a g a i n s t I/R i n j u r y . T h i s p o s s i b i l i t y has been e x p l o r e d by e x a m i n i n g t h e e f f e c t s o f a l l o p u r i n o l p r e t r e a t r n e n t i n a swine model o f h e a r t - l u n g t r a n s p l a n t a t i o n . 29 1.3.3.4 Metal Chelating; and Other Antioxidant Properties of  A l l o p u r i n o l The c a t a l y t i c r ole of t r a n s i t i o n metals i n mediating oxy-radical induced tiss u e damage i s well established [13,194]. The iron chelator deferoxamine and a l l o p u r i n o l have both been shown to reduce c e l l u l a r damage i n is o l a t e d rabbit hearts subjected to a period of hypoxia followed by reoxygenation [195]. In addition, u r i c acid, whose chemical structure i s s i m i l a r to that of a l l o p u r i n o l , has been shown to be capable of chelating copper and iron ions [50,51]. These findings suggest that another possible mechanism of a l l o p u r i n o l protection against I/R injury might involve chelation of t r a n s i t i o n metal ions. This idea has been explored by investigating the actions of a l l o p u r i n o l and i t s metabolite, oxypurinol, on cupric ion- and f e r r i c ion-catalysed oxidation of ascorbate and oxidation of erythrocyte membrane l i p i d s . Although pharmacological interventions involving pretreatrnent with a l l o p u r i n o l [155,156] or iron chelators [139,196] have been shown to reduce the extent of I/R-induced t i s s u e damage, the extent of protection i s incomplete. This suggests that xanthine oxidase- and iro n -dependent systems are l i k e l y not the only sources of r a d i c a l production involved i n the development of myocardial I/R injury. Since myoglobin i s present i n cardiac muscle at a high concentration [197], i t s pro-oxidant action i n ischemic tissu e [198-200] might also be an important determinant in 30 t h e t r i g g e r i n g o f u n c o n t r o l l e d o x i d a t i v e p r o c e s s e s . I n t h e i s c h e m i c myocardium, i n c r e a s e d p r o d u c t i o n o f hydrogen p e r o x i d e from t h e enhanced a u t o x i d a t i o n o f oxy-myoglobin [200] o r abnormal m e t a b o l i s m o f oxygen [81,201] would s t i m u l a t e t h e o x i d a t i o n o f m y o g l o b i n , w i t h t h e c o n c o m i t a n t p r o d u c t i o n o f r e a c t i v e f e r r y l heme o x i d a n t s [198,199]. These m y o g l o b i n - d e r i v e d o x i d a n t s c o u l d r e s u l t i n t h e p e r o x i d a t i o n o f b i o l o g i c a l membrane l i p i d s [202] and o x i d a t i v e damage t o p r o t e i n m o l e c u l e s [ 2 0 3 ] , s u b s e q u e n t l y l e a d i n g t o t i s s u e i n j u r y . The use of p h a r m a c o l o g i c a l agents c a p a b l e of a t t e n u a t i n g t h e p o t e n t i a l l y h a r m f u l e f f e c t s of m y o g l o b i n would r e p r e s e n t a r a t i o n a l approach t o p r o t e c t i n g a g a i n s t m y o c a r d i a l I/R i n j u r y . I n o r d e r t o e x p l o r e o t h e r a c t i o n s o f a l l o p u r i n o l r e l a t i n g t o i t s p r o t e c t i v e e f f e c t s on I/R i n j u r y , e f f e c t s o f a l l o p u r i n o l and o x y p u r i n o l on t h e m y o g l o b i n - t - b u t y l h y d r o p e r o x i d e - c a t a l y s e d o x i d a t i o n o f u r i c a c i d and p e r o x i d a t i o n of e r y t h r o c y t e membrane l i p i d s were examined. I n c o n c l u s i o n , t h e p r e s e n t i n v e s t i g a t i o n was aimed a t e l u c i d a t i n g t h e m o l e c u l a r b a s i s i n t h e development o f m y o c a r d i a l I/R i n j u r y and e x p l o r i n g m e c h a n i s t i c a l t e r n a t i v e s t o x a n t h i n e o x i d a s e i n h i b i t i o n i n t h e p r o t e c t i o n by a l l o p u r i n o l a g a i n s t I/R i n j u r y . I n f o r m a t i o n o b t a i n e d from t h e a f o r e m e n t i o n e d s t u d i e s may be u s e f u l i n d e v i s i n g e f f e c t i v e t h e r a p y i n t h e p r e v e n t i o n o f I/ R - i n d u c e d c l i n i c a l l y r e l e v a n t t i s s u e damage. On t h e b a s i s of o b s e r v e d changes i n a n t i o x i d a n t c a p a c i t y o f e r y t h r o c y t e s , we a l s o 31 i n t e n d e d t o e x p l o r e the p o s s i b i l i t y o f o p t i m i z i n g the e f f e c t i v e n e s s o f t h e r a p e u t i c i n t e r v e n t i o n s ( i n c l u d i n g a l l o p u r i n o l ) u n d e r t a k e n i n p a t i e n t s p r i o r t o c a r d i o p u l m o n a r y bypass s u r g e r y o r organ t r a n s p l a n t a t i o n i n o r d e r t o m i n i m i z e the r i s k o f damage r e s u l t i n g from p o s t - i s c h e m i c t i s s u e r e p e r f u s i o n . 32 2 . MATERIALS AND METHODS 2 . 1 Surgical Procedures 2 . 1 . 1 Animal Care A n i m a l s r e c e i v e d f o o d and wat e r ad l i b i t u m and were m a i n t a i n e d i n a c o n s t a n t t e m p e r a t u r e (22°C) e n v i r o n m e n t w i t h a c o n s t a n t 12-hr l i g h t s c h e d u l e ( l i g h t on a t 0700 h r , o f f a t 1900 h r ) . 2 . 1 . 2 Experimental Model of Ischemia/Reperfusion Injury 2 . 1 . 2 . 1 Pentobarbital Anesthetized Open-Chest Rabbit Male New Z e a l a n d w h i t e r a b b i t s (2.5-3.0 kg) were a n e s t h e t i z e d w i t h p e n t o b a r b i t a l ( a d m i n i s t e r e d v i a a m a r g i n a l e a r v e i n as an i n i t i a l 30 mg/kg b o l u s w i t h s u p p l e m e n t a r y doses g i v e n p e r i o p e r a t i v e l y as r e q u i r e d ) . A t r a c h e o t o m y was q u i c k l y p e r f o r m e d and t h e r a b b i t s were v e n t i l a t e d m e c h a n i c a l l y u s i n g room a i r supplemented w i t h 100% O2 f o r th e d u r a t i o n o f t h e e x p e r i m e n t . The r a t e o f t h i s a r t i f i c i a l v e n t i l a t i o n was a d j u s t e d t o m a i n t a i n normal v a l u e s of a r t e r i a l b l o o d pH and p a r t i a l p r e s s u r e of oxygen ( P O 2 ) i n r a b b i t s . The c h e s t was opened and t h e exposed h e a r t was t h e n suspended i n a p e r i c a r d i a l c r a d l e . The l e f t c i r c u m f l e x c o r o n a r y a r t e r y was i d e n t i f i e d and a 4-0 s i l k l i g a t u r e was passed, under i t . The ends o f t h e l i g a t u r e were t h r e a d e d t h r o u g h a s h o r t l e n g t h o f t u b i n g t o form a s n a r e . The a n i m a l was a l l o w e d t o s t a b i l i z e f o r 15 min b e f o r e t h e i n d u c t i o n o f i s c h e m i a . 33 2.1.2.2 I n d u c t i o n o f R e g i o n a l I s c h e m i a Ischemia was induced by tightening the snare around the coronary artery and maintained by clamping the tubing with a hemostat. After the desired period of l i g a t i o n , the snare was released to i n i t i a t e reperfusion. If ve n t r i c u l a r f i b r i l l a t i o n occurred during the l i g a t i o n or reperfusion period, sinus rhythm was restored by the application of 0.5 Watt-second countershocks. 2.1.2.3 I d e n t i f i c a t i o n o f t h e Oc c l u d e d Zone At the end of the reperfusion period, the heart was excised and put into a cold (4°C) solution of 50 mM Tris-HCl buffer, containing 0.1 mM EDTA, pH 7.6. The snare was pulled t i g h t to ree s t a b l i s h the l i g a t i o n and the heart was perfused with a solution of Fast Green FCF (0.5 mg/ml i n iso t o n i c saline) v i a the a o r t i c root [204]. The ischemic or ischemic/reperfused l e f t v e n t r i c u l a r t i s s u e was i d e n t i f i e d by the absence of staining and was excised for biochemical analysis. 2.1.2.4 Sham-Operated A n i m a l s Sham-operated animals, which served as non-ischemic controls, were subjected to the same su r g i c a l procedures except for the tightening of the coronary artery l i g a t u r e . 34 2.1.3 Isolated Lanqendorff-Perfused Rabbit Heart Male New Zealand white rabbits (2.5-3.0 kg) were anesthetized and subjected to the same su r g i c a l procedures as described for open-chest rabbits. A f t e r putting the li g a t u r e around the l e f t circumflex coronary artery, the heart was excised and placed i n Krebs-Henseleit buffer solution (see below for the composition). The heart was quickly mounted on a Langendorff apparatus and perfused retrogradely (100 cm H2O) through the a o r t i c root with Krebs-Henseleit buffer, containing NaCl (112 mM) , NaHCC>3 (20 mM) , KC1 (5.7 mM) , EDTA (0.03 mM) , MgCl 2 (1 .2 mM) and glucose (11 mM), and gassed with a mixture of O2/CO2 (95/5, v/v) at 37°C. 2.1.3.1 Induction of Regional Ischemia i n the Isolated Rabbit Heart Regional ischemia was induced and reperfusion was i n i t i a t e d as described for the open-chest rabbits. At the end of the reperfusion period, the li g a t u r e was retightened and the heart was perfused for 1 min with Fast Green FCF solution (0.5 mg/ml i n Krebs-Henseleit b u f f e r ) . The ischemic /reperfused tiss u e was i d e n t i f i e d and excised for biochemical analysis. 2.1.3.2 Sham-Control for Isolated Rabbit Heart Isolated Langendorff-perfused rabbit hearts, which served as non-ischemic controls, were prepared by the same 35 procedures described i n the previous section, but without coronary artery l i g a t i o n . I 2.1.3.3 Induction of Regional Ischemia In Vivo Followed by  In V i t r o Reperfusion A f t e r a 30 min period of coronary artery l i g a t i o n i n open-chest rabbits, hearts were excised and perfused i n the Langendorff apparatus for 10 min, with the l i g a t u r e s t i l l i n t a c t . Thereafter, the hearts were reperfused for 60 min with Krebs-Henseleit buffer. The ischemic/reperfused tiss u e was i d e n t i f i e d as described previously. 2.1.4 Heart-Lung Transplantation i n Pigs The inves t i g a t i o n of ef f e c t s of chronic a l l o p u r i n o l treatment (in pigs) on the antioxidant capacity of erythrocytes was performed i n connection with a p a r a l l e l heart-lung transplantation study examining the e f f e c t s of pretreating donor and re c i p i e n t animals with a l l o p u r i n o l on the post-operative functional v i a b i l i t y of transplanted heart and lung ti s s u e s . These experiments were c a r r i e d out i n c o l l a b o r a t i o n with a cardiovascular research team at Vancouver General Hospital (VGH). The measurement of erythrocyte s u s c e p t i b i l i t y to oxidative challenge was done by the author. A l l su r g i c a l procedures and cardiorespiratory functional measurements were performed by our collaborators at VGH, as described i n d e t a i l i n a recent report [ 2 0 5 ] . B r i e f l y , male pigs (20 to 22 kg) were treated with 36 a l l o p u r i n o l ( t a b l e t s ) g i v e n o r a l l y a t a d a i l y dose of 50 mg/kg f o r 3 o r 5 days. The f i n a l dose was g i v e n i m m e d i a t e l y b e f o r e s u r g e r y . The h e a r t - l u n g t r a n s p l a n t a t i o n p r o c e d u r e s were performed i n p i g s a n e s t h e t i z e d w i t h ketamine (20 mg/kg) and m a i n t a i n e d w i t h i s o f l u r a n e ( 0 . 5 - 2 . 0 % ) . H a r v e s t i n g o f h e a r t - l u n g organs from donor a n i m a l s and t h e o r t h o t o p i c t r a n s p l a n t a t i o n i n r e c i p i e n t a n i m a l s were done u s i n g s t a n d a r d t e c h n i q u e s . H e a r t and l u n g t i s s u e s were p e r f u s e d w i t h c o l d i s o - o s m o l a r c a r d i o p l e g i c s o l u t i o n (8-10°C and 16-18°C, r e s p e c t i v e l y ) , and p l a c e d i n hypothermic s t o r a g e . The t o t a l i s c h e m i c t i m e , w h i c h i n c l u d e d t h e p e r i o d o f o r t h o t o p i c t r a n s p l a n t a t i o n , was 6 h o u r s . Assessments of c a r d i a c and pulmonary f u n c t i o n were performed a t t h r e e t i m e i n t e r v a l s , namely p r e - i s c h e m i a (T-^), 30 min ( T 2 ) and 2 hr (T3) p o s t - t r a n s p l a n t a t i o n . P r e - i s c h e m i c (T^) measurements (which were made i n t h e donor a n i m a l ) were t a k e n as c o n t r o l f o r t h o s e measured a f t e r t r a n s p l a n t a t i o n ( i n t h e r e c i p i e n t a n i m a l ) . S e r i a l measurements of c a r d i a c i n d e x ( C I ) , s t r o k e i n d e x ( S I ) , l u n g w a t e r c o n t e n t (LW), a r t e r i a l b l o o d p a r t i a l p r e s s u r e of oxygen ( p 0 2 ) , pulmonary v a s c u l a r r e s i s t a n c e (PVR), a l v e o l a r -a r t e r i a l oxygen g r a d i e n t (AaG) and a l v e o l a r - a r t e r i a l oxygen t e n s i o n r a t i o (AaR) were performed. 37 2.2 Drug Treatment Protocols 2.2.1 Chronic A l l o p u r i n o l Treatment i n Rabbits In the chronic treatment regimen, a l l o p u r i n o l (Sigma Chemical Co.) was given o r a l l y i n a l k a l i n i z e d (with NaOH) drinking water (1 mg/ml at an estimated d a i l y dose of 75 mg/kg) for 7 days. The drinking water of the animals used as controls i n these experiments was a l k a l i n i z e d to an equivalent degree as that required to maintain a l l o p u r i n o l i n solution (pH 9.0). 2.2.2 Acute A l l o p u r i n o l and Oxypurinol Treatment i n Rabbits Rabbits were treated with a l l o p u r i n o l or oxypurinol given intravenously at a dose of 50 mg/kg 1 hr p r i o r to coronary artery l i g a t i o n . A l l o p u r i n o l or oxypurinol was dissolved i n saline (50 mg/ml) by the addition of concentrated NaOH (to pH 11.5). The drug solution was made up to a f i n a l volume of 5 ml with saline and administered v i a the marginal ear vein over 5 min. 2.2.3 Acute U74006F Treatment i n Rabbits Rabbits were treated with the 21-aminosteroid antioxidant U74006F (Upjohn Co.) (3 mg/kg) given as an intravenous bolus (via the marginal ear vein) over 5 min beginning eit h e r 15 min p r i o r to coronary artery l i g a t i o n or 15 min before the onset of reperfusion. Control animals were given drug vehicle. 38 2.3 Biochemical and Chemical Analyses 2.3.1 Tissue S u s c e p t i b i l i t y to In V i t r o Peroxide Challenge The antioxidant capacity of tiss u e was assessed i n terms of s e n s i t i v i t y to glutathione (GSH) depletion and l i p i d peroxidation following incubation of t i s s u e homogenate in v i t r o with increasing concentrations of t -butylhydroperoxide (TBHP). 2.3.1.1 Preparation of Tissue Homogenates Anesthetized animals were s a c r i f i c e d by cardiac excision. Tissue samples from the occluded zone (or a comparable region of l e f t v e n t r i c l e i n control animals) were placed i n 50 mM Tris-0.1 mM EDTA, pH 7.6 (10%, w/v) and homogenised using two 15 sec bursts of a Polytron (Brinkmann, Westbury, N.Y.) at 2 5% maximal speed. 2.3.1.2 S u s c e p t i b i l i t y of Tissues to TBHP-Induced Depletion  of GSH Aliquots of tissue homogenate (0.3 ml) were combined with an equal volume of saline/azide (isotonic saline with 2 mM sodium azide) containing increasing concentrations of TBHP ( f i n a l concentrations ranged from 0 to 0.5 mM for heart tissues or 0 to 1.0 mM for kidney or l i v e r t i s s u e s ) . Samples were incubated for 30 min at 37°C and the reaction was terminated with 0.15 ml cold 25% (w/v) TCA solution. Following centrifugation, the supernatants were analysed for GSH (acid soluble-sulfhydryl group) content using 5,5'-39 d i t h i o - b i s - ( 2 - n i t r o b e n z o i c a c i d ) (DTNB). The a s s a y m i x t u r e c o n t a i n e d 2.4 ml of 0.1 M phosphate b u f f e r (pH 8.0) and 0.3 ml o f s u p e r n a t a n t , and t h e r e a c t i o n was i n i t i a t e d by a d d i n g 0.1 ml DTNB s o l u t i o n (3 mM, f r e s h l y p r e p a r e d i n phosphate b u f f e r ) . Absorbance a t 412 nm o f t h e r e a c t i o n m i x t u r e was measured a t 10 min. The GSH c o n t e n t was e s t i m a t e d u s i n g a s t a n d a r d c a l i b r a t i o n c u r v e . T i s s u e GSH c o n t e n t ( b a s a l GSH l e v e l ) , e x p r e s s e d as nmoles/mg t i s s u e , was measured i n t h e absence of TBHP. TBHP-induced GSH d e p l e t i o n was e x p r e s s e d as % d e c r e a s e i n GSH c o n t e n t when compared w i t h t h e b a s a l l e v e l ( i e . i n t h e absence of TBHP). 2.3.1.3 S u s c e p t i b i l i t y of Tissues to L i p i d Peroxidation A l i q u o t s o f t i s s u e homogenate (0.5 ml) were i n c u b a t e d w i t h an e q u a l volume of TBHP i n s a l i n e / a z i d e ( f i n a l c o n c e n t r a t i o n s ranged from 0 t o 10 mM) f o r 30 min a t 37°C. R e a c t i o n s were t e r m i n a t e d by t h e a d d i t i o n of 0.5 ml c o l d 28% (w/v) TCA c o n t a i n i n g 0.1 M sodium a r s e n i t e . F o l l o w i n g c e n t r i f u g a t i o n , a o n e - m i l l i t e r a l i q u o t of t h e s u p e r n a t a n t was combined w i t h 0.5 ml TBA s o l u t i o n (0.5% (w/v) 2-TBA i n 0.025 M NaOH) and b o i l e d f o r 15 min. The f o r m a t i o n of t h i o b a r b i t u r i c a c i d - r e a c t i v e s u b s t a n c e s (TBARS) was e s t i m a t e d from t h e absorbance a t 532 nm, as d e s c r i b e d by T a p p e l & Z a l k i n [ 2 0 6 ] . 40 2.3.2 Erythrocyte S u s c e p t i b i l i t y to In V i t r o Peroxide  Challenge The antioxdant capacity of erythrocytes was assessed i n term of s e n s i t i v i t y to l i p i d peroxidation following i n v i t r o incubation with increasing concentrations of TBHP. 2.3.2.1 Preparation of Packed Erythrocytes Heparinized blood samples were obtained from animals and erythrocytes were separated by centrifugation at 3,000 xg for 5 min at 4°C using a c l i n i c a l centrifuge (Int. Equip. Co, Needham, Mass.), with removal of the plasma and white c e l l s . Red c e l l s were then washed twice with isotonic saline containing 2.0 mM sodium azide. 2.3.2.2 S u s c e p t i b i l i t y of Erythrocytes to TBHP-Induced L i p i d  Peroxidation Aliquots (50 /xl) of erythrocytes were weighed and combined with 0.45 ml of saline-azide solution (to give a 10% suspension), and preincubated for 5 min at 37°C. Peroxidative challenge was induced by the addition of an equal volume (0.5 ml) of TBHP in saline-azide ( f i n a l TBHP concentrations ranged from 0.25 to 10 mM) . After a 30 min incubation at 37°C, the reaction was terminated by addition of 0.5 ml 28% TCA-0.1 M sodium arsenite. The reaction mixture was centrifuged and a 1.0 ml aliquot of the supernatant was combined with 0.5 ml TBA solution (0.5% (w/v) 2-TBA in 0.025 M NaOH) and boiled for 15 min. The 41 extent of malondialdehyde (MDA) production, expressed as nmoles MDA/mg Hb, was estimated from absorbances at 532 nm and 453 nm of the reaction mixture using acid-hydrolysed malondialdehyde b i s ( d i e t h y l a c e t a l ) (Aldrich) as a standard, according to the method of Stocks & Dormandy [207] and G i l b e r t et. a l . [208]. 2.3.2.3 Hemoglobin Assay Hemoglobin was measured using the cyanomethemoglobin method of Drabkin & Austin [209]. An aliquot (50 fll) of erythrocytes was weighed and d i l u t e d with 4.95 ml H2O, and the r e s u l t i n g hemolysate was centrifuged to remove the membrane debris. An aliquot of the supernatant was mixed with 0.5 ml of 1.8 mM K 3Fe(CN) 6, 0.5 ml of 2.5 mM KCN and s u f f i c i e n t H2O to make up a f i n a l volume of 1.5 ml. Absorbance at 540 nm of the reaction mixture was measured at 3 0 min and hemoglobin concentration (mg/ml) was estimated using a standard c a l i b r a t i o n curve. The hemoglobin concentration of erythrocytes was expressed as mg Hb/g RBC. 2.3.3 Tissue Antioxidant Enzyme A c t i v i t i e s Tissue antioxidant enzyme a c t i v i t i e s were measured using c y t o s o l i c fractions prepared from tiss u e homogenates. Spectrophotometry measurements were performed using a Perkin-Elmer model Lambda 6B spectrophotometer or a Beckman ACTA C2 spectrophotometer. A l l assays were c a r r i e d out at room temperature (22°C - 25°C) unless otherwise indicated. 42 2.3.3.1 Preparation of Cytosolic Fractions Tissue homogenates (prepared as described in the previous section) were d i l u t e d 1:3 with the homogenising buffer and centrifuged for 15 min, either at 16,000 xg in an Eppendorf 5415 microcentrifuge (heart tissues) or at 105,000 xg i n a Beckman L2-65 ultracentrifuge ( l i v e r or kidney t i s s u e s ) , to obtain the c y t o s o l i c f r a c t i o n . 2.3.3.2 Catalase The a c t i v i t y of catalase (CAT) was measured according to the method described by Aebi [210]. An aliquot (0.9 ml) of c y t o s o l i c f r a c t i o n was mixed with 18 pi of d i l u t e d ethanol (95% ethanol/H^O, 1:1 (v/v)). After incubating for 30 min on ice, 0.1 ml of cold T r i t o n X-100 solution (10% (v/v) i n 50 mM Tris-0.1 mM EDTA, pH 7.6) was added and 0.4 ml of t h i s mixture was d i l u t e d with 9.6 ml of 50 mM phosphate buffer (pH 7.0) immediately p r i o r to assay. A two m i l l i l i t e r - a l i q u o t of t h i s d i l u t e d sample was put into a cuvette and the reaction was i n i t i a t e d by adding 1 ml of 30 mM H2O2 (freshly prepared i n 50 mM phosphate buffer, pH 7.0). A f t e r mixing, absorbance at 240 nm was measured at 15 sec and 3 0 sec, and enzyme a c t i v i t y was expressed as K/mg t i s s u e , where K i s the rate constant [210]. 43 2.3.3.3 Cu,Zn-Superoxide Dismutase The a c t i v i t y of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) was measured according to the method described by Winterbourne et. a l . [211]. An aliquot (2.0 ml for heart or 0.5 ml for l i v e r and kidney tissues) of c y t o s o l i c f r a c t i o n was combined with s u f f i c i e n t H2O to make up a volume of 2.0 ml. Following addition of 0.5 ml ethanol and 0.3 ml chloroform, the mixture was vortexed thoroughly and centrifuged at 3,000 xg for 5 min using a c l i n i c a l centrifuge. The r e s u l t i n g supernatant was again centrifuged in an Eppendorf centrifuge (at maximal speed) for 5 min to obtain a c l e a r extract. Assay mixtures contained 1.0 ml of 75 mM phosphate buffer (pH 7.8), 0.2 ml of 0.1 M Na2EDTA-1.5 mg% NaCN, 0.1 ml of 1.5 mM Nitro blue tetrazolium, varying aliquots of the clear extract (0 - 500 /zl) and s u f f i c i e n t H2O to make up a f i n a l volume of 2.95 ml. The reaction was i n i t i a t e d by adding 50 /xl of 0.12 mM r i b o f l a v i n and reaction mixtures were mixed and illuminated with fluorescent l i g h t for 2.5 min twice (with vortexing between and at the end of the illumination) . Absorbance at 560 nm of the reaction mixtures was measured and enzyme a c t i v i t y was expressed as Units/mg tissu e [211]. 2.3.3.4 Glutathione Peroxidase The a c t i v i t y of glutathione peroxidase (GPX) was measured using the method of Paglia & Valentine [212] as modified by Lawrence & Burke [213]. An aliquot (0.5 ml) of 44 c y t o s o l i c f r a c t i o n was combined with an equal volume of double-strength Drabkin's reagent (0.0016 M KCN-0.0012 M K 3Fe (CN) 6-0.0238 M NaHC03), and the mixture was kept on ice u n t i l used for the assay. Reaction mixtures contained 2.0 ml of 75 mM phosphate buffer (pH 7.0), 50 /xl of 60 mM GSH, 100 /xl of glutathione reductase enzyme solution (30 Units/ml, Sigma Chemical Co), 50 /xl of 0.12 M sodium azide, 100 /xl of 15 mM Na2EDTA, 100 1 of 3 mM NADPH, varying aliquots of sample (100-300 iii for heart or 50-100 /xl for l i v e r and kidney tissues) and s u f f i c i e n t H 20 to make up a f i n a l volume of 2.9 ml. The reaction was i n i t i a t e d by addition of 100 /xl 7.5 mM H 20 2 solution and absorbance changes of the reaction mixtures were monitored spectrophotometrically at 34 0 nm for 5 min. Enzyme a c t i v i t y was calculated using an e x t i n c t i o n c o e f f i c i e n t for NADPH at 340 nm of 6.22 x 10a/M/cm and expressed as nmoles NADPH/min/mg ti s s u e . 2.3.3.5 G l u t a t h i o n e Reductase The a c t i v i t y of glutathione reductase (GRD) was measured using the method of Long & Carson [214]. Reaction mixtures contained 0.5 ml of 18 mM glutathione d i s u l f i d e (GSSG), 1 ml of 0.45 M Tris-90 mM EDTA (pH 7.6), an aliquot (0.4 ml for heart or 0.1 ml for l i v e r and kidney tissues) of c y t o s o l i c f r a c t i o n and s u f f i c i e n t H 20 to make up a f i n a l volume of 2.9 ml. The reaction was i n i t i a t e d by addition of 100 /i l 3 mM NADPH solution and absorbance changes at 340 nm 45 o f t h e r e a c t i o n m i x t u r e s were m o n i t o r e d s p e c t r o p h o t o m e t r i c a l l y f o r 5 min. Enzyme a c t i v i t y was c a l c u l a t e d u s i n g an e x t i n c t i o n c o e f f i c i e n t f o r NADPH a t 340 nm o f 6.22 x 10^/M/cm and e x p r e s s e d as nmoles NADPH/min/mg t i s s u e . 2.3.3.6 Hemoglobin Assay The hemoglobin c o n t e n t o f c y t o s o l i c f r a c t i o n s was measured by t h e method d e s c r i b e d p r e v i o u s l y . 2.3.3.7 Correction for Enzyme A c t i v i t y Contributed by Blood A c t i v i t i e s o f t i s s u e c y t o s o l i c a n t i o x i d a n t enzymes were c o r r e c t e d f o r t h e c o r r e s p o n d i n g enzyme a c t i v i t i e s c o n t r i b u t e d by t h e b l o o d c o n t a m i n a n t s . The c o r r e c t i o n was e s t i m a t e d from t h e hemoglobin c o n t e n t o f t h e c y t o s o l i c f r a c t i o n and t h e a c t i v i t y o f enzymes measured i n e r y t h r o c y t e s o b t a i n e d from t h e same a n i m a l . 2.3.4 Erythrocyte Antioxidant Enzyme A c t i v i t i e s A c t i v i t i e s o f a n t i o x i d a n t enzymes i n e r y t h r o c y t e s were measured i n hemolysates p r e p a r e d as d e s c r i b e d below. 2.3.4.1 Preparation of Hemolysates A 0.4 ml a l i q u o t o f packed e r y t h r o c y t e s ( p r e p a r e d as d e s c r i b e d p r e v i o u s l y ) was combined w i t h 3.6 ml H2O and t h e m i x t u r e was s u b j e c t e d t o t h r e e f r e e z e and thaw c y c l e s u s i n g d r y i c e / a c e t o n e t o o b t a i n t h e hemolysate. 46 2.3.4.2 Catalase An aliquot (20 ill) of hemolysate was added to 10 ml phosphate buffer (50 mM, pH 7.0) immediately p r i o r to assay. This d i l u t e d hemolysate was assayed as described for the tis s u e c y t o s o l i c f r a c t i o n s . Catalase a c t i v i t y was expressed as K/mg Hb. 2.3.4.3 Cu #Zn-Superoxide Dismutase An aliquot (0.5 ml) of hemolysate was extracted and assayed as described for the tissue c y t o s o l i c f r a c t i o n s . Cu,Zn-S0D a c t i v i t y was expressed as Units/mg Hb. 2.3 .4.4 Glutathione Peroxidase An aliquot (0.4 ml) of hemolysate was mixed with 3.6 ml H2O, and 0.5 ml of t h i s d i l u t e d hemolysate was then combined with an equal volume of double-strength Drabkin's solution (prepared as described previously), and the mixture was kept on i c e u n t i l used for the assay. Varying aliquots (50 -150 fll) of sample were assayed as described for the tissue c y t o s o l i c f r a c t i o n s . Glutathione peroxidase a c t i v i t y was expressed as nmoles NADPH/min/mg Hb. 2.3.4.5 Glutathione Reductase Diluted hemolysate (prepared as described for the GPX assay) was used for t h i s determination. Reaction mixtures contained the same constituents as outlined for the ti s s u e c y t o s o l i c f r a c t i o n s except that 100, 300 and 500 ill aliquots of hemolysate were assayed. After adding 100 ill of 3 mM NADPH, reaction components were quickly mixed, and absorbances at 340 nm were measured. Reaction mixtures were incubated at 37°C for 15 min and absorbances at 340 nm were again measured. Glutathione reductase a c t i v i t y , expressed as nmoles NADPH/min/mg Hb, was determined from absorbance changes during the incubation period, using an extincti o n c o e f f i c i e n t for NADPH at 340 nm of 6.22 x 106/M/cm. 2.3 . 4 . 6 Hemoglobin Assay An aliquot of d i l u t e d hemolysate (prepared as described for the GPX assay) was centrifuged and the supernatant was analysed for hemoglobin content as described previously section. 2.3.5 Mitochondrial ATPase A c t i v i t y 2.3.5.1 I s o l a t i o n of Mitochondria Samples of control or ischemic l e f t v e n t r i c u l a r tissue (0.8 g) were homogenised i n 8 ml buffer (0.25 M sucrose-50 mM T r i s , pH 7.4) using a Polytron (with one 5 sec burst at 25% maximal speed). The homogenate was centrifuged at 1,2 00 xg for 10 min. The supernatant was decanted and centrifuged at 11,000 xg for 15 min. The p e l l e t was resuspended i n 8 ml homogenising buffer and centrifuged again at 11,000 xg for 15 min. The f i n a l p e l l e t was resuspended i n 0.6 ml homogenisation buffer. The protein 48 content of t h i s mitochondrial f r a c t i o n was determined by the Lowry method using bovine serum albumin as a standard [215]. 2.3.5.2 M i t o c h o n d r i a l A z i d e - S e n s i t i v e ATPase A c t i v i t y ATPase a c t i v i t y of the mitochondrial f r a c t i o n was determined using the procedure described by Moore & Godin [216] with only s l i g h t modification. Assay mixtures, i n a f i n a l volume of 2.8 ml, contained the following at the f i n a l concentrations given i n the brackets: Tris-HCl buffer, pH 7.4 (55 mM) , MgCl 2 (3 mM) , EGTA (0.3 mM) , ATP (3 mM) and Tr i t o n X-100 (0.005%,v/v) and were incubated at 37°C for 5 min. The reaction was i n i t i a t e d by adding 0.2 ml of membrane suspension (containing 75 /ig membrane protein i n the presence of sodium azide (5 mM) or 7.5 /zg membrane protein i n the absence of sodium azide). After a 15 min incubation at 37°C, the reaction was terminated by adding 1 ml of cold 10% TCA, followed by centrifugation using a c l i n i c a l centrifuge. The r e s u l t i n g supernatant was assayed for inorganic phosphate (Pi) using the method decribed by Fiske & Subbarow [217]. A 3.0 ml aliquot of the supernatant was mixed with 1.4 ml H2O and 0.4 ml of 5% ammonium molybdate, and the reaction was i n i t i a t e d by adding 0.2 ml ANS solution prepared as follows. A mixture of l-amino-2-naphthol-4-sulfonic acid (0.125 g) and sodium s u l f i t e (0.25 g) i n ~30 ml of freshly prepared sodium b i s u l f i t e solution (15% (w/v) f i n a l concentration) was heated u n t i l the solution turned yellow, and was made up to 49 a f i n a l volume o f 50 ml. A f t e r an i n c u b a t i o n f o r 15 min a t room t e m p e r a t u r e , a b s o r b a n c e a t 660 nm was measured. The P i c o n t e n t was e s t i m a t e d w i t h r e f e r e n c e t o a phosphate s t a n d a r d . ATPase a c t i v i t i e s were e x p r e s s e d as /moles Pi/mg membrane p r o t e i n / h r . The a z i d e - s e n s i t i v e m i t o c h o n d r i a l ATPase a c t i v i t y was e s t i m a t e d by s u b t r a c t i n g t h e v a l u e measured i n t h e p r e s e n c e o f sodium a z i d e from t h a t measured i n t h e absence o f sodium a z i d e . 2.3.6 T i s s u e ATP T r a n s m u r a l samples o f c o n t r o l o r i s c h e m i c v e n t r i c u l a r t i s s u e ( a p p r o x i m a t e l y 100 mg) were q u i c k - f r o z e n i n l i q u i d n i t r o g e n and s t o r e d a t -70°C f o r p e r i o d s n o t e x c e e d i n g 1 week. T i s s u e s were ground w i t h 6% p e r c h l o r i c a c i d (4 i i l / m g t i s s u e ) under l i q u i d n i t r o g e n and t h e n thawed on i c e . A f t e r c e n t r i f u g a t i o n , an a l i q u o t (80 /zl) o f t h e s u p e r n a t a n t was n e u t r a l i z e d w i t h 60 / i l o f 1.4 M KHCO3, l e t s t a n d on i c e f o r 15 min and r e c e n t r i f u g e d . The r e s u l t i n g s u p e r n a t a n t was a n a l y s e d f o r ATP c o n t e n t as d e s c r i b e d by Jaworek e t . a l . [ 2 1 8 ] . An a l i q u o t (100 / i l ) o f t h e s u p e r n a t a n t was mixed w i t h 1 ml o f N A D H - c o n t a i n i n g p h o s p h o g l y c e r i c a c i d b u f f e r ( p r e p a r e d by a d d i n g 1 ml o f 3-p h o s p h o g l y c e r i c a c i d b u f f e r (Sigma) t o a v i a l c o n t a i n i n g 0.3 mg NADH (Sigma) and m i x i n g w i t h 2 ml o f H2O) and 80 (il o f H2O. A b s o r b a n c e a t 340 nm was measured a f t e r 3 min, 20 (il o f p h o s p h o g l y c e r a t e k i n a s e / g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r ogenase enzyme s o l u t i o n (Sigma C h e m i c a l Co.) was added 50 and t h e absorbance was measured a g a i n 5 min l a t e r . The ATP c o n t e n t was e s t i m a t e d from t h e change i n absorbance o f t h e r e a c t i o n m i x t u r e u s i n g a s t a n d a r d c a l i b r a t i o n c u r v e . 2.3.7 P r e p a r a t i o n o f E r y t h r o c y t e Membranes E r y t h r o c y t e membranes were p r e p a r e d from o u t d a t e d human b l o o d by s t e p w i s e h y p o t o n i c l y s i s as d e s c r i b e d by Godin & S c h r i e r [ 2 1 9 ] . One u n i t o f o u t d a t e d human b l o o d ( o b t a i n e d from t h e Red C r o s s ) was d i l u t e d t o 1,200 ml w i t h c o l d s a l i n e , and c e n t r i f u g e d a t 600 xg, f o l l o w e d by t h e c a r e f u l r emoval o f plasma and b u f f y c o a t m a t e r i a l . Red c e l l s were washed ( w i t h c e n t r i f u g a t i o n a t 15,000 xg) w i t h i s o t o n i c s a l i n e and an a l i q u o t (100 ml) o f t h e s e washed e r y t h r o c y t e s was d i l u t e d t o 1,200 ml w i t h c o l d 0.8 M NaCl s o l u t i o n . The m i x t u r e was s t i r r e d f o r 10 min a t 4°C, and c e n t r i f u g e d a t 15,000 xg f o r 5 min. The r e s u l t i n g p e l l e t w h i c h i n c l u d e d l i g h t - c o l o r e d m a t e r i a l ( e r y t h r o c y t e membranes) was resuspended and d i l u t e d t o 1,200 ml w i t h c o l d 0.06 M N a C l . T h i s p r o c e d u r e was r e p e a t e d t h r e e t i m e s u s i n g NaCl s o l u t i o n s o f d e c r e a s i n g c o n c e n t r a t i o n s (0.04 M, 0.02 M and 0.009 M, r e s p e c t i v e l y ) . When 0.02 M o r 0.009 M NaCl was used, t h e pH of m i x t u r e was c a r e f u l l y a d j u s t e d t o 7.4 w i t h 1 M T r i s (pH 7.4), and t h e n o n - l y s e d e r y t h r o c y t e s (which formed a d i s c r e e t p e l l e t u nderneath t h e membrane l a y e r ) were removed a f t e r each c e n t r i f u g a t i o n . A f t e r t h e s t e p w i s e h y p o t o n i c l y s i s , t h e r e s u l t i n g membrane p r e p a r a t i o n was resuspended i n 10 mM T r i s - H C l (pH 7.4), w i t h f i n a l volumes r a n g i n g from 51 600 to 1,200 ml, depending on the extent of v i s i b l e hemoglobin contamination. A f t e r s t i r r i n g for 10 min at 4°C, the mixture was centrifuged at 3 0,000 xg for 10 min, and the membranes were removed and pooled. An aliquot of t h i s membrane preparation was used for protein determination using the Lowry method [215], and the remainder was quick-frozen i n dry ice/acetone p r i o r to storage at -20°G and used within one week. 2.3.8 F e r r i c Chloride-Induced Oxidation of Erythrocyte  Membrane Lip i d s The f e r r i c chloride-induced oxidation of erythrocyte membrane l i p i d s was measured i n isoton i c s a l i n e adjusted to pH 7.4 with 1 M T r i s , pH 7.4. Reactions were performed eithe r i n the absence or presence of various concentrations of the t e s t compounds. A l l chemicals were dissolved i n buffered s a l i n e except butylated hydroxytoluene (BHT), which was dissolved i n isopropanol; 10 til of t h i s isopropanol solu t i o n was used i n the reaction mixture. The reaction was i n i t i a t e d by adding 250 izl of f e r r i c chloride s o l u t i o n i n a mixture containing 0.3 mg membrane protein i n a f i n a l volume of 1 ml. Reaction mixtures were incubated for 3 0 min at 37°C. When time-course studies were performed, GSH or ascorbic acid, at a f i n a l concentration of 50 /zM, was added (in 10 /il) before the reaction was i n i t i a t e d . Following increasing periods of incubation at 37°C, the reactions were terminated by adding 0.5 ml cold 28% TCA, containing 0.1 M 52 sodium a r s e n i t e . F o l l o w i n g c e n t r i f u g a t i o n , t h e s u p e r n a t a n t was a s s a y e d f o r TBARS by m i x i n g 1 ml s u p e r n a t a n t w i t h 0.5 ml TBA r e a g e n t (0.5% (w/v) 2-TBA i n 0.025 M NaOH), h e a t i n g i n a b o i l i n g w a t e r b a t h f o r 15 min and m e a s u r i n g t h e absorbance a t 532 nm. None o f t h e t e s t compounds was found t o i n t e r f e r e w i t h t h e TBA c o l o r r e a c t i o n . 2.3.9 Cupric Chloride-TBHP-Induced Oxidation of Erythrocyte  Membrane L i p i d s The c u p r i c c h l o r i d e - T B H P - i n d u c e d o x i d a t i o n o f e r y t h r o c y t e membrane l i p i d s was measured i n i s o t o n i c s a l i n e a d j u s t e d t o pH 7.4 ( w i t h 1 M T r i s , pH 7.4). The r e a c t i o n was i n i t i a t e d by a d d i n g 250 / l l c u p r i c c h l o r i d e - T B H P s o l u t i o n (0.2 mM - 10 mM, f i n a l c o n c e n t r a t i o n s ) t o a m i x t u r e c o n t a i n i n g 0.75 mg membrane p r o t e i n , i n a f i n a l volume o f 1.0 m l , f o l l o w e d by a 30 min i n c u b a t i o n a t 37°C. R e a c t i o n s were t e r m i n a t e d by a d d i n g 3 ml c o l d 1% p h o s p h o r i c a c i d . F o l l o w i n g t h e a d d i t i o n o f 1 ml TBA r e a g e n t (0.6% (w/v) 2-TBA i n 0.05 M NaOH), t h e m i x t u r e was b o i l e d f o r 45 min. A f t e r c o o l i n g , t h e samples were e x t r a c t e d w i t h 5 ml of n-b u t a n o l / p y r i d i n e (15/2, v/v) and c e n t r i f u g e d t o a c h i e v e phase s e p a r a t i o n . The TBARS c o n t e n t o f t h e b u t a n o l l a y e r was d e t e r m i n e d by m e a s u r i n g t h e ab s o r b a n c e a t 532 nm. 53 2.3.10 Myoglobin-TBHP-Induced Oxidation of Erythrocyte  Membrane Lipids The myoglobin-TBHP-induced oxidation of erythrocyte membrane l i p i d s was measured i n 50 mM phosphate buffer, pH 7.0. Reaction mixtures, i n a f i n a l volume of 1 ml, contained 2 6 [M myoglobin and 0.3 mg membrane protein, eit h e r i n the absence or presence of the t e s t compounds at increasing concentrations. A l l chemicals were dissolved i n phosphate buffer, except BHT, which was dissolved i n isopropanol; 10 [il of t h i s isopropanol solution was used i n the reaction mixture. The reaction was i n i t i a t e d by adding 100 ill TBHP at a f i n a l concentration of 1.0 mM. Reaction mixtures were incubated for 3 0 min or varying periods (10 -110 min) at 37°C and the reactions were terminated by adding 0.5 ml cold 28% (w/v) TCA, containing 0.1 M sodium arsenite. The content of TBARS i n the reaction mixtures was assayed as described for f e r r i c chloride-stimulated oxidative reactions. 2.3.11 Transition Metal Ion-Catalysed Oxidation of Ascorbic  Acid Ascorbate oxidation was monitored spectro-photometrically by the decrease i n absorbance at 280 nm. This wavelength was chosen rather than 2 65 nm, the absorption maximum for ascorbate, i n order to minimize the contribution by a l l o p u r i n o l to the absorption. The cupric or f e r r i c ion-catalysed oxidation of ascorbate was assayed 54 i n 10 mM phosphate buffer, pH 7.4, either i n the absence or presence of 0.1 mM EDTA used to reduce the basal oxidation of ascorbate catalysed by t r a n s i t i o n metal contaminants present i n the reaction medium. The reaction mixture, i n a f i n a l volume of 3 ml, contained 100 /M ascorbate i n the absence or presence of the t e s t compounds. The reaction was i n i t i a t e d by adding 100 ill of cupric or f e r r i c chloride solu t i o n at f i n a l concentrations ranging from 10 to 100 juM. The change i n absorbance at 280 nm of the reaction mixture was monitored continuously when the assay was performed i n the absence of EDTA. When 0.1 mM EDTA was present i n the reaction mixture, the absorbance at 280 nm was measured 1 min a f t e r the addition of cupric or f e r r i c chloride. Afte r a period of incubation at room temperature (15 or 40 min for cupric or f e r r i c ion-catalysed reactions, r e s p e c t i v e l y ) , the absorbance was again measured. The rate of ascorbate oxidation, expressed as nmoles/min, was calculated using a standard c a l i b r a t i o n curve and was constant over the incubation period. The rate of cupric or f e r r i c ion-catalysed ascorbate oxidation was corrected for the basal oxidation rate i n the absence of exogenous added metal ions. 2.3.12 UV Absorption Spectroscopy of Ascorbate/Allopurinol/  Copper Ion UV absorption spectra of reaction mixtures were recorded with a Perkin-Elmer model Lambda 6B 55 spectrophotometer. Various combinations of a l l o p u r i n o l / a s c o r b i c acid/cupric chloride or EDTA were mixed i n the following manner: 1.0 ml of 250 /xM a l l o p u r i n o l ; 1.0 ml of 125 /zM ascorbic acid; 0.5 ml of 500 /ZM EDTA; 0.5 ml of 500 iM cupric chloride and s u f f i c i e n t H2O to make up a f i n a l volume of 3 ml. Spectra of the reaction mixtures were recorded 2 min a f t e r the constituents had been mixed. 2.3.13 Myoglobin-TBHP-Catalysed Oxidation of Uric Acid Oxidation of u r i c acid was monitored spectrophotometrically by the decrease i n absorbance at 292 nm, the absorption maximum of u r i c acid, as described by Howell & Wyngaarden [220]. The myoglobin-TBHP-catalysed oxidation of u r i c acid was assayed i n 50 mM phosphate buffer, pH 7.0, containing 2.0 mM sodium azide. Reaction mixtures, i n a f i n a l volume of 3 ml, contained 100 /zM u r i c acid and 2 6 /zM myoglobin, either i n the absence or presence of the t e s t compounds at increasing concentrations. The reaction was i n i t i a t e d by adding 100 /zl TBHP at a f i n a l concentration of 0.5 mM. Absorbance of the reaction mixture at 292 nm was measured 1 min a f t e r the addition of TBHP. After 40 min of incubation at room temperature, the absorbance was measured again. The rate of u r i c acid oxidation, expressed as nmoles/min, was calculated using a standard c a l i b r a t i o n curve and was constant over the incubation period. 56 2.3.14 S t a t i s t i c a l Analyses Comparisons between two means were performed u s i n g a 2-t a i l e d u n p a i r e d Student's t t e s t . M u l t i p l e comparisons among groups were done u s i n g one-way a n a l y s i s of v a r i a n c e (ANOVA) f o l l o w e d by Duncan's t e s t t o assess s p e c i f i c group d i f f e r e n c e a t P < 0.05. C o r r e l a t i o n between parameters was a s s e s s e d by c o r r e l a t i o n a n a l y s i s . A l t e r a t i o n s i n p i g e r y t h r o c y t e MDA over the treatment p e r i o d were a n a l y s e d by ANOVA with r e p e a t e d measures f o l l o w e d by Duncan's t e s t t o assess s p e c i f i c group d i f f e r e n c e a t P < 0.05. 57 3 . RESULTS 3 . 1 S u s c e p t i b i l i t y o f T i s s u e s t o P e r o x i d e C h a l l e n g e The s u s c e p t i b i l i t y of tissues (heart, l i v e r and kidney) to peroxide challenge was assessed i n terms of s e n s i t i v i t y to GSH depletion and the formation of TBARS, an i n d i r e c t index of l i p i d peroxidation, following i n v i t r o exposure of tis s u e homogenates to increasing concentrations of TBHP. As shown i n Figs. 1 and 2, the concentration dependence of these two processes, regardless of the tissue being examined, d i f f e r e d markedly, with GSH depletion being more se n s i t i v e and formation of TBARS occurring at concentrations of TBHP higher than those inducing maximal GSH depletion. Homogenates prepared from l i v e r tissues showed s i g n i f i c a n t l y higher GSH l e v e l s than those prepared from kidney or myocardial tissues, with the l a t t e r having the lowest values either i n the absence or presence of TBHP at a l l concentrations tested (Fig. 1). The s u s c e p t i b i l i t y of tissue homogenates to TBHP-induced formation of TBARS also d i f f e r e d markedly among tissues; l e v e l s of TBARS produced by l i v e r homogenates were greater than those of kidney or myocardial tissue, with the l a t t e r having the lowest values at f i n a l concentrations of TBHP greater than 0.5 mM (Fig. 2) . A c t i v i t i e s of antioxidant enzymes, namely catalase (CAT), Cu,Zn-superoxide dismutase (Cu,Zn-S0D), glutathione peroxidase (GPX) and glutathione reductase (GRD) 58 F i g . 1 TBHP-induced depletion of GSH i n t i s s u e homogenates prepared from non-ischemic t i s s u e s from r a b b i t . Assays were performed by in vitro incubation of tissue homogenate (10%, w/v) with increasing concentrations of TBHP, as described in Materials and Methods. Values were expressed as nmoles GSH/mg tissue. Each point represents mean ± SEM, with n = 5 animals for each tissue. The three tissues showed statistically significant (P < 0.05) differences from each other at all TBHP concentrations tested. 59 - • - HEART - • - LIVER - A - KIDNEY 10 i w • I 1 1 1 0.00 0.10 0.20 0.30 0.40 0.50 TBHP Concentration ( mM ) 60 F i g . 2 TBHP-induced formation of TBARS i n tissue homogenates prepared from non-ischemic tissues from rabbit. Assays were performed by in vitro incubation of tissue homogenate (10%, w/v) with increasing concentrations of TBHP, as described in Materials and Methods. TBARS content was expressed as absorbance at 532 nm. Each point represents mean ± SEM, with n = 5 animals for each tissue. The three tissues showed statistically significant (P < 0.05)a differences from each other at all TBHP concentrations tested with the exception of the liver and kidney at the lowest concentration of TBHP. - • - HEART - • - LIVER - A - KIDNEY 0.80 n 0 1 2 3 4 5 TBHP Concentration ( mM ) 62 were measured i n rabbit tissues. A l l the a c t i v i t i e s of myocardial antioxidant enzymes were s i g n i f i c a n t l y lower than those of l i v e r or kidney (Fig. 3-6) . The a c t i v i t i e s of CAT and Cu,Zn-SOD i n l i v e r and kidney were 7- and 2-fold, respectively, higher than those of myocardial tissue (Fig. 3,4). The highest a c t i v i t i e s of GPX and GRD were found i n l i v e r and kidney tissues, respectively, with the values s i g n i f i c a n t l y d i f f e r i n g from those of other tissues examined (Fig. 5,6). 3.2 Altered Antioxidant Capacity i n Ischemic/Reperfused  Myocardial Tissues The antioxidant capacity of myocardial tissue homogenates prepared from control, ischemic or ischemic/reperfused tissues, was assessed in terms of s u s c e p t i b i l i t y to GSH depletion and the formation of TBARS following in v i t r o oxidative challenge with increasing concentrations of TBHP. Myocardial tiss u e from animals subjected to a 40 min period of ischemia induced by l e f t circumflex coronary artery l i g a t i o n showed s i g n i f i c a n t decreases i n GSH le v e l s at a l l concentrations of TBHP tested (Fig. 7) . The small increases ( r e l a t i v e to SHAM-CON samples) in formation of TBARS were s t a t i s t i c a l l y s i g n i f i c a n t at the highest concentrations of TBHP tested ( i . e . , 5 and 10 mM) (Fig. 8). The foregoing ischemia-induced changes i n GSH and TBARS were greatly i n t e n s i f i e d by a subsequent 60 min period of reperfusion, with a l l values 63 F i g . 3 Catalase a c t i v i t i e s i n rabbit tissues. The a c t i v i t y of catalase (CAT) was measured using c y t o s o l i c f r a c t i o n prepared from t i s s u e homogenate, as described in Materials and Methods. CAT a c t i v i t y was expressed as K/mg t i s s u e , where K i s the rate constant. Values given are mean ± SEM, with n = 5 animals for each t i s s u e . The heart showed a s t a t i s t i c a l l y s i g n i f i c a n t (P < 0.05) difference from the l i v e r and kidney. 64 CAT M 4) 3 W V) Ui £ 0 . 8 0 n 0 . 4 0 -0 .00 Heart Liver Kidney 65 F i g . 4 Cu,Zn-Superoxide dismutase a c t i v i t i e s i n rabbit t i s s u e s . The activity of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) was measured using cytosolic fraction prepared from tissue homogenate, as described in Materials and Methods. Cu,Zn-SOD activity was expressed as Units/mg tissue. Values given are mean ± SEM, with n = 5 animals for each tissue. The heart showed a statistically significant (P < 0.05) difference from the liver and kidney. 66 3n Cu,Zn-SOD Ui £ <0 2 -1 -T Heart Liver Kidney 67 F i g . 5 G l u t a t h i o n e p e r o x i d a s e a c t i v i t i e s i n r a b b i t t i s s u e s . The a c t i v i t y of glutathione peroxidase (GPX) was measured using c y t o s o l i c f r a c t i o n prepared from t i s s u e homogenate, as described in Materials and Methods. GPX a c t i v i t y was expressed as nmoles/NADPH/min/mg t i s s u e . Values given are mean ± SEM, with n = 5 animals for each t i s s u e . A l l the three tissues showed s t a t i s t i c a l l y s i g n i f i c a n t (P < 0.05) differences from each other. 68 CP 3 CO CO 20n GPX O ) E 1 5 " c E 1 0 -CL Q < CO o E Heart Liver Kidney 69 F i g . 6 G l u t a t h i o n e r e d u c t a s e a c t i v i t i e s i n r a b b i t t i s s u e s . The a c t i v i t y of glutathione reductase (GRD) was measured using c y t o s o l i c f r a c t i o n prepared from t i s s u e homogenate. GRD a c t i v i t y was expressed as nmoles NADPH/min/mg t i s s u e . Values given are mean ± SEM, with n = 5 animals for each t i s s u e . A l l the three tissues showed s t a t i s t i c a l l y s i g n i f i c a n t (P < 0.05) differences from each other. 70 GRD 3 (0 09 O ) £ OL Q < 0) 4) O £ c 8 n 6 -4 -2 " Heart Liver Kidney 71 F i g . 7 TBHP-induced depletion of GSH i n myocardial t i s s u e homogenates following a 40 min period of ischemia with or without subsequent reperfusion for 60 min i n ra b b i t s . Assays were performed by in vitro incubation of tissue homogenate (10%, w/v) prepared from control (non-ischemic, sham-operated), ischemic and/or reperfused myocardial tissue with increasing concentrations of TBHP. Values were expressed as nmoles GSH/mg tissue. Each point represents mean ± SEM, with n = 5 animals in each case. ' The three experimental groups showed statistically significant (P < 0.05) differences from each other at all TBHP concentrations tested with the exception of the SHAM-CON (non-ischemic) and ISC/NON (ischemic/non-reperfused) samples in the absence of TBHP. 72 TBHP Concentration ( mM ) 73 F i g . 8 TBHP-induced TBARS formation i n myocardial tissue homogenates following a 40 min period of ischemia with or without subsequent reperfusion for 60 min i n rabbits. Assays were performed by in v i t r o incubation of t i s s u e homogenate (10%, w/v) prepared from control (non-ischemic, sham-operated), ischemic and/or reperfused myocardial t i s s u e with increasing concentrations of TBHP, as described in Materials and Methods. TBARS content was expressed as absorbance at 532 nm. Each point represents mean ± SEM, with n = 5 animals in each case. The three experimental groups showed s t a t i s t i c a l l y s i g n i f i c a n t (P < 0.05) differences from each other at a l l TBHP concentrations tested with the exception of the SHAM-CON and ISC/NON samples at the three lowest concentrations of TBHP. 74 S H A M - O ISC / ISC / C O N NON REP 0.80 -1 TBHP Concentration ( mM ) 75 d i f f e r i n g s i g n i f i c a n t l y from those for non-ischemic and ischemic, non-reperfused tissues both i n the absence and presence of TBHP challenge (Fig. 7,8). 3.3 A l t e r a t i o n s i n M y o c a r d i a l A n t i o x i d a n t C a p a c i t y i n  R a b b i t s S u b j e c t e d t o I n c r e a s i n g P e r i o d s o f I s c h e m i a The time-course of al t e r a t i o n s i n myocardial antioxidant capacity was examined i n animals subjected to increasing periods of coronary artery l i g a t i o n with or without subsequent reperfusion (60 min). F i f t y - f i v e animals were randomly assigned to one of the following groups consisting of 5 animals each: SHAM-CON : sham-operated animals, no ischemia; ISC/NON (5 groups) : 5, 10, 20, 40 and 60 min period of ischemia, respectively, without reperfusion; ISC/REP (5 groups) : 5, 10, 20, 40 and 60 min of ischemia, respectively, followed by 60 min of reperfusion. GSH depletion and formation of TBARS were estimated following i n v i t r o exposure of tissue homogenates to TBHP at f i n a l concentrations of 0.05 and 2.5 mM, respectively. During the course of ischemia (Fig. 9, s o l i d l i n e s ) , an enhanced GSH s u s c e p t i b i l i t y to oxidation was present from the e a r l i e s t time point examined (5 min), while the increase in the formation of TBARS did not become s i g n i f i c a n t u n t i l 60 min p o s t - l i g a t i o n (Fig. 10, s o l i d line) . These e f f e c t s , suggestive of an impairment in myocardial antioxidant capacity, were greatly i n t e n s i f i e d by post-ischemic reperfusion (Fig. 9,10, dotted l i n e s ) . After 76 F i g . 9 Changes i n s u s c e p t i b i l i t y of myocardial t i s s u e to TBHP-induced depletion of GSH a f t e r varying periods of coronary artery l i g a t i o n with or without 60 min of reperfusion i n ra b b i t s . Assays were performed by in vitro incubation of tissue homogenate (10%, w/v) prepared from ischemic and/or reperfused myocardial tissue, as described in Materials and Methods. GSH depletion was expressed as % decrease when compared with control incubation (i.e., in the absence of TBHP). Values given are %GSH depletion measured at a final concentration of TBHP of 0.05 mM. Each point represents mean ± SEM, with n = 5 animals in each case. * denotes significant (P < 0.05) difference when compared with the corresponding ISC/NON animals. GSH Ligation time ( min ) 78 F i g . 10 Changes i n s u s c e p t i b i l i t y of myocardial tissue to TBHP-induced formation of TBARS af t e r varying periods of coronary artery l i g a t i o n with or without 60 min of reperfusion i n rabbits. Assays were performed by in v i t r o incubation of t i s s u e homogenate (10%, w/v) prepared from ischemic and/or reperfused myocardial t i s s u e with increasing concentrations of TBHP. TBARS content was expressed as absorbance at 532 nm. Values given are TBARS formation measured at a f i n a l concentration of TBHP of 2.5 mM. Each point represents mean ± SEM, with n = 5 animals in each case. denotes s i g n i f i c a n t (P < 0.05) difference when compared with the corresponding ISC/NON animals. 79 TBARS ISC /NON I S C / R E P M CO 0.80*1 0 .60 * T (0 4> o c <0 £k k. o <0 < 0 .40 i 0 .20 0 .00 2 0 4 0 —i 6 0 Ligation time ( min ) 80 an i n i t i a l period suggesting an enhancement of tissue antioxidant capacity, TBHP-induced GSH depletion showed a s t r i k i n g increase above the value i n the presence of ischemia alone between 20 and 40 min of ischemia (Fig. 9). In t h i s same time i n t e r v a l , the formation of TBARS also increased dramatically (Fig. 10). 3.3.1 E f f e c t s of Chronic A l l o p u r i n o l Pretreatrnent The e f f e c t s of chronic a l l o p u r i n o l pretreatrnent (1 mg/ml i n drinking water at an estimated d a i l y dose of 75 mg/kg) on I/R-induced alt e r a t i o n s i n myocardial s u s c e p t i b i l i t y to in v i t r o oxidative challenge with TBHP were examined. S i g n i f i c a n t protection against GSH depletion (Fig. 11) and l i p i d peroxidation (Fig. 12) was seen in the absence of any detectable a l t e r a t i o n s i n tissue l e v e l s of ATP (Fig. 13) and GSH (data not shown). A l l o p u r i n o l treatment did not a l t e r the myocardial GSH level s of control ( i . e . , non-ischemic) tissue or i t s s e n s i t i v i t y to TBHP-induced GSH depletion and formation of TBARS (Table I ) . 3.3.2 Eff e c t s of Acute A l l o p u r i n o l and Oxypurinol  Pretreatrnent A l l o p u r i n o l administered acutely as a single intravenous bolus (50 mg/kg) 1 hr p r i o r to the induction of ischemia was found to have no s i g n i f i c a n t protective e f f e c t on the enhanced GSH depletion or formation of TBARS resu l t i n g from 30 min of coronary artery l i g a t i o n followed 81 F i g . 11 E f f e c t of chronic a l l o p u r i n o l treatment on the s u s c e p t i b i l i t y of myocardial t i s s u e to TBHP-induced depletion of GSH a f t e r a 30 min period of coronary artery l i g a t i o n followed by 60 min of reperfusion i n r a b b i t s . Animals were treated with allopurinol (given orally 1 mg/ml in drinking water for 7 days at an estimated daily dose of 75 mg/kg) or vehicle prior to coronary artery ligation. GSH depletion, expressed as % decrease, was measured at a final concentration of TBHP of 0.05 mM, as described in Materials and Methods. Values are given as mean ± SEM, with n = 8 animals in each case. * denotes significant (P < 0.05) difference when compared with the corresponding ISC/REP animals in the absence of drug treatment. 83 F i g . 12 E f f e c t of chronic a l l o p u r i n o l treatment on the s u s c e p t i b i l i t y of myocardial t i s s u e to TBHP-induced formation of TBARS a f t e r a 30 min period of coronary artery l i g a t i o n followed by 60 min of reperfusion i n ra b b i t s . Animals were treated with allopurinol or vehicle as described in Fig. 11. TBARS formation, expressed as absorbance at 532 nm, was measured at a final concentration of TBHP of 2.5 mM. Values are given as mean ± SEM, with n = 8 animals in each case. * denotes significant (P < 0.05) difference when 84 TBARS 0.60-1 85 F i g . 13 E f f e c t of chronic a l l o p u r i n o l treatment on the t i s s u e ATP l e v e l a f t e r a 30 min period of coronary ar t e r y l i g a t i o n followed by 60 min of reperfusion i n r a bbits Animals were treated with allopurinol or vehicle as described in Fig. 11. ATP content, expressed in nmoles/mg tissue, was measured in transmural myocardial tissue, as described in Materials and Methods. Values are given as mean ± SEM, with n = 8 animals in each case. 86 4 i ATP 4> 3 <0 O ) £ 0> 4) O £ 2" S H A M - C O N ISC/REP ISC/REP ALP-Treated 87 by a 60 min period of reperfusion. Oxypurinol, the active metabolite of a l l o p u r i n o l , was also devoid of protective a c t i v i t y under these conditions (Table I I ) . 3.3.3 E f f e c t s of Chronic A l l o p u r i n o l Treatment on the  A c t i v i t y of Myocardial Antioxidant Enzymes As an approach to elucidating the molecular basis of the protective e f f e c t s of chronic a l l o p u r i n o l pretreatment, the e f f e c t s of t h i s treatment regimen on the a c t i v i t y of myocardial antioxidant enzymes, namely CAT, Cu,Zn-SOD, GPX and GRD, were examined. As shown i n Table III, no s i g n i f i c a n t changes were apparent except for GRD. The a c t i v i t y of myocardial GRD i n the a l l o p u r i n o l - t r e a t e d animals was s i g n i f i c a n t l y increased (by approximately 30% r e l a t i v e to vehicle-treated c o n t r o l s ) . 3.3.4 E f f e c t s of Acute U74006F Treatment on Ischemia/  Reperfusion-Induced Alterations i n Rabbit Myocardium Twenty-five rabbits were randomly assigned to one of the following groups consisting of 5 animals each: Group 1 : no ischemia, treated with drug vehicle only; Group 2 : no ischemia, treated with U74006F; Group 3 : ischemia (40 min) and reperfusion (60 min), treated with drug vehicle only; Group 4 : ischemia and reperfusion, treated with U74006F p r i o r to coronary l i g a t i o n ; Group 5 : ischemia and reperfusion, treated with U74006F p r i o r to the onset of reperfusion. 8 8 T a b l e I E f f e c t s o f c h r o n i c a l l o p u r i n o l t r e a t m e n t on m y o c a r d i a l a n t i o x i d a n t c a p a c i t y . B a s a l GSH (nmoles/mg t i s s u e ) % GSH D e p l e t i o n TBARS F o r m a t i o n ( O D 5 3 2 ) c o n t r o l 2.18 + 0.02 67 + 5.0 0.23 + 0.006 ALP-t r e a t e d 2.08 + 0.06 63 + 4.7 0.24 + 0.013 Animals were treated with a l l o p u r i n o l or vehicle, as described in Fig. 11. GSH depletion and TBARS formation were measured at f i n a l concentrations of TBHP of 0.15 and 2.5 mM, respectively. A l l values are given as mean ± SEM, with n = 5 in each group. 8 9 Table II Ef f e c t s of acutely administered a l l o p u r i n o l or oxypurinol (50 mg/kg, i.v.) on I/R injury. Treatment Basal GSH % GSH TBARS (nmoles/mg tissue) Depletion Formation (OD 5 3 2) Sham-control 2.39+0.12 23+6.0 0.21 ± 0.009 (no ischemia) none 1.56 + 0.10 51 + 8.7 0.38 + 0.047 a l l o p u r i n o l 1.67 + 0.10 45 + 12.5 0.31 + 0.058 oxypurinol 1.84+0.12 54+8.9 0.32+0.04 6 Drugs were administered as a single dose 1 hr prior to a 30 min period of coronary artery ligation followed by 60 min of reperfusion. Measurement of GSH depletion and TBARS formation used final TBHP concentrations of 0.075 and 2.5 mM, respectively. All values are given as mean ± SEM, with n = 9 in all experimental groups except for sham-control with n = 5. Table III E f f e c t s of chronic a l l o p u r i n o l treatment on the a c t i v i t y of myocardial antioxidant enzymes. CAT Cu,Zn-SOD GPX GRD control 0.0006 +0.0002 0. 59 + 0.03 2 + 0 7 2 1.5 + 0.04 ALP-treated 0.0004 +0.0001 0.62 + 0. 15 3 + 0 1 + 0 The a c t i v i t y of CAT (K/mg t i s s u e ) , Cu,Zn-SOD (units/mg t i s s u e ) , GPX (nmoles NADPH/min/mg tissue) and GRD (nmoles NADPH/min/mg tissue) were measured in l e f t v e n t r i c u l a r tissues. A l l values are given as mean ± SEM, with n - 5 in each group. * s i g n i f i c a n t l y d i f f e r e n t from control. 91 In the absence of coronary artery l i g a t i o n , treatment with U74006F had no d i s c e r n i b l e e f f e c t s on any of the biochemical c h a r a c t e r i s t i c s examined (Table IV). A 40 min period of ischemia followed by 60 min of reperfusion produced s i g n i f i c a n t decreases in mitochondrial ATPase a c t i v i t y and in t i s s u e l e v e l s of ATP and GSH as well as a marked reduction i n myocardial antioxidant capacity ( i . e . , enhanced s u s c e p t i b i l i t y to TBHP-induced GSH depletion and formation of TBARS). With the exception of mitochondrial ATPase i n a c t i v a t i o n , none of these I/R-induced a l t e r a t i o n s were influenced by U74006F pretreatment. When U74006F was administered p r i o r to the induction of ischemia, the mitochondrial ATPase showed a small degree of protection which did not quite a t t a i n s t a t i s t i c a l s i g n i f i c a n c e . However, when U74006F was given shortly before the onset of reperfusion, the a c t i v i t y of t h i s s u bcellular marker enzyme did not d i f f e r from that i n non-ischemic samples (Table IV). 3 . 4 Time-Course of Alterations i n Myocardial Antioxidant  Capacity During Post-ischemic Reperfusion In order to investigate whether or not I/R-related a l t e r a t i o n s i n myocardial antioxidant capacity correlate temporally with the burst of oxygen r a d i c a l formation which occurs within the f i r s t few minutes of post-ischemic reperfusion [78,80], the time-course of a l t e r a t i o n s in myocardial antioxidant capacity was examined in rabbits subjected to a fixed (40 min) period of coronary artery Table IV Effects of U74006F pretreatment on I/R-induced a l t e r a t i o n s i n rabbit myocardium. Experimental M-ATPase ATP GSH % GSH TBARS group (umol Pi/hr/ (nmol/mg tissue ) Depletion Formation mg Protein) (OD532) Group 1 266+20 3 . 83 + 0. 21 2 . 07 + 0. 04 19 + 4 . 5 0 . 21 + 0. 008 Group 2 2 7 5 + 9 4 . 06 + 0 . 16 2 . 13 + 0 . 08 22 + 5 . 8 0 . 21 + 0. 005 Group 3 2 0 6 + l l a 0 . 54 + 0. l l a 1. 06+0 . 0 6 a 72 + 5 . 2 a 0 . 57 + 0 . 0 6 2 a Group 4 2 3 0 + l l a 0 . 4 9 ± 0 . 0 8 a 1. 06 + 0 . 0 6 a 72 + 1. 8 a 0 . 49 + 0. 0 5 1 a Group 5 252 + 7 0 . 67 + 0 . 0 7 a 1. 23 + 0 . 0 9 a 6 6+6 . 2 a 0 . 51 + 0 . 0 6 3 a Measurements of GSH depletion and TBARS formation in v i t r o used f i n a l TBHP concentrations of 0.075 and 2.5 mM, respectively. Values are given as mean ± SEM ,for n = 5 in each group. See Materials and Methods for i d e n t i f i c a t i o n s of Group 1 to Group 5 . a - s i g n i f i c a n t l y d i f f e r e n t from corresponding non-ischemic value (i.e. Group 1 and Group 2) 93 l i g a t i o n followed by increasing periods of reperfusion. F i f t y - f o u r rabbits were randomly assigned to one of the following groups consisting of 6 animals each: SHAM- CON : sham-operated, no ischemia; ISC/NON : ischemia without reperfusion; ISC/REP : (7 groups) - ischemia followed by reperfusion for 1, 5, 15, 30, 60, 90 and 120 min, respectively. Myocardial antioxidant capacity was not altered with time i n sham-operated animals, even though the duration of the experiment was between 2 and 3 hours (data not shown). Figures 14, 15 and 16 show the basal tissue GSH le v e l s , GSH depletion at 0.075 mM ( f i n a l concentration) TBHP and TBARS formation at 2.5 mM TBHP as a function of reperfusion time. As seen i n these figures, myocardial GSH lev e l s and s u s c e p t i b i l i t y to TBHP-induced GSH depletion and formation of TBARS changed only s l i g h t l y a f t e r 40 min of ischemia and none of these alt e r a t i o n s attained s t a t i s t i c a l s i g n i f i c a n c e when compared with non-ischemic (SHAM-CON) samples. During the i n i t i a l 5 min of reperfusion, basal GSH lev e l s remained unchanged ( r e l a t i v e to SHAM-CON or ischemic/non-reperfused (ISC/NON) samples), but decreased s i g n i f i c a n t l y below those of SHAM-CON and ISC/NON tissues after 15 min of reperfusion (Fig. 14). A progressive decrease i n basal glutathione l e v e l s occurred a f t e r periods of reperfusion greater than 15 min, with a l l values being s i g n i f i c a n t l y d i f f e r e n t from those of SHAM-CON and ISC/NON tissues (Fig. 14). Moreover, during the i n i t i a l 15 min of reperfusion, the s u s c e p t i b i l i t y of myocardial tissue to 94 F i g . 14 Time-course of a l t e r a t i o n s i n myocardial GSH l e v e l s during the course of post-ischemic reperfusion i n ra b b i t s . Animals were subjected to increasing periods of reperfusion following a 40 min period of coronary artery ligation. Myocardial GSH level, expressed as nanomoles/mg tissue, was measured as described in Materials and Methods. Each point represents the mean ± SEM of values' from 6 animals. + - significantly different from SHAM-CON or ISC/NON animals * - significantly different from both SHAM-CON and ISC/NON animals. -40 0 40 80 120 Ligation Post-ligation reperfusion time (min) 96 F i g . 15 Time-course of alt e r a t i o n s i n GSH depletion of myocardial tissue during the course of post-ischemic reperfusion i n rabbits. Animals were subjected to increasing periods of reperfusion following a 40 min period of coronary artery l i g a t i o n . GSH depletion, expressed as % depletion, was measured as described in Materials and Methods. Measurement of GSH depletion used a f i n a l concentration of TBHP of 0.075 mM. Each point represents the mean ± SEM of values from 6 animals. + and * denote are as described in Fig. 14. 100 98 F i g . 16 Time-course of al t e r a t i o n s i n formation of TBARS i n myocardial tissue during the course of post-ischemic reperfusion i n rabbits. Animals were subjected to increasing periods of reperfusion following a 40 min period of coronary artery l i g a t i o n . TBARS formation, expressed as absorbance at 532 nm, was measured as described in Materials and Methods. Measurement of TBARS formation used a f i n a l concentration of TBHP of 2.5 mM. Each point represents the mean ± SEM of values from 6 animals. + and * are as described in Fig. 14. Ligation Post-ligation reperfusion time (min) 100 TBHP-induced GSH depletion was altered i n a complex manner (Fig. 15) . After reperfusion for 5 min, myocardial tissues became less susceptible to GSH depletion ( r e l a t i v e to ISC/NON samples); however, an abrupt increase in s u s c e p t i b i l i t y occurred 15 min af t e r the i n i t i a t i o n of reperfusion (Fig. 15). Reperfusion for periods longer than 15 min resulted i n a further enhancement in the s u s c e p t i b i l i t y to GSH depletion; maximal depletion was not attained u n t i l 90 min of reperfusion, with a l l values being s i g n i f i c a n t l y d i f f e r e n t from those of SHAM-CON and ISC/NON tissues (Fig. 15) . F i n a l l y , the i n i t i a l 5 min of reperfusion had minimal e f f e c t s on the formation of TBARS induced by TBHP, but reperfusion for periods longer than 5 min resulted i n a d r a s t i c increase; the maximal l e v e l of TBARS formed was not attained u n t i l 60 min af t e r the i n i t i a t i o n of reperfusion, with a l l values d i f f e r i n g s i g n i f i c a n t l y from those of SHAM-CON and ISC/NON tissues (Fig. 16) . 3.4.1 A l t e r a t i o n s i n t h e A c t i v i t i e s o f A n t i o x i d a n t Enzymes  i n I s c h e m i c / R e p e r f u s e d M y o c a r d i a l T i s s u e s As an approach to investigating the p o s s i b i l i t y of in a c t i v a t i o n or a transient impairment in the functioning of antioxidant enzymes during post-ischemic reperfusion, the a c t i v i t i e s of three antioxidant enzymes, namely, Cu,Zn-S0D, GPX and GRD were examined in ischemic/reperfused myocardial tissu e i n connection with the reperfusion time-course study. 101 Myocardial CAT was not examined because i t s a c t i v i t y in rabbit hearts (when correction i s made for contamination by blood-derived catalase) i s v i r t u a l l y undetectable under the assay conditions used. As shown in Table V , mean a c t i v i t i e s of Cu,Zn-S0D, GPX and GRD did not show any s i g n i f i c a n t change ( r e l a t i v e to SHAM-CON samples) following a 4 0 min period of ischemia, with or without subsequent reperfusion for periods up to 120 min. However, when co r r e l a t i o n analysis was performed using data from a l l the ischemic/reperfused rabbits (7 ISC/REP groups, n=42), the a c t i v i t i e s of Cu,Zn-SOD and GRD were found to correlate p o s i t i v e l y with basal GSH l e v e l s , but negatively with TBHP-induced GSH depletion and the formation of TBARS. No s i g n i f i c a n t correlations were found for GPX (Table VI). 3.4.2 pH Dependence of Antioxidant Enzyme A c t i v i t i e s The i n t r a c e l l u l a r pH of rabbit myocardium has been reported to decrease from 7.0 to 6.0 a f t e r 30 min of ischemia [177]. This might be an important factor in causing I/R injury, i f transient functional impairment i n the a c t i v i t y of antioxidant enzymes were to occur under these conditions. In order to explore t h i s p o s s i b i l i t y , the e f f e c t s of a c i d o t i c pH on antioxdant enzyme a c t i v i t i e s were examined. The a c t i v i t i e s of CAT, GPX and GRD were measured in hemolysates and homogenates of myocardial and l i v e r t issue, using buffers adjusted to pH 6.0 and pH 7.0, respectively. Because the measurement of Cu,Zn-SOD a c t i v i t y Table V Time-course of post-ischemic reperfusion injury i n rabbit myocardium: e f f e c t s on antioxidant enzymes. Reperfusion time (min) SHAM-CON ISC/NON 1 5 15 30 60 90 120 SOD 0.64 0.67 0.70 0.66 0.66 0.63 0.65 0.63 0.61 +0.03 +0.05 +0.03 +0.07 +0.05 +0.03 +0.03 +0.02 +0.04 GPX 2.5 2.5 2.3 2.4 2.5 2.4 2.4 2.3 2.8 +0.2 +0.1 +0.1 +0.2 +0.1 +0.2 +0.1 +0.1 +0.2 GRD 1.6 1.8 1.6 1.7 1.6 1.7 1.4 1.4 1.4 +0.2 +0.1 +0.2 +0.2 +0.1 +0.1 +0.1 +0.1 +0.1 The activities of Cu,Zn-SOD (Units/mg tissue), GPX ,• (nmoles NADPH/min/mg tissue) and GRD (nmoles NADPH.min/mg tissue) were measured as described in Materials and Methods. Values are given as mean ± SEM for n = 6. SHAM-CON and ISC/NON are the non-ischemic and ischemic/non-reperfused groups, respectively. 1 0 3 Table VI Correlations between the a c t i v i t i e s of antioxidant enzymes and antioxidant capacity of ischemic/reperfused rabbit myocardium. Basal TBHP-induced GSH l e v e l s GSH depletion Formation of TBARS Cu,Zn-SOD +0.48 -0.50 -0.43 (P < 0.001) (P < 0.001) (P < 0.004) GRD +0.54 -0.51 -0.55 (P <0.0005) (P < 0.001) (P < 0.0005) GPX +0.05 -0.09 -0.01 (P < 0. 750) (P<0.600) (P < 0.970) The r e l a t i o n s h i p between a c t i v i t i e s of antioxidant enzymes and antioxidant status was q u a n t i t a t i v e l y assessed by c o r r e l a t i o n analysis. The antioxidant capacity of ischemic/reperfused myocardial tissues was assessed in terms of s u s c e p t i b i l i t y to TBHP-induced GSH depletion and formation of TBARS (at f i n a l concentrations of TBHP of 0.075 and 2.5 mM, respectively) , and antioxidant enzyme a c t i v i t i e s were measured by in v i t r o assays, as described in Materials and Methods. Data given are c o r r e l a t i o n c o e f f i c i e n t s with corresponding p r o b a b i l i t i e s in parentheses. 104 was not f e a s i b l e under conditions of low pH, the e f f e c t of a c i d o t i c pH on the a c t i v i t y of t h i s enzyme was not examined. As shown in Table VII, the a c t i v i t i e s of antioxidant enzymes (except erythrocyte CAT) were suppressed to varying degrees. 3 . 5 B r i e f Episodes of Ischemia It has been shown that b r i e f episodes of ischemia can cause prolonged a l t e r a t i o n s i n myocardial u l t r a s t r u c t u r e , high energy phosphates and regional function [221]. Experimental and c l i n i c a l evidence concerning the occurrence of myocardial necrosis following such b r i e f episodes of ischemia (less than 20 min i n duration) has been inconsistent [178-180, 221]. The e f f e c t s of b r i e f episodes of ischemia on myocardial antioxidant capacity was examined in rabbits subjected to coronary artery l i g a t i o n . 3 . 5.1 E f f e c t s of Cumulative B r i e f Episodes of Ischemia on  Myocardial Antioxidant Capacity and ATP Levels Twenty-four rabbits were randomly assigned to one of the following groups consisting 6 animals each: SHAM- CON : sham-operated animals, no ischemia; Brief ISC/REP (1  cycle) : 10 min period of coronary artery l i g a t i o n followed by 15 min of reperfusion; Brief ISC/REP (4 cycles) : 10 min period of coronary artery l i g a t i o n followed by 15 min of reperfusion, 4 times; Data for ISC/REP ( 40 min period of coronary artery l i g a t i o n followed by 60 min of reperfusion) Table VII Effects of a c i d o t i c pH on antioxidant enzyme a c t i v i t i e s . Erythrocyte Liver Heart pH 7.0 6.0 % deer. 7.0 6.0 % deer. 7.0 6.0 % deer. CAT 0.080 0.081 -1.9 0.0085 0.0074* -13.4 N.D. N.D. N.D. +0.004 +0.004 +0.8 +0.0004 +0.0004 +2.3 GPX 38 14** 64 22 4.1** 82 2.3 1.6* 28 +2.7 +1.2 +1.5 +1.3 +0.1 +1.0 +0.1 +0.1 +5.0 GRD 4.8 2.8** 42 5.9 3.6** 39 1.4 0.90** 37 +0.3 +0.2 +3.0 +0.2 +0.2 +2.0 +0.1 +0.08 +0.4 The activities of CAT (K/mg Hb or mg tissue), GPX and GRD (nmoles NADPH/min/mg Hb or mg tissue) were measured in buffers adjusted to pH 7.0 and pH 6.0, respectively, using hemolysates, non-ischemic liver and left ventricular tissues. Values are given as mean ± SEM for n = 5. The effect of acidotic pH on enzyme activity is expressed as a percentage decrease relative to that at pH 7.0. N.D.= "Not-Determined" - CAT activity in the heart was undetectable under the assay conditions used. * = P < 0.01, ** = p < 0.001 when compared with the values at pH 7.0 106 animals were obtained from the previous study (see Table IV) Repetitive b r i e f episodes of ischemia (up to 4 cycles) had no d i s c e r n i b l e e f f e c t on myocardial GSH l e v e l s or on s u s c e p t i b i l i t y to TBHP challenge ( r e l a t i v e to SHAM-CON), except for a small but s i g n i f i c a n t decrease in s u s c e p t i b i l i t y to GSH depletion a f t e r one ischemic episode (Table VIII), while a prolonged ischemic i n s u l t followed by 60 min of reperfusion caused a s i g n i f i c a n t decrease in myocardial GSH l e v e l s , and an enhanced s u s c e p t i b i l i t y to TBHP-induced depletion of GSH and formation of TBARS. However, myocardial ATP l e v e l s were s i g n i f i c a n t l y decreased ( r e l a t i v e to SHAM-CON samples) a f t e r repeated exposure to b r i e f episodes of ischemia, with the extent of depletion depending on the number of exposures (Table VIII). Moreover, myocardial ATP l e v e l s measured following the prolonged ischemic i n s u l t were s i g n i f i c a n t l y lower than those measured a f t e r r e p e t i t i v e b r i e f episodes of ischemia, although the t o t a l duration of time during the period of ischemia and reperfusion was the same in a l l cases. No s i g n i f i c a n t changes i n the a c t i v i t i e s of myocardial Cu,Zn-SOD, GPX or GRD were seen following either b r i e f or prolonged episodes of ischemia followed by reperfusion (Table IX). Table VIII E f f e c t s of b r i e f cumulative episodes of ischemia on myocardial antioxidant capacity and ATP l e v e l s . Basal GSH % GSH TBARS ATP le v e l Depletion Formation (nmoles/mg tissue) (nmoles/mg tissue) (OD532) SHAM-CON 2.13+0.10 21+3.5 0.23+0.007 3.96+0.01 Brief ISC/REP 1.93+0.09 7+2.0a 0.24+0.016 2.71+0.23 (1 cycle) Brief ISC/REP 1.87+0.12 15+5.7 0.25±0.018 1.64+0.48a'b (4 cycles) ISC/REP 1.06+0.07° 73+4.7C 0.57+0.062° 0.54+0.11° Rabbits were subjected either to a 10 min period of coronary artery l i g a t i o n followed by 15 min of reperfusion (Brief ISC/REP, 1 or 4 cycles) or a 40 min period of l i g a t i o n with 60 min of reperfusion (ISC/REP) . GSH depletion and TBARS formation were measured at f i n a l concentrations of TBHP of 0.075 and 2.5 mM, respectively. Values are given as mean ± SEM for n = 6 in each group. a - s i g n i f i c a n t l y d i f f e r e n t from SHAM-CON. - s i g n i f i c a n t l y d i f f e r e n t from Brief ISC/REP (1 c y c l e ) . ° - s i g n i f i c a n t l y d i f f e r e n t from a l l groups. Table IX Ef f e c t s of b r i e f cumulative episodes of ischemia on myocardial antioxidant enzyme a c t i v i t i e s . Cu,Zn-SOD GPX GRD (Units/mg tissue) (nmoles NADPH/min/mg tissue) SHAM-CON 0.83+0.02 2.4+0.1 2.2+0.3 Brie f ISC/REP 0.76+0.03 2.6+0.2 1.6+0.2 (1 cycle) Brief ISC/REP 0.74+0.02 3.1+0.4 2.0+0.1 (4 cycles) ISC/REP 0.79+0.02 2.5+0.1 1.8+0.1 Myocardial a c t i v i t i e s of Cu,Zn-super oxide dismutase (Cu,Zn-SOD), glutathione peroxidase (GPX) and glutathione reductase (GRD) were measured as described in Materials and Methods. Values are given as mean ± SEM for n = 6 in each group. 109 3.5.2 E f f e c t s of Ischemic Preconditioning on I/R-related  Myocardial Alterations Preconditioning of pig myocardium by b r i e f episodes of ischemia has been shown to reduce the extent of I/R injury following a subsequent prolonged period of ischemia and reperfusion [181]. In order to investigate whether ischemic preconditoning produces any ef f e c t s on the I/R-related a l t e r a t i o n i n myocardial antioxidant capacity, the following study was undertaken. Eighteen rabbits were randomly assigned to one of the following groups consisting of 6 animals each: SHAM-CON : sham-operated animals, no ischemia; PRECON : r e p e t i t i v e b r i e f episodes of ischemia (5 min period of coronary artery l i g a t i o n followed by 5 min of reperfusion), 4 times; PRECON+ISC/REP : r e p e t i t i v e b r i e f episodes of ischemia (as i n the preceding group) followed by a 40 min period of coronary artery l i g a t i o n with 60 min of reperfusion. ISC/REP : 40 min period of coronary artery l i g a t i o n followed by 60 min of reperfusion. Repetitive episodes of ischemia produced no detectable e f f e c t s on myocardial GSH level s or on s u s c e p t i b i l i t y to TBHP challenge; however, tissue ATP le v e l s were s i g n i f i c a n t l y decreased ( r e l a t i v e to SHAM-CON samples) (Table X). When the ischemic preconditioning was followed by a 40 min period of coronary artery l i g a t i o n with 60 min of reperfusion, a s i g n i f i c a n t decrease ( r e l a t i v e to SHAM-CON and PRECON samples) i n myocardial GSH le v e l s and an enhanced Table X Ef f e c t s of ischemic preconditioning on I/R-induced myocardial a l t e r a t i o n s . Basal GSH % GSH TBARS ATP le v e l Depletion Formation (nmoles/mg tissue) (nmoles /mg tissue) (OD532) SHAM-CON 2 . 19 + 0 . 10 10 0.23 + 4.7 +0.007 3.96 + 0 . 01 PRECON 1. 95 + 0 . 11 10 0.23 + 3.0 +0.009 2 . 47c + 0. 29 PRECON + ISC/REP 1. 57 + 0 . 16 a, b 58 a' b 0.50 a' b +12.0 +0.102 0.4 3 a ' b + 0.21 ISC/REP 1.44a''b -0 . 05 55a,b + 11. 8 0 . 34 0 . 040 0.S2 a ' b + 0. 11 GSH depletion and TBARS formation were measured at f i n a l concentrations of TBHP of 0.1 and 2.5 mM, respectively. Values are given as mean ± SEM for n = 6 in each group. a - s i g n i f i c a n t l y d i f f e r e n t from SHAM-CON. b - s i g n i f i c a n t l y d i f f e r e n t from PRECON. Table XI Effects of ischemic preconditioning on myocardial antioxidant enzyme a c t i v i t i e s . Cu,Zn-SOD GPX GRD (Units/mg tissue) (nmoles NADPH/min/mg tissue) SHAM-CON 0 . 8 5 5 + 0 . 0 3 2 2 . 4 2 + 0 . 0 6 2 . 1 8 + 0 . 2 5 PRECON 0 . 8 8 0 + 0 . 0 3 2 2 . 6 3 + 0 . 0 7 1 . 7 3 + 0 . 2 2 PRECON+ISC/REP 0.809±0.034 2 . 3 7 + 0 . 0 9 2.10+0.24 ISC/REP 0 . 8 6 1 + 0 . 0 6 7 2 . 5 1 + 0 . 1 2 1 . 7 5 + 0 . 1 1 Myocardial a c t i v i t i e s of Cu,Zn-superoxide dismutase (Cu,Zn-SOD), glutathione peroxidase (GPX) and glutathione reductase (GRD) were measured as described in Materials and Methods. Values are given as mean ± SEM for n=6 in each group. 112 s u s c e p t i b i l i t y to TBHP-induced depletion of GSH arid formation of TBARS occurred (Table X) . Myocardial ATP le v e l s were further reduced, with a l l values being s i g n i f i c a n t l y lower than those of SHAM-CON and PRECON samples. Ischemic preconditioning did not seem to a f f e c t the I/R-induced myocardial a l t e r a t i o n s when compared with those of non-preconditioned animals (Table X). No s i g n i f i c a n t changes in myocardial antioxidant enzyme a c t i v i t i e s were seen in PRECON or PRECON+ISC/REP tissues (Table XI). 3.6 I/R-Induced Alterations i n Antioxidant Capacity i n Isolated Lancrendorff-Perfused Hearts As an approach to investigating the role of blood elements involved in causing impairment i n myocardial antioxidant capacity during ischemia and reperfusion, i s o l a t e d buffered-perfused rabbit hearts were subjected to a 40 min period of coronary artery l i g a t i o n followed by 60 min of reperfusion. Eighteen rabbits were randomly assigned to one of the following groups consisting of 6 animals each: SHAM-CON (IN VITRO) : sham-operated Langendorff-perfused hearts, no ischemia; ISC/REP (IN VITRO) : Langendorff-perfused hearts, a 40 min period of coronary artery l i g a t i o n followed by 60 min of reperfusion; ISC/REP (IN  VIVO/IN VITRO) : animals subjected to a 30 min period of coronary artery l i g a t i o n i n vivo, with an additional 10 min of ischemia during Langendorff perfusion ( i . e . , in vitr o ) that was followed by 60 min of reperfusion i n v i t r o . Data 113 for SHAM-CON (IN VIVO) and ISC/REP (IN VIVO) (40 min period of coronary l i g a t i o n followed by 60 min of reperfusion) were obtained from the ischemia time-course studies (see Figs. 9 and 10). In is o l a t e d and Langendorff-perfused hearts, tissue GSH l e v e l s were s i g n i f i c a n t l y decreased by 40% (r e l a t i v e to SHAM-CON (IN VIVO) samples). This was associated with an enhancement i n tissue s u s c e p t i b i l i t y to TBHP-induced depletion of GSH but not to the formation of TBARS (Table XII). Following a 40 min period of ischemia with 60 min of reperfusion in v i t r o , myocardial GSH lev e l s as well as tissue s u s c e p t i b i l i t y to oxidative challenge remained unchanged ( r e l a t i v e to SHAM-CON (IN VITRO) samples). In contrast, a s i g n i f i c a n t decrease in myocardial GSH l e v e l s and an increase i n tissue s u s c e p t i b i l i t y to TBHP challenge occurred following ischemia and reperfusion in vivo ( i . e . , ISC/REP (IN VIVO) samples). When the hearts were subjected to a 3 0 min period of ischemia in vivo followed by an additional 10 min of ischemia i n v i t r o , reperfusion with buffer for 60 min i n v i t r o caused a s i g n i f i c a n t increase ( r e l a t i v e to SHAM-CON (IN VITRO) samples) in tissue s u s c e p t i b i l i t y to TBHP-induced formation of TBARS, while the tissue GSH lev e l s and t h e i r s u s c e p t i b i l i t y to TBHP-induced depletion remained r e l a t i v e l y unchanged when compared with those of SHAM-CON (IN VITRO) samples (Table XII). 114 Table XII I/R-induced al t e r a t i o n s i n antioxidant capacity i n in t a c t or i s o l a t e d Langendorff-perfused rabbit hearts, Basal GSH % GSH TBARS (nmoles/mg tissue) Depletion Formation (OD 5 3 2) SHAM-CON (IN VIVO) 2 .39 + 0 .12 13 + 3.9 0.21 +0.009 SHAM-CON (IN VITRO) 1.44c + 0.06 59 a + 2.1 0.20 +0.007 ISC/REP (IN VITRO) 1.33 + 0. 10 51 + 5.9 0.20'-+0.009 ISC/REP 1.14 (IN VIVO/ +0.13 IN VITRO) 58 + 1.6 0 . 21a' b ' C / ^ +0.022 ISC/REP (IN VIVO) 1. 00' + 0. 08 62 a + 6.3 0 . 53 a +0.043 Measurements of GSH depletion and TBARS formation used f i n a l TBHP concentrations of 0.05 and 2.5 mM, respectively. Values are given as mean ± SEM, with n = 6 in SHAM-CON, ISC/REP (IN VITRO) and ISC/REP (IN VIVO/IN VITRO) groups. a - s i g n i f i c a n t l y d i f f e r e n t from SHAM-CON (IN VIVO). b - s i g n i f i c a n t l y d i f f e r e n t from SHAM-CON (IN VITRO). c - s i g n i f i c a n t l y d i f f e r e n t from ISC/REP (IN VIVO). - s i g n i f i c a n t l y d i f f e r e n t from ISC/REP (IN VITRO). 115 3.7 Heart-Lung Transplantation As shown i n the previous section, chronic a l l o p u r i n o l treatment protected against I/R-induced impairment in myocardial antioxidant capacity i n rabbits. This observation and the fact that protective actions of a l l o p u r i n o l are demonstrable in a var i e t y of tissues [150,187,188] suggest i t s e f f e c t s on antioxidant status may be generalized and widespread. The e f f e c t s of chronic a l l o p u r i n o l treatment on the antioxidant capacity of erythrocytes and the functional consequences of ischemia and reperfusion were examined using a swine model of heart-lung transplantation. 3.7.1 E f f e c t s of A l l o p u r i n o l Pretreatment on the  Antioxidant Capacity of Pig Erythrocytes Donor and rec i p i e n t pigs were treated with a l l o p u r i n o l given o r a l l y at a d a i l y dose of 50 mg/kg for 5 days. The l a s t dose was given on the day of surgery. The s u s c e p t i b i l i t y of pig erythrocytes to in v i t r o oxidative challenge, as shown by the decrease in TBHP-induced MDA production, was s i g n i f i c a n t l y reduced by a l l o p u r i n o l pretreatment (Fig. 17). The pooled data shown in t h i s figure indicate that the protective e f f e c t of a l l o p u r i n o l was time- (or dose-) dependent, so that the mean reduction in MDA production (induced by TBHP at a f i n a l concentration of 0.5 mM) did not at t a i n s t a t i s t i c a l s i g n i f i c a n c e u n t i l day 3 (P < 0.02). I n h i b i t i o n of l i p i d peroxidation by 116 Fig. 17 Effect of allopurinol treatment on susceptibility of pig erythrocytes to l i p i d peroxidation. Measurement of erythrocyte MDA used a f i n a l concentration of TBHP of 0.5 mM. Each point represents the mean + SEM of values from 12 animals. * P < 0.02 r e l a t i v e to control (i.e. p r i o r to i n i t i a t i o n of drug administration). 117 0.80-O X 0.70-X 0.30 I 1 i 1 — — i Day 1 Day 2 Day 3 Day 4 Day 5 Time 118 a l l o p u r i n o l reached a maximal value of 33% by day 4 (P < 0.02 r e l a t i v e to baseline, but not s i g n i f i c a n t l y d i f f e r e n t from value at day 3). By day 5, erythrocyte MDA le v e l s were no longer s i g n i f i c a n t l y d i f f e r e n t from baseline l e v e l s ( i . e . , at day 1). When patterns from i n d i v i d u a l experimental animals are examined, however, the considerable degree of inter-animal v a r i a b i l i t y i n the time-course of allopurinol-induced protection against l i p i d peroxidation becomes immediately apparent (Table XIII). Thus, although maximal protection was usually attained by day 3 or 4 (7 out of 12 animals), some (3 out of 12) responded maximally on day 2 ( i . e . , a f t e r a single dose of allopurinol) while substantial decreases i n red c e l l l i p i d peroxidation were observed i n 2 animals on day 5. The differences observed could not be accounted for by variatio n s in the forced peroxidation assay, since r e p l i c a t e determinations agreed within + 5%. On the f i f t h day following the i n i t i a t i o n of a l l o p u r i n o l treatment, pigs were allocated to donor or re c i p i e n t categories for the heart-lung transplantation study. A convenient measure of lung functional i n t e g r i t y assessed 2 hr post-transplantation was the estimation of lung water content. This index of functional v i a b i l i t y correlated s i g n i f i c a n t l y with the extent of a l l o p u r i n o l protection against TBHP-induced l i p i d peroxidation in red c e l l s from ind i v i d u a l , donor (P < 0.005) and rec i p i e n t (P < 0.05) animals (Fig. 18). The lung water contents were Table XIII Inter-animal v a r i a t i o n i n allopurinol-induced protection against l i p i d peroxidation i n pig erythrocytes. [Heparinized venous blood samples were drawn before oral a l l o p u r i n o l administration at a dai l y dose of 50 mg/kg. The s u s c e p t i b i l i t y of erythrocytes to TBHP-induced l i p i d peroxidation was measured. Values given are percentage of control ( i . e . values at Day 1)]. Day 2 Day 3 Day 4 Day 5 1 56. 3 22 . 9 68 . 8 47 . 9 2 109 . 5 71.4 95 . 5 63 . 5 3 87 . 1 85.7 34.3 .. 75 . 7 4 78 . 0 60.0 32.0 74 . 0 5 60 . 0 80 . 0 85 . 5 49 . _3 6 76.2 104 . 8 100 . 0 90 . 5 7 87 . 2 76.6 29.8 74 . 5 8 97 . 2 93 . 0 32.4 83 . 1 9 66 . 2 73.5 95 . 6 108 . 8 10 63 . 5 71.4 95 . 2 103 . 2 11 122 . 4 70.1 53 . 7 108 . 9 12 116.9 77.9 84 . 9 105. 1 Susceptibility of erythrocytes to TBHP-induced lipid peroxidation was assessed as described in Materials and Methods. Underlined figures represent maximal protection. 120 F i g . 18 Correlation between erythrocyte MDA and lung water (LW) l e v e l s . The regression lines of donor ( % • ) and recipient pigs ( o M I I I M I I I I O ) erythrocyte MDA levels against LW levels. Red cell MDA was measured prior to surgery (i.e., on day 5) and LW was measured at 2 hr following post-transplantation reperfusion. 121 Donors r = 0.95, P < 0.005 Recipients r = 0.82, P < 0.05 1.00i 0.20 1 1 1 1 • , 0 200 400 600 800 1000 LW ( ml ) 122 found to correlate well with other indices of pulmonary-function, such as a r t e r i a l blood p0 2, a l v e o l a r - a r t e r i a l gradient (AaG) and a l v e o l a r - a r t e r i a l r a t i o (AaR) (data not shown). 3.8 Metal Chelating Properties of A l l o p u r i n o l A number of a l t e r n a t i v e mechanisms to xanthine oxidase i n h i b i t i o n in the protection by a l l o p u r i n o l against I/R injury have been proposed [162,164,165]. However, the chelation of t r a n s i t i o n metal ions, whose c a t a l y t i c actions in oxy-radical-mediated reactions are also important determinants i n the pathogenesis of oxidative t i s s u e damage [13,194], has not yet been considered. The metal chelating actions of a l l o p u r i n o l and i t s active metabolite oxypurinol were, therefore, examined i n i n v i t r o systems measuring the t r a n s i t i o n metal ion-catalysed oxidation of ascorbic acid and oxidation of erythrocyte membrane l i p i d s . 123 3 . 8 . 1 E f f e c t s of A l l o p u r i n o l and Oxypurinol on Transition  Metal Ion-Catalysed Oxidation of Ascorbic acid Since the mechanism of t r a n s i t i o n metal ion-catalysed ascorbate oxidation involves formation of an ascorbate-t r a n s i t i o n metal ion complex [225], t r a n s i t i o n metal chelators would be expected to i n t e r f e r e with t h i s i n t e r a c t i o n , thereby decreasing the reaction rate. Ascorbate oxidized spontaneously i n phosphate buffer in the absence of added t r a n s i t i o n metal ions (Fig. 19a). This basal ascorbate oxidation was decreased by a l l o p u r i n o l (100 f i n a l concentration). Cupric chloride, at a f i n a l concentration of 10 u-M, dramatically increased the rate of ascorbate oxidation (Fig. 19b) . A l l o p u r i n o l also i n h i b i t e d t h i s cupric ion-catalysed oxidation, in a concentration-dependent manner, with almost complete suppression at a f i n a l concentration of 100 izM. On the other hand, the addition of f e r r i c chloride, up to a f i n a l concentration of 100 juM, did not s i g n i f i c a n t l y a l t e r the rate of ascorbate oxidation (data not shown). When the oxidation of ascorbate was measured i n the presence of 100 /LAM EDTA, the oxidation rate was decreased from the basal value of 3.40 + 0.11 (SEM) nmoles/min (Fig. 19a) to 1.01 + 0.12 nmoles/min (Fig. 20). The addition of cupric chloride, at a f i n a l concentration of 10 and 30 xzM, did not stimulate ascorbate oxidation under these conditions (data not shown) , but when present at a f i n a l concentration of 100 iiM, cupric chloride s i g n i f i c a n t l y 124 F i g . 19 C u p r i c i o n - c a t a l y s e d o x i d a t i o n of a s c o r b a t e : e f f e c t s o f a l l o p u r i n o l . Ascorbate oxidation, as indicated by the decrease in absorbance at 280 nm, was monitored for 5 min in reaction mixtures containing 100 \xM a s c o r b a t e in 10 mM phosphate buffer (pH 7.4), either in the absence or presence of allopurinol, as described in Materials and Methods. Each point represents the mean value of three experiments, with the SEM < 5% of the mean. 125 £ c o 00 CM CO 4> O c (0 JQ o < 1.0-0.8 -0.6 0.4 0.2 H 0.0 1.0 0.8 0.6 H 0.4 0.2 0.0 0 uM CuCI - A - - A -2 a 100 uM ALP — • • c°ntroi 10 uM CuCI 1 ° 0 uM ALP ^ 0 ^ 7 *Lf> rot 60 120 180 240 300 Time (sec) 126 F i g . 20 C u p r i c and f e r r i c i o n - c a t a l y s e d o x i d a t i o n o f a s c o r b a t e i n t h e p r e s e n c e o f EDTA. Assays were performed as described in Materials and Methods. Rates of ascorbate oxidation, expressed in nanomoles/min, are given as mean ± SEM of three experiments. *, ** and *** denote significant (P < 0.05, P < 0.01 and P < 0.001, respectively) differences when compared with appropriate controls. * * • None SH ALP (50 uM) ^ ALP (500 uM) OXYP (50 uM) OXYP (500 uM) Uric acid (50 uM) Control 100 uM 10 uM 30 uM 100 uM Cupric chloride Ferric chloride 128 increased the rate of ascorbate oxidation to 17.2 ,+ 1.05 nmoles/min (Fig. 20). Both a l l o p u r i n o l and oxypurinol (500 /iM f i n a l concentrations) s i g n i f i c a n t l y i n h i b i t e d t h i s cupric ion-catalysed oxidation of ascorbate, reducing the oxidation rate to 22% and 16% of control, respectively. However, when a l l o p u r i n o l and oxypurinol were added at a f i n a l concentration of 50 tzM, only oxypurinol produced a s i g n i f i c a n t degree of i n h i b i t i o n . F e r r i c chloride, when added in the presence of 100 txM E D T A , stimulated the oxidation of ascorbate in a concentration-dependent manner to a maximum of 5.88 + 0.49 nmoles/min (Fig. 20). Both a l l o p u r i n o l and oxypurinol at f i n a l concentrations of 500 /LIM s l i g h t l y i n h i b i t e d the oxidation of ascorbate catalysed by 10 /iM f e r r i c chloride, but at increased concentrations of f e r r i c chloride, t h i s i n h i b i t o r y e f f e c t of a l l o p u r i n o l and oxypurinol was abolished. Uric acid, at a f i n a l concentration of 50 u-M, in h i b i t e d the cupric ion-catalysed oxidation of ascorbate, reducing the rate of oxidation to 63% of control, but had no s i g n i f i c a n t i n h i b i t o r y e f f e c t on f e r r i c ion-catalysed oxidation. 3.8.2 Transition Metal Ion-Catalysed Oxidation of  Erythrocyte Membrane Lipids Recently, considerable evidence has emerged suggesting the important role of l i p i d peroxidation i n the pathogenesis of many diseases, drug t o x i c i t i e s and I/R injury [3]. In the heart, l i p i d peroxidation of cardiomyocyte membranes induced 129 by reactive oxy-radicals generated during the reperfusion of ischemic t i s s u e may be responsible for the development of i r r e v e r s i b l e damage [169]. The involvement of iron i n both i n i t i a t i o n [222] and propagation [223] phases of l i p i d peroxidation reactions within b i o l o g i c a l membranes i s well established. Moreover, iron-dependent l i p i d peroxidation i s thought to play a c r u c i a l r ole i n pathologically relevant oxy-radical-induced tissue damage i n vivo [224]. Therapeutic interventions involving the use of agents that i n h i b i t iron-dependent l i p i d peroxidation may therefore represent a r a t i o n a l approach to the management of oxy-r a d i c a l - r e l a t e d diseases. The f e r r i c ion or cupric ion-TBHP-stimulated l i p i d peroxidation was examined in v i t r o using erythrocyte membranes as a l i p i d source. The ef f e c t s of a l l o p u r i n o l and oxypurinol on the t r a n s i t i o n metal ion-catalysed oxidation of membrane l i p i d s were also investigated. 3.8.2.1 F e r r i c Chloride-Induced Oxidation of Erythrocyte  Membrane Lipids When human erythrocyte membranes were incubated with increasing concentrations of f e r r i c chloride, an enhancement of l i p i d peroxidation was observed, as indicated by the formation of TBARS, an i n d i r e c t measure of l i p i d peroxidation (Fig. 21). The concentration-dependent increase reached a maximum value at 0.3 mM, which was 130 F i g . 21 F e r r i c ion - induced formation of TBARS i n erythrocyte membranes. Assays were performed as described in Materials and Methods. TBARS content was expressed as absorbance at 532 nm. Each point represents the mean ± SD of triplicate samples from one representative experiment. 131 0.50i 0.00 1 1 1 1 i . 0.00 0.10 0.20 0.30 0.40 0.50 Ferric Chloride Cone. ( mM ) 132 followed by a progressive decrease at higher concentrations of f e r r i c ion. 3 + The time-course of Fe -stimulated production of TBARS showed a t y p i c a l autocatalytic pattern, wherein the i n i t i a t i o n of l i p i d peroxidation was preceded by a 1 0 min lag phase a f t e r which l e v e l s of TBARS increased rapidly, a t t a i n i n g maximal le v e l s a f t e r approximately 9 0 min of incubation (Fig. 22). When GSH or ascorbic acid, at a f i n a l concentration of 5 0 /LIM, was added to the reaction mixture, an increased rate of TBARS formation was observed, with a shortening of the lag phase to less than 5 min (Fig. 22) . The stimulatory e f f e c t of ascorbic acid on the Fe -induced l i p i d peroxidation was greater than that of GSH, with the former causing a s i g n i f i c a n t increase in maximal TBARS production. 3.8.2.2 E f f e c t s of A l l o p u r i n o l and Oxypurinol on F e r r i c  Chloride-Stimulated Oxidation of Erythrocyte  Membrane Lipids Incubation of erythrocyte membranes with f e r r i c chloride increased the production of TBARS in a concentration-dependent manner (Fig. 23). Butylated hydroxytoluene (BHT) reduced the extent of f e r r i c ion-induced l i p i d oxidation, with almost complete i n h i b i t i o n at a f i n a l concentration of 4 /iM at a l l f e r r i c ion concentrations tested. Isopropanol, the solvent i n which the BHT was dissolved, had no e f f e c t i n t h i s system (data 133 F i g . 22 E f f e c t s of GSH and ascorbic acid on the time-course of f e r r i c ion - induced formation of TBARS i n erythrocyte membranes. Assays were performed as described in Materials and Methods. TBARS content was expressed as absorbance at 532 nm. GSH or ascorbic acid (ASC) was added to the reaction mixture at a f i n a l concentration of 50 / i M . 134 - • - CON - • - +GSH - A - +ASC 2.50T Incubation time ( min ) 135 F i g . 23 F e r r i c ion - induced formation of TBARS i n erythrocyte membranes: eff e c t s of a l l o p u r i n o l and oxypurinol. Assays were performed as described in Materials and Methods. TBARS content was expressed as absorbance at 532 nm. Values are given as mean ± SEM of at least three experiments. * and *** denote significant (P < 0.05 and P < 0.001, respectively) differences when compared with the control. 0.25 0.20 0.15 0.10 0.05 0 I I None II BHT (10 uM) ^ BHT (100 uM) • Uric acid (250 uM) • Uric acid (500 uM) • OXYP (500 uM) (9 ALP (500 uM) w Hi i i II i 1111 1111 1111 III 1 1 1 III III Tl'l i;; s" III Control Cupric tBHP chloride Cupric + tBHP chloride 137 not shown). EDTA at a f i n a l concentration of 0.4 mM strongly suppressed l i p i d oxidation induced by 0.2 mM f e r r i c chloride, but the i n h i b i t o r y action was almost completely overcome by increasing concentration of f e r r i c chloride to 0.4 mM (Fig. 23). Uric acid, when added at a f i n a l concentration of 2 mM, had a comparable i n h i b i t o r y e f f e c t to EDTA, but the higher concentration of f e r r i c chloride only p a r t l y overcame the i n h i b i t i o n (Fig. 23). A l l o p u r i n o l (4 mM) or oxypurinol (3 mM) s i g n i f i c a n t l y i n h i b i t e d l i p i d peroxidation induced by 0.2 mM f e r r i c chloride; however, at the higher concentration of f e r r i c chloride, the i n h i b i t o r y e f f e c t s of both these compounds were no longer s i g n i f i c a n t (Fig. 23) . None of these compounds interfered with the TBA color reaction (data not shown). 3.8.2.3 E f f e c t s of A l l o p u r i n o l and Oxypurinol on Cupric Ion- TBHP-Induced Oxidation of Erythrocyte Membrane  Lipids The peroxidation of erythrocyte membrane l i p i d s was stimulated by TBHP, as indicated by the production of TBARS (Fig. 24). This peroxidation was not affected by either a l l o p u r i n o l or oxypurinol. When TBHP was combined with cupric chloride, there was a marked increase in the extent of l i p i d peroxidation, even though cupric chloride alone produced no s i g n i f i c a n t e f f e c t on peroxidation (Fig. 24). A l l o p u r i n o l and oxypurinol at f i n a l concentrations of 500 /iM reduced t h i s enhanced peroxidation to 87% and 88% of 138 F i g . 24 Cupric ion-TBHP - induced formation of TBARS in erythrocyte membranes: ef f e c t s 'of a l l o p u r i n o l and oxypurinol. Assays were performed as described in Materials and Methods. Cupric chloride and TBHP were added at final concentrations of 0.2 mM and 10 mM, respectively. TBARS content was expressed as absorbance at 532 nm. The basal level of TBARS production in the reaction mixture was measured in the absence of both cupric chloride and TBHP. Values are mean ± SEM of at least three experiments. *, ** and *** denote significant (P < 0.05, P < 0.01 and P < 0.001, respectively) differences when compared with the control. E c eg CO LO •4—' a CD o c CTJ _Q v_ O CO JQ < 0.6 0.4 0.2 0 I I None i l BHT (0.4 uM) H BHT (4 uM) • EDTA (0.4 mM) Uric acid (2 mM) ALP (4 mM) OXYP (3 mM) Control 0.2 mM 0.4 mM Ferric chloride M W VO 140 control, respectively (Fig. 24), but no further decreases in TBARS l e v e l were observed at concentrations of a l l o p u r i n o l up to 2 mM. BHT strongly i n h i b i t e d l i p i d peroxidation induced by the cupric chloride-TBHP mixture, in a concentration-dependent manner, reducing the TBARS l e v e l in the presence of 100 juM BHT to below that produced by TBHP alone (Fig. 24). Uric acid also s i g n i f i c a n t l y i n h i b i t e d the cupric chloride-TBHP-induced peroxidation i n a concentration-dependent manner, with the production of TBARS being reduced to 72% of control at a f i n a l concentration of 500 izM (Fig. 24) . 3.8.3 UV Absorption Spectra of Allopurinol/Ascorbic Acid/  Copper ion UV spectral analysis has been used to demonstrate the formation of a complex between u r i c acid and copper ion involved in the i n h i b i t i o n by u r i c acid of the cupric ion-catalysed oxidation of ascorbate [50]. The possible complexation among a l l o p u r i n o l , t r a n s i t i o n metal ions and ascorbic acid was, therefore, examined in the present study using UV spectral analysis. The spectrum generated from a mixture of a l l o p u r i n o l and ascorbate (spectrum B i n Fig. 25a) was very s i m i l a r to the sum of the ind i v i d u a l spectra of a l l o p u r i n o l and ascorbic acid (data not shown). Cupric chloride, although i t did not appreciably a l t e r the spectrum of a l l o p u r i n o l (data not shown), generated a d i s t i n c t i v e spectrum in the 14.1 F i g . 25 UV absorption spectrum of the a l l o p u r i n o l -ascorbate-cupric c h l o r i d e mixture: e f f e c t s of EDTA. Spectra were recorded as described in Materials and Methods. Spectra in (a) were recorded from the allopurinol-ascorbate-cupric chloride (A, • ), allopurinol-ascorbate (3, ; and cupric chloride (C, — — ) mixtures, respectively. Spectra in (b) represent the allopurinol-ascorbate- cupric chloride-EDTA (A1, a l l o p u r i n o l (B', ) and ascorbate-cupric chloride-EDTA (C, — ; mixtures, r e s p e c t i v e l y . 142 206 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 Wavelength (nm) 143 presence of a l l o p u r i n o l and ascorbate (spectrum A in Fig. 25a). This spectrum showed a decrease i n absorption between 250 nm and 280 nm, presumably r e s u l t i n g from the oxidation of ascorbate. Moreover, there was a s l i g h t decrease and a moderate increase i n absorbances at 2 06 nm and 231 nm, respectively, and a s l i g h t increase in absorption between 280 nm and 340 nm, which became apparent when absorbances of the allopurinol-ascorbate and cupric chloride spectra were subtracted from those of the allopurinol-ascorbate-cupric chloride spectrum (see Table XIV). The addition of EDTA to the a l l o p u r i n o l -ascorbate-cupric chloride mixture abolished these spectral changes (Table XIV), with the production of a spectrum (lab e l l e d A 1 i n Fig. 25b) comparable to that expected for the simple addition i n d i v i d u a l absorbances for a l l o p u r i n o l and an ascorbate-cupric chloride-EDTA mixture (spectra B' and C in Fig. 25b). Similar changes in spectral c h a r a c t e r i s t i c s were observed following the addition of cupric chloride to an oxypurinol-ascorbate mixture (data not shown). In contrast, the addition of f e r r i c chloride to an allopurinol-ascorbate mixture merely resulted i n a spectrum s i m i l a r to that produced by a mixture of a l l o p u r i n o l and f e r r i c chloride (data not shown). 144 Table XIV Absorbance changes of the a l l o p u r i n o l - ascorbate cupric chloride mixture: e f f e c t s of EDTA. Changes i n absorbance 9 206 nm 231 nm 300 nm ALP-ASC-CU -0.1339 +0.0947 +0.0494 (+0.0370) (+0.0091) (+0.0126) ALP-ASC-CU-EDTA +0.0189* -0.0060* +0.0011* (+0.0610) (+0.0032) (±0.0012) - changes in absorbances were calculated by subtracting the absorbances both of B or B' and C or C spectra from those of A or A' spectrum, respectively (see also Fig. 31). Values are for given as mean ± SD for n = 3. * - P < 0.005 when compared with the ALP-ASC-Cu mixture. 145 3.9 Myoglobin-TBHP-Catalysed Oxidation of Uric Acid and  Erythrocyte Membrane l i p i d s Since myoglogin i s present i n cardiac muscle at a high concentration [197], i t s pro-oxidant action i n ischemic tiss u e [198-200] might also be important in t r i g g e r i n g uncontrolled oxidative processes. The use of pharmacological agents capable of attenuating the p o t e n t i a l l y harmful e f f e c t s of myoglobin might represent a r a t i o n a l approach to protecting against myocardial I/R injury. Myoglobin reacts with hydroperoxides with r e s u l t i n g formation of reactive oxidants which can oxidize u r i c acid [220] and b i o l o g i c a l membrane l i p i d s [202]. These myoglobin-dependent oxidative reactions were examined in  v i t r o by measuring the oxidation of u r i c acid and erythrocyte membrane l i p i d s , and the e f f e c t s of a l l o p u r i n o l and oxypurinol on these oxidative reactions were also investigated. 3.9.1 E f f e c t s of A l l o p u r i n o l and Oxypurinol on Myoglobin- TBHP-Catalysed Oxidation of Uric Acid In the presence of myoglobin (26 ziM f i n a l concentration), TBHP (0.5 mM) stimulated the rate of u r i c acid oxidation to 1.73 ± 0.07 (SEM) nmoles/min (Fig. 26). No s i g n i f i c a n t oxidation of u r i c acid was observed when TBHP was added in the absence of myoglobin, and vice versa (data not shown). This myoglobin-TBHP-catalysed oxidation of u r i c acid was strongly i n h i b i t e d by ascorbic acid and GSH in a 146 F i g . 26 Myoglobin-TBHP - catalysed oxidation of u r i c acid: e f f e c t s of a l l o p u r i n o l and oxypurinol. Assays were performed as described in Materials and Methods. Rate of uric acid oxidation was expressed as nmoles/min. Values are given as mean ± SEM for n = 3. *, ** and *** denote significant ( P < 0.05, P < 0.01 and P < 0.001, respectively) differences when compared with the control. ASC - ascorbic acid GSH - reduced glutathione ALP - allopurinol OXYP - oxypurinol n m o l e s / m i n o o ro > cn o H * * * 0.1 mM H * * * LOmM w X * * * 0.1 mM H * * * LOmM > r-H * 0.5mll H * * LOmM O X •< -I # O.SmM H * * LOmM 148 concentration-dependent manner (Fig. 26). Both a l l o p u r i n o l and oxypurinol i n h i b i t e d the rate of u r i c acid oxidation, reducing the oxidation rate to 80% and 82% of control, respectively, at f i n a l concentrations of 1 mM (Fig. 26). 3.9.2 E f f e c t s of A l l o p u r i n o l on Myoqlobin-TBHP-Induced  Oxidation of Erythrocyte Membrane Lipids Incubation of erythrocyte membranes with the myoglobin-TBHP mixture (26 /zM and 1.0 mM f i n a l concentrations, respectively) resulted in a time-dependent increase in the formation of TBARS, an i n d i r e c t index of l i p i d peroxidation (Fig. 27) . This myoglobin-TBHP-induced formation of TBARS approached a maximum after approximately 110 min of incubation. Neither myoglobin nor TBHP, when added alone, caused any detectable formation of TBARS (data not shown). The i n h i b i t o r y e f f e c t s of the test compounds on the myoglobin-TBHP-induced peroxidation of erythrocyte membrane l i p i d s were examined in reaction mixtures incubated for a period of 30 min (during which time period the rate of TBARS formation in control samples was l i n e a r , see Fig. 27). BHT in h i b i t e d the peroxidation of membrane l i p i d s in a concentration-dependent manner, s i g n i f i c a n t l y reducing the TBARS produced to 21% of control at a f i n a l concentration of 0.1 mM (Fig. 28). Al l o p u r i n o l , when added at a f i n a l concentration of 2 mM, s i g n i f i c a n t l y reduced the formation of TBARS to 90% of control, but the i n h i b i t i o n was not 149 F i g . 27 Time-course of myoglobin-TBHP-induced formation of TBARS i n erythrocyte membranes. Assays were performed as described in Materials and Methods. TBARS content was expressed as absorbance at 532 nm. Each point represents the mean for n = 3, with the SD < 5% of the mean. 150 Incubation time (min) 151 F i g . 28 Myoglobin-TBHP-induced formation of TBARS i n erythrocyte membranes: e f f e c t of a l l o p u r i n o l . Assays were performed as described in Materials and Methods. TBARS content was expressed as absorbance at 532 nm. Values are given as mean ± SEM from three experiments using d i f f e r e n t membrane preparations. *, ** and *** denote s i g n i f i c a n t (P < 0.05, P < 0.01 and P < 0.001, respectively) differences when-, compared with the control. BHT - butylated hydroxytoluene ALP - a l l o p u r i n o l URC - u r i c acid Absorbance at 5 32 nm o • o o p io _JL_ O cn o - j o o z m z H H 0 .01mM * * * 0.1mM > r-H I .OmM H * 2 .0mM C 30 O * * 0.1mM fo 153 s i g n i f i c a n t when added at a f i n a l concentration of 1 mM (Fig. 28). Uric acid also i n h i b i t e d the l i p i d peroxidation at a f i n a l concentration of 0.1 mM, reducing the TBARS produced to 83% of control (Fig. 28). 154 4. DISCUSSION 4.1 Assessment of Tissue Antioxidant Capacity Tert-butylhydroperoxide (TBHP) has previously been used as a model hydroperoxide i n studying the e f f e c t s of oxidative stress i n in t a c t c e l l s or organs, such as heart, l i v e r and kidney [226]. Its s t a b i l i t y during storage and high s o l u b i l i t y i n aqueous phases have permitted i t s ap p l i c a t i o n in a variety of in v i t r o systems involving the study of free radical-mediated processes [227-229]. One such a p p l i c a t i o n was exploited i n the present investigation in that the antioxidant capacity of tissues was assessed by measuring s e n s i t i v i t y of tissue homogenates to glutathione (GSH) depletion and formation of t h i o b a r b i t u r i c a c i d -reactive substances (TBARS) following i n v i t r o incubation with TBHP. Presumably, oxy-radicals generated from the hemoprotein-catalysed decomposition of TBHP molecules [229,230] can react with antioxidant molecules present in the t i s s u e homogenate, and i n i t i a t e free radical-chain reactions, such as the peroxidation of polyunsaturated l i p i d components of b i o l o g i c a l membranes. The oxidative reactions i n i t i a t e d by TBHP could induce GSH oxidation, as indicated by the concentration-dependent decrease i n GSH content i n tissue homogenates prepared from heart, l i v e r and kidney tissues (Fig. 1). Increasing concentrations of TBHP, which presumably overwhelmed the antioxidant capacity of tissue homogenates, caused' an 155 increase i n formation of TBARS, an i n d i r e c t index of l i p i d peroxidation (Fig. 2). Under the conditions employed in t h i s in v i t r o assay, glutathione peroxidase (GPX) present in the t i s s u e homogenate would probably be inactivated in the presence of TBHP [300]. It i s , therefore, u n l i k e l y that the depletion of GSH i s a r e s u l t of GPX-dependent enzymatic degradation of TBHP. This i s strengthened by the finding that l i v e r t i s s u e homogenates, although possessing the highest GPX a c t i v i t y among the tissues studied (Fig. 5), did not show a greater extent of TBHP-induced GSH depletion (Fig. 1) . In contrast, TBARS production in l i v e r homogenates was greater than those in other tissues at a l l TBHP concentrations tested (Fig. 2). This might indicate that l i v e r , i n fact, i s more susceptible to l i p i d damage than heart and kidney. It has been shown that hepatotoxicity induced by environmental pollutants (eg. carbon tetrachloride) and drug metabolites (eg. acetaminophen) l i k e l y involves oxy-radical-mediated l i p i d peroxidation processes [301,302]. Both high tissue GSH le v e l s and a c t i v i t i e s of antioxidant enzymes in l i v e r , e s p e c i a l l y catalase (CAT) and GPX, may suggest a compensatory mechanism for protecting against oxidative injury induced by endogenously generated oxidants in t h i s vulnerable tissue. In t h i s regard, the depletion of l i v e r GSH content by dimethyl maleate in mice was associated with an enhanced s u s c e p t i b i l i t y to hepatotoxicity produced by agents such as acetaminophen and carbon t e t r a c h l o r i d e [302,303]. Thus, 156 measurements of s u s c e p t i b i l i t y of tissue homogenates to TBHP-induced GSH depletion and formation of TBARS may be able to r e f l e c t , to a certa i n extent, the antioxidant capacity of tissue with regard to GSH protection and s u s c e p t i b i l i t y of membrane l i p i d s to oxidative challenge. A si m i l a r approach has been used to assess the antioxidant capacity of plasma by measuring the decomposition of various non-enzymatic antioxidants using a water-soluble and heat-l a b i l e compound, 2,2'-azo-bis (2-amidinopropane) as a free r a d i c a l generating agent [39]. 4.2 Al t e r a t i o n s i n Antioxidant Capacity i n Ischemic/  Reperfused Rabbit Myocardium Changes in the s e n s i t i v i t y of tissue homogenates to in v i t r o oxidative challenge have been used as an indicati o n of altered t i s s u e s u s c e p t i b i l i t y to oxidative injury in vivo [227,228]. A recent study by Fe r r e i r a et. a l . [189] has shown that myocardial biospies from patients undergoing revascularization surgery exhibited s i g n i f i c a n t increases in oxidative stress, as measured in v i t r o by hydroperoxide-induced chemiluminescence, the magnitude of which p a r a l l e l e d biochemical and u l t r a s t r u c t u r a l indices of myocardial damage. Our finding that an abrupt increase in s e n s i t i v i t y of myocardial tissue homogenates to TBHP-induced oxidative challenge ( i n d i c a t i v e of impairment in antioxidant capacity) occurred a f t e r 20 - 40 min period of ischemia with 60 min of reperfusion which p a r a l l e l e d the onset of i r r e v e r s i b l e 157 injury [155,156] suggests the involvement of oxidative processes i n these pathological changes. GSH i s a c r u c i a l determinant of tissue s u s c e p t i b i l i t y to oxidative damage [231-233] and impaired GSH homeostasis has been shown to be a feature of myocardial I/R injury, p a r t i c u l a r l y during the post-ischemic reperfusion phase [129,171]. The decrease i n GSH l e v e l s of tissues subjected to oxidative stress i s believed to r e f l e c t the formation of mixed d i s u l f i d e s with protein sulfhydryls and/or of glutathione d i s u l f i d e (GSSG), the l a t t e r being capable in some systems of undergoing ATP-dependent e x t r a c e l l u l a r t r anslocation [171], thereby diminishing the c e l l u l a r supply of GSH from the NADPH-dependent reduction of GSSG by glutathione reductase (GRD) [234]. The r e s u l t s presented here suggest the existence of a c e l l u l a r GSH pool showing increased s u s c e p t i b i l i t y to oxidative depletion which becomes maximal early in the course of ischemia (about 10 min) and remains constant (in the absence of reperfusion) with periods of ischemia up to 60 min (Fig. 9). Under these conditions, minimal increases in l i p i d s u s c e p t i b i l i t y to oxidative challenge are observed (Fig. 10). Reperfusion following ischemia of short duration ( i . e , 5 min) revealed evidence for a transient enhancement of tiss u e antioxidant capacity as r e f l e c t e d i n a s i g n i f i c a n t decrease in s u s c e p t i b i l i t y of GSH to oxidative depletion (Fig. 9). As also noted in the reperfusion time-course study, an increased s t a b i l i t y of myocardial GSH to peroxide-induced 158 depletion was observed 5 min af t e r the i n i t i a t i o n of post-ischemic reperfusion (see Fig. 15). One possible explanation i s suggested by re s u l t s of a study reported by Zimmer et.  a l . [235] who showed that oxidative stress produced by peroxide challenge can cause stimulation of the NADPH-generating hexose monophosphate shunt i n is o l a t e d perfused rat hearts. An increased production of NADPH, which i s l i k e l y to be l i m i t i n g i n the GRD-mediated regeneration of GSH from the d i s u l f i d e form, might therefore explain the transient increase in antioxidant capacity seen in our study. The enhanced GSH status may be b e n e f i c i a l for scavenging reactive oxidants by redox c y c l i n g of myoglobin in the myocardium, thereby protecting against oxidative damage i n cardiac muscle [54]. However, t h i s early protective e f f e c t showed a s t r i k i n g reversal with increasing duration of ischemia or reperfusion which was characterized by a marked increase i n s u s c e p t i b i l i t y of tissue GSH and l i p i d s to oxidation. The greater s e n s i t i v i t y of GSH to TBHP-induced depletion (when compared with TBHP-induced peroxidation of l i p i d s , see Fig. 7,8) and the higher s u s c e p t i b i l i t y of myocardial GSH to I/R-related a l t e r a t i o n s (Fig. 9) than that of TBARS formation during the course of ischemia and reperfusion suggest that GSH may play a p i v o t a l role in protecting against t i s s u e oxidative injury. Our demonstration of the high GSH l e v e l i n l i v e r which i s a major s i t e of reactive oxidant production i n vivo [304] i s consistent with t h i s 159 hypothesis. This view i s further strengthened by r e s u l t s of recent studies by Ceconi et. a l . [129] who showed that N-acetyl-cysteine pretreatrnent leading to a preservation of GSH l e v e l s in rabbit hearts subjected to ischemia and reperfusion also decreases the extent of c e l l u l a r damage as characterized by creatine phosphokinase release and impaired mitochondrial function. Moreover, Chatham et. a l . [236] and Singh et. a l . [237] have recently demonstrated that the depletion of myocardial GSH by buthionine sulfoximine pretreatrnent can exacerbate the extent of I/R injury i n rats and pigs. 4.3 Time-Course of Alterations i n Myocardial Antioxidant Capacity and Antioxidant Enzyme A c t i v i t i e s During Post- Ischemic Reperfusion A progressive impairment i n myocardial antioxidant capacity was observed in rabbits during the course of post-ischemic reperfusion. The protracted time-course of these changes dissociated them from the early burst of r a d i c a l formation shown to occur at the onset of post-ischemic reperfusion of the myocardium [78,80]. Mullane et. a l . [130] have shown that the maximal degree of myocardial necrosis induced by a short period of coronary occlusion in dogs i s not attained u n t i l several hours a f t e r the i n i t i a t i o n of reperfusion. This observation and our r e s u l t s , as presented here, suggest that secondary oxidative processes occurring during the prolonged period of post-ischemic reperfusion are 160 involved in the development of i r r e v e r s i b l e I/R injury. Oxidants derived from the activated neutrophils that i n f i l t r a t e the previously ischemic myocardium [182,183] may be one factor i n causing such oxidative damage. This i s further supported by results obtained in our study using i s o l a t e d Langendorff-perfused rabbit hearts subjected to ischemia and reperfusion, namely that I/R-induced al t e r a t i o n s in myocardial antioxidant capacity seen in i n t a c t hearts were not d i s c e r n i b l e i n blood-free-perfused hearts (see Table XII). The high s u s c e p t i b i l i t y of the myocardium to damage by ischemia and reperfusion and by free radical-generating drugs such as adriamycin [238] may rel a t e , in part, to the r e l a t i v e l y low a c t i v i t y of antioxidant enzymes in t h i s t i s s u e [27]. As noted i n preliminary studies from our laboratory, a 40 min period of ischemia with 60 min of reperfusion, despite causing i r r e v e r s i b l e tissue damage, did not produce any detectable changes in the a c t i v i t y of myocardial antioxidant enzymes. The r e s u l t s obtained in the present study, which are i n agreement with t h i s early observation, indicate that mean antioxidant enzyme a c t i v i t i e s in ischemic/reperfused myocardial tiss u e did not change s i g n i f i c a n t l y during the course of post-ischemic reperfusion, when compared with those of non-ischemic or ischemic/non-reperfused tissue (Table V). When the a c t i v i t i e s of antioxidant enzymes (Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and GRD) were viewed in r e l a t i o n to 161 functional indices of myocardial antioxidant status in animals undergoing ischemia and reperfusion, s i g n i f i c a n t c o r r e l a t i o n s which suggested functional impairment of antioxidant enzymes became apparent. The use of multiple c o r r e l a t i o n analyses with a large number of animals has allowed the emergence of information which was not apparent from simple group averaging. The lack of such a c o r r e l a t i o n for GPX may indicate that i t s a c t i v i t y in the myocardium i s not l i m i t i n g . In addition, GPX of both myocardial and brain tissue has been reported to be less susceptible than Cu,Zn-SOD to ischemia-induced i n a c t i v a t i o n [239,240]. While the assignment of cause/consequence re l a t i o n s h i p to the I/R-induced suppression of antioxidant enzyme a c t i v i t i e s and a l t e r a t i o n s i n antioxidant capacity s t i l l remains to be determined, i t seems unlikely, i n the absence of any detectable changes in GPX a c t i v i t i e s , that the suppression of enzyme a c t i v i t i e s i s caused by I/R-induced i r r e v e r s i b l e t i s s u e necrosis whose e f f e c t s on enzyme i n a c t i v a t i o n would l i k e l y be non-specific. This i s further strengthened by the finding i n rat myocardium that while the a c t i v i t y of SOD was decreased immediately following the induction of ischemia, s i g n i f i c a n t reduction in the a c t i v i t y of GPX was not seen u n t i l t i s s u e necrosis was apparent [239]. Therefore, changes i n Cu,Zn-SOD and GRD a c t i v i t i e s may precede, rather than accompany, i r r e v e r s i b l e t i s s u e injury. F i n a l l y , a r e v e r s i b l e impairment i n the functioning of antioxidant enzymes, as suggested by both pH-dependence studies and 162 c o r r e l a t i o n analyses, may occur during the early phase of reperfusion when the i n t r a c e l l u l a r pH has not returned to normal. In t h i s regard, the i n t r a c e l l u l a r pH of rabbit myocardium has been reported to decrease from 7.0 to 6.0 a f t e r a 30 min period of ischemia [177]. I f such transient enzyme i n h i b i t i o n coincided with the burst of oxy-radicals in the early post-ischemic reperfusion phase [78,80], i t could be c r i t i c a l i n the t r i g g e r i n g of i r r e v e r s i b l e myocardial I/R injury. 4.4 The E f f e c t s of Cumulative B r i e f Episodes of Ischemia on  Myocardial Antioxidant Capacity and ATP Levels Much evidence has accumulated supporting the involvement of oxy-radicals i n causing c o n t r a c t i l e dysfunction i n myocardial "stunning" [6]. Pretreatment with oxy-radical scavengers and other antioxidants have consistently been shown to improve post-ischemic dysfunction following a b r i e f period of ischemia [241-246]. However, the protection afforded by oxy-radical scavenger pretreatment i s often not associated with any improvement in myocardial high-energy phosphate stores [241-243]. This suggests that the oxy-radical-mediated c o n t r a c t i l e dysfunction may be independent of high-energy phosphate status i n the ischemic myocardium. Our demonstration that the p a r t i a l depletion of myocardial ATP content aft e r r e p e t i t i v e b r i e f episodes of ischemia was not associated with any detectable changes i n myocardial antioxidant 163 capacity i s consistent with t h i s hypothesis. The i n a b i l i t y of c e l l s to maintain adequate l e v e l s of ATP i s a c h a r a c t e r i s t i c feature of i r r e v e r s i b l e damaged c e l l s [247]. The impairment of mitochondrial function and/or the loss of t o t a l adenine nucleotides i n the c e l l s have been attributed to the f a i l u r e of ATP resynthesis, thereby causing i r r e v e r s i b l e c e l l damage [247-249]. Our r e s u l t s which demonstrated the i n a b i l i t y of hearts to restore the normal ATP l e v e l s even a f t e r one b r i e f episode of ischemia might be at t r i b u t a b l e to the ischemia-induced mitochondrial dysfunction and adenine nucleotide depletion. The absence of detectable changes i n myocardial antioxidant capacity following 4 cycles of b r i e f episodes of ischemia suggest that the depletion of ATP (to 40% of the control level) by ischemia could not t r i g g e r i r r e v e r s i b l e t i s s u e damage. However, reperfusion following a prolonged period of ischemia (40 min) which reduced tissue ATP l e v e l by 75% could lead to i r r e v e r s i b l e damage [50]. It has also been shown i n is o l a t e d rat hearts that the abrupt reduction in ATP l e v e l s (to 40% of control values) following a 20 min period of global ischemia was associated with depletion of adenine nucleotides which p a r a l l e l e d the onset of i r r e v e r s i b l e damage [247]. Since the preservation of adenine nucleotides a r i s i n g from ischemia-induced ATP degradation i s important for post-ischemic ATP resynthesis [254], the f a i l u r e of r e p e t i t i v e b r i e f episodes of ischemia, but not the prolonged period of ischemia and reperfusion, to produce 164 i r r e v e r s i b l e tissue damage might be related to the severity of adenine nucleotide depletion i n the ischemic t i s s u e . An extended period of ischemia and reperfusion l i k e l y resulted i n a greater degree of tissue wash-out of adenine nucleotides, thereby causing i r r e v e r s i b l e injury. The increased s t a b i l i t y of GSH to TBHP-induced depletion (Table VIII) was probably caused by oxidative stress a r i s i n g from one b r i e f period of ischemia. This concurs with r e s u l t s obtained in studies involving rabbits subjected to 5 min period of ischemia with 60 min of reperfusion (Fig. 9) or a 40 min period of ischemia followed by 5 min of reperfusion (Fig. 14). In both cases, a stimulation of the hexose monophosphate shunt i n response to oxidative stress may be involved. Although the r e p e t i t i v e b r i e f episodes of ischemia decreased myocardial ATP l e v e l s , the severity of subsequently induced I/R injury, as indicated by the increased t i s s u e s u s c e p t i b i l i t y to TBHP-induced GSH depletion and formation of TBARS, did not appear to be altered by t h i s ischemic preconditioning process (Table X). These r e s u l t s suggest that changes in myocardial antioxidant capacity and ATP le v e l s can occur independently, although ATP depletion usually accompanies the development of i r r e v e r s i b l e I/R-induced injury [156,247]. 165 4.5 I / R - I n d u c e d A l t e r a t i o n s i n M y o c a r d i a l A n t i o x i d a n t C a p a c i t y i n I s o l a t e d L a n g e n d o r f f - p e r f u s e d R a b b i t H e a r t s The involvement of neutrophils i n the pathogenesis of myocardial I/R injury now seems well established [130,162,183]. Under the influence of chemotactic substances produced from ischemic tissues, neutrophils i n f i l t r a t e into the ischemic myocardium and become activated [162]. The generation of cytotoxic oxidants and the release of p r o t e o l y t i c enzymes from activated neutrophils can cause damage to the vascular endothelium and to the cardiac myocytes [183]. These processes are greatly i n t e n s i f i e d when oxygen i s reintroduced to the ischemic tiss u e during the reperfusion phase. In addition to the deleterious e f f e c t s produced by neutrophil-derived oxidants and p r o t e o l y t i c enzymes, the aggregation of activated neutrophils in myocardial c a p i l l a r i e s can impair coronary blood flow, thereby further exacerbating the ischemic injury and r e s u l t i n g i n the "nb-reflow" phenomenon af t e r the i n i t i a t i o n of reperfusion [162]. Results obtained in the present study using i s o l a t e d Langendorff-perfused rabbit hearts subjected to ischemia and reperfusion suggest that blood elements, possibly activated neutrophils, may be a c r u c i a l factor in causing I/R-related oxidative injury in the myocardium. Although the antioxidant capacity was depressed in buffer-perfused hearts (as indicated by the decrease i n tiss u e GSH l e v e l s and an enhanced s u s c e p t i b i l i t y to GSH depletion), manifestations of oxidative injury 166 ( i . e . , increased s u s c e p t i b i l i t y to TBHP-induced GSH depletion and formation of TBARS) were not observed following a 40 min period of ischemia with 60 min of reperfusion under these neutrophil-free perfusion conditions (Table XII). When the induction of myocardial ischemia was performed in vivo ( i . e . , with blood components present in the ischemic t i s s u e ) , reperfusion with buffer solution could also cause oxidative injury, as indicated by an enhancement in s u s c e p t i b i l i t y to TBHP-induced formation of TBARS, although the extent of oxidative damage was less than that observed in animals undergoing both ischemia and reperfusion in vivo (Table XII). This suggests that oxy-radical-mediated processes involved i n the development of I/R injury may, at leas t i n part, be i n i t i a t e d by oxidants produced from activated neutrophils which accumulate i n the ischemic tissue. In t h i s regard, i n f l u x of neutrophils into the myocardium has been observed a f t e r a 45 min to 3 hour period of transient coronary occlusion in the canine myocardium [305,306]. 4.6 The Eff e c t s of A l l o p u r i n o l Pretreatment on Myocardial  I/R Injury The I/R-induced impairment in myocardial antioxidant capacity was attenuated by a chronic a l l o p u r i n o l treatment regimen previously shown to prevent u l t r a s t r u c t u r a l and biochemical a l t e r a t i o n s i n ischemic/reperfused rabbit 167 myocardium [155,156]. In contrast to N-acetyl-cysteine [129], which can act as a d i r e c t precursor i n c e l l u l a r glutathione synthesis, the protective actions of a l l o p u r i n o l , as described here, were not associated with any s i g n i f i c a n t e f f e c t on the depressed GSH content of myocardial tissues following ischemia and reperfusion (data not shown) . This r e s u l t d i f f e r s from that reported by Peterson et. a l . [154] who showed that the markedly reduced myocardial GSH leve l s in dogs subjected to coronary artery l i g a t i o n (60 min) followed by reperfusion (30 or 120 min) are f u l l y restored to control l e v e l s a f t e r 120 min of reperfusion i n animals treated o r a l l y with a l l o p u r i n o l (50 mg/kg) for 2 days p r i o r to the experiment and as an intravenous infusion beginning 30 min p r i o r to coronary occlusion. Although a d i r e c t comparison between the present study and the foregoing one by Peterson et. a l . [154] i s precluded by the m u l t i p l i c i t y of differences in experimental conditions, an e f f e c t of chronic a l l o p u r i n o l treatment on myocardial antioxidant capacity i s apparent in both cases. While the exact role of antioxidant enzymes in the development of myocardial I/R injury i s yet to be determined, the reperfusion time-course studies suggest the functionally relevant impairment in myocardial Cu,Zn-SOD and GRD a c t i v i t i e s during the course of post-ischemic reperfusion. The protection against myocardial I/R injury afforded by exogenously administered SOD and CAT [73,146] also indicates that the a c t i v i t y of these enzymes may be 168 inadequate under conditions of increased oxidative stress. In t h i s regard, the a b i l i t y of c e r t a i n substances such as acetyl-homocysteine-thiolactone [251], the antioxidant 6,6'-methylene-bis 2,2 1-dimethyl-4-methane s u l f o n i c acid sodium 1,2-dihydroquinoline [252] and b a c t e r i a l endotoxin [253] to protect tissues against oxidative injury has been attributed to the enhancement or preservation of antioxidant enzyme a c t i v i t i e s . Our finding that myocardial GRD a c t i v i t y was increased by a chronic a l l o p u r i n o l treatment regimen which can reduce the extent of I/R injury suggests that such a mechanism might be involved in a l l o p u r i n o l protection against myocardial I/R injury. The protection afforded by a l l o p u r i n o l pretreatment in ischemic/reperfused rabbit myocardium was not associated with any detectable preservation of t i s s u e ATP l e v e l s (Fig. 13). The ATP-sparing e f f e c t of a l l o p u r i n o l observed in some models of I/R injury has been attributed to a reduction i n the degradation of nucleotide precursors, thereby allowing t h e i r more e f f i c i e n t r e u t i l i z a t i o n for ATP synthesis [162,254]. However, the rapid t i s s u e wash-out of these ATP degradation products casts considerable doubt on t h i s p o s s i b i l i t y [255]. Furthermore, the independence of a l l o p u r i n o l protective e f f e c t s and a l t e r a t i o n s in tissue ATP content has been repeatedly demonstrated [242,256]. This i s further strengthened by our findings i n rabbits subjected to b r i e f episodes of myocardial ischemia that I/R-induced a l t e r a t i o n s i n myocardial antioxidant capacity were not 169 influenced by substantial depletion of tissu e ATP l e v e l s . I t , therefore, seems u n l i k e l y that the protection against I/R injury afforded by a l l o p u r i n o l treatment i s d i r e c t l y related to processes involved i n ATP homeostasis. 4.7 The E f f e c t s of Acute U74006F Treatment on I/R-Induced  Alt e r a t i o n s i n Rabbit Myocardium Although the marked manifestations of myocardial I/R injury appear during the post-ischemic reperfusion phase (see F i g . 9,10), acutely administered protective agents often must be present throughout the ischemic phase in order to be maximally e f f e c t i v e [129,257]. In the present study, U74006F was found to abolish I/R-induced i n a c t i v a t i o n of mitochondrial ATPase when injected shortly before the onset of myocardial reperfusion (Table IV). Although some in d i c a t i o n of cytoprotection was also seen when U74006F was administered p r i o r to coronary artery l i g a t i o n , t h i s did not a t t a i n s t a t i s t i c a l s i g n i f i c a n c e . The decreased effectiveness of U74006F when given early in the experimental period could relate, at lea s t i n part, to i t s rapid rate of hepatic metabolism [258]. The b e n e f i c i a l e f f e c t s of U74006F on mitochondrial i n t e g r i t y were not associated with any detectable increase in t i s s u e ATP l e v e l s . Similar r e s u l t s were observed from previous studies in our laboratory involving membrane-active agents such as the verapamil derivative D-600, propranolol and halothane [216,259]. A recent report by Simidzhiev et. 170 a l . [260] has shown that short-chain derivatives of a-tocopherol are able to stimulate mitochondrial ATPase a c t i v i t y and uncouple oxidative phosphorylation by membrane modifying actions that are unrelated to t h e i r antioxidant capacity. When taken together, these findings suggest that U74006F may have prevented I/R-induced decreases in mitochondrial ATPase a c t i v i t y by membrane perturbational e f f e c t s rather than by v i r t u e of i t s well documented r a d i c a l scavenging properties. The absence of detectable e f f e c t s of U74006F on d i r e c t manifestations of myocardial oxidative injury, namely the depletion of tissue GSH and the increased s u s c e p t i b i l i t y to in v i t r o oxidative challenge, was somewhat unexpected. The intravenous dose of U74006F used (3 mg/kg) was comparable to or greater than doses previously shown to afford s i g n i f i c a n t protection in experimental models of concussive injury to the head [166] or spinal cord [167], subarachnoid hemorrhage and associated cerebral vasospasm [168,261] as well as in global cerebral ischemia [262]. The foregoing studies demonstrating b e n e f i c i a l effects of U74006F have been performed in several d i f f e r e n t types of animals, including rabbits [261], and using a variety of anesthetic agents, including pentobarbital [262]. It i s therefore somewhat unli k e l y that the negative r e s u l t s reported here can be attributed to our choice of the pentobarbital-anesthetized rabbit as experimental model. Rather, our findings may be an i n d i c a t i o n that responsiveness to U74006F treatment i s 171 c r i t i c a l l y dependent on the p a r t i c u l a r type of injury involved. U74006F has been found most e f f e c t i v e i n reducing the extent of CNS injury induced by physical trauma in which ischemia i s only a secondary consequence i n association with vasospastic responses to injury. I t has been suggested that trauma-induced hemorrhage i n the CNS favours hemoglobin-catalysed production of reactive oxidants with r e s u l t i n g injury to vascular endothelial c e l l s [263]. Given the i n t r i n s i c r a d i c a l generating c a p a b i l i t y of these c e l l s [264] and the r e l a t i v e deficiency of r a d i c a l scavenging systems in the cerebrospinal f l u i d [265], necrosis would spread rapidly in an autocatalytic fashion. The fact that the probable i n i t i a t i n g stimulus, namely r a d i c a l attack on the endothelium, i s e x t r a c e l l u l a r in o r i g i n may account for the r e l a t i v e l y high s u s c e p t i b i l i t y of such processes to protection by U74006F administered immediately before or shortly a f t e r the onset of injury. The s i t u a t i o n with myocardial I/R damage d i f f e r s from that described above in that both i n t r a c e l l u l a r and e x t r a c e l l u l a r sources of oxygen r a d i c a l production are l i k e l y to be important in the i n i t i a t i o n process [266]. Under these conditions, optimal protection by antioxidants might, therefore, require t h e i r repeated administration p r i o r to the ischemic i n s u l t i n order to achieve adequate i n t r a c e l l u l a r l e v e l s of drug. This could also explain the need for a chronic treatment regimen rather than acute 172 administration i n order to demonstrate protection against I/R injury by a l l o p u r i n o l (Fig. 11,12 & Table 2). 4.8 The E f f e c t s of A l l o p u r i n o l Pretreatment on the Antioxidant Capacity of Erythrocytes and Functional  V i a b i l i t y of Transplanted Luncr Tissue The protective actions of a l l o p u r i n o l in I/R injury have been documented in studies involving a var i e t y of tissues [147-151,156,267]. The r e s u l t s obtained from the present study examining the e f f e c t s of a l l o p u r i n o l pretreatment i n a swine model of heart-lung transplantation indicate that a l l o p u r i n o l treatment produced a b e n e f i c i a l e f f e c t on the antioxidant capacity of erythrocytes, as r e f l e c t e d i n the reduction i n s u s c e p t i b i l i t y of red c e l l s to i n v i t r o peroxidative challenge (Fig. 17). These re s u l t s and the finding that the extent of red c e l l protection in both donor and rec i p i e n t animals correlated s i g n i f i c a n t l y with the functional v i a b i l i t y of the transplanted lung (assessed by tissue' water content) suggest that generalized a l t e r a t i o n s i n tissu e antioxidant capacity may be di s c e r n i b l e from measurements of TBHP-induced formation of TBARS (including MDA) i n red c e l l s . Recent studies from our laboratory have shown that rats with chemically-induced diabetes exhibit increased s u s c e p t i b i l i t y of red c e l l l i p i d s to oxidative damage i n v i t r o and marked a l t e r a t i o n s in tissue antioxidant enzyme a c t i v i t i e s [268]. This abnormality i n red c e l l antioxidant capacity based on 173 measurements of TBARS i s also demonstrable in c l i n i c a l diabetes to an extent that p a r a l l e l s the severity of secondary d i a b e t i c complications present [269]. It seems unl i k e l y , based on a recent study by Zimmerman et. a l . [270], that the observed a l t e r a t i o n s in red c e l l s induced by a l l o p u r i n o l treatment r e f l e c t changes in the antioxidant capacity of plasma. These investigators have shown that the antioxidant capacity of e x t r a c e l l u l a r f l u i d s (plasma and i n t e s t i n a l lymph) was unaltered in cats given a l l o p u r i n o l o r a l l y for two days at a dose of 50 mg/kg, the same as that used i n the present study. The authors suggested that the powerful antioxidant properties of e x t r a c e l l u l a r f l u i d a t t r i b u t a b l e to the presence of numerous endogenous non-enzymatic scavengers may have precluded the demonstration of any further increase by a l l o p u r i n o l treatment. If, however, a l t e r a t i o n s in red c e l l antioxidant capacity can be taken as a general i n d i c a t i o n of the s i t u a t i o n i n tissues, our findings concerning the time/dose dependence of allopurinol-induced protection may have an important bearing on the v a r i a b i l i t y of reported re s u l t s obtained with t h i s agent in a variety of experimental models of I/R injury. In most investigations documenting protection by a l l o p u r i n o l , the drug was administered for varying periods of time p r i o r to the ischemic episode [153-156, 255]. Treatment with a l l o p u r i n o l beginning 24 hours before the induction of myocardial ischemia i n two studies 174 using dogs has yielded inconsistent r e s u l t s [132,271], while acute administration of a l l o p u r i n o l has usually f a i l e d to e l i c i t myocardial protective e f f e c t s [272-274]. The foregoing experimental results are i n general accord with the time-course of allopurinol-induced protection of red c e l l s observed i n the present study, further strengthening the view that red c e l l s u s c e p t i b i l i t y to in v i t r o oxidative challenge may provide a useful functional measure of generalized a l t e r a t i o n s in tissue antioxidant status produced by a l l o p u r i n o l and possibly other pharmacological agents. The inter-animal differences in response to al l o p u r i n o l treatment might involve v a r i a b i l i t y in the absorption of a l l o p u r i n o l following oral administration or in the metabolism of a l l o p u r i n o l which involves xanthine oxidase-mediated oxidation with resultant formation of superoxide r a d i c a l s . Given that xanthine oxidase a c t i v i t y i s also present i n plasma [279], an increased oxy-radical production may occur i n plasma following oral a l l o p u r i n o l administration with r e s u l t i n g a l t e r a t i o n s i n red c e l l antioxidant status. The high s e n s i t i v i t y of lung tissue to I/R-induced damage has greatly c u r t a i l e d the use of heart-lung transplantation in the management of patients with end-stage pulmonary disease [275]. Several techniques for lung preservation during ischemia have been evaluated, including hypothermic storage, continuous perfusion and autoperfusion [191-193], However, none of these has been e n t i r e l y 175 successful i n preventing vulnerable lung tiss u e from I/R injury following prolonged periods of ischemia. In contrast to other organ systems, l i t t l e work has been done to elucidate the mechanism of reperfusion injury in the lung following an episode of ischemia. Preliminary studies i n our laboratory indicated that xanthine oxidase a c t i v i t y was undetectable i n pig lungs (data not shown), suggesting that xanthine oxidase might not be an important source of r a d i c a l production i n ischemic lung tissue. While the role of xanthine oxidase i n the development of tissue damage in pig lung i s yet to be determined, i t has been shown that xanthine oxidase i n h i b i t i o n by lodoximide can ameliorate I/R injury in iso l a t e d rat lungs [276]. The functional i n t e g r i t y of transplanted lungs as assessed in terms of lung water content as well as other indices of pulmonary function, such as a r t e r i a l blood p a r t i a l pressure of oxygen (p0 2), a l v e o l a r - a r t e r i a l oxygen gradient (AaG), a l v e o l a r - a r t e r i a l oxygen tension r a t i o (AaR) and pulmonary vascular resistance (PVR) (data not shown). The c l i n i c a l usefulness of lung water measurements in the assessment of lung functional i n t e g r i t y has recently been commented upon by Staub [277]. The increase in lung water content, as observed in the present study, was often associated with an increased AaG, a decreased AaR as well as an increased PVR (data not shown) . A l l these changes are suggestive of extensive c a p i l l a r y endothelial damage, possibly due to an increase i n oxy-radical a c t i v i t y . 176 The observed p a r a l l e l i s m between the preservation of pulmonary function and the protection of red c e l l s against oxidative challenge suggests that a l l o p u r i n o l may exert b e n e f i c i a l e f f e c t s by v i r t u e of a generalized e f f e c t on tissue antioxidant capacity. This i s consistent with our demonstration of a l l o p u r i n o l protection against the impairment in antioxidant capacity which accompanies myocardial I/R injury in open-chest anesthetized rabbits. On the other hand, antioxidant properties of erythrocytes have been shown to be b e n e f i c i a l in protecting against I/R-or oxidant-induced tissue injury [307,312]. Brown et. a l . [312] have demonstrated that reperfusion with human erythrocytes increased v e n t r i c u l a r function and decreased myocardial hydrogen peroxide l e v e l s in is o l a t e d rat hearts subjected to a 20 min period of normothermic global ischemia. In contrast, reperfusion with erythrocytes that were deprived of catalase a c t i v i t y and/or GSH did not produce any protective e f f e c t s . Thus, the enhancement of antioxidant capacity of erythrocytes by a l l o p u r i n o l pretreatrnent may, at least in part, be d i r e c t l y responsible for observed protection against I/R-induced damage in the myocardium and lung. 177 4.9 Inhibitory E f f e c t s of A l l o p u r i n o l and Oxypurinol on  T r a n s i t i o n Metal Ion-Catalysed Ascorbate Oxidation  and L i p i d Peroxidation A number of possible mechanisms other than xanthine oxidase i n h i b i t i o n by a l l o p u r i n o l have been implicated in i t s protection against I/R injury, including an increased e f f i c i e n c y of ATP salvage [162,163], f a c i l i t a t i o n of mitochondrial electron transfer [164] and d i r e c t i n a c t i v a t i o n of endogenously formed reactive species, such as hydroxyl r a d i c a l s or myeloperoxidase-derived hypochlorous acid [165]. One mode of action not previously considered i s the chelation of t r a n s i t i o n metal ions whose c a t a l y t i c actions i n oxy-radical mediated reactions have been shown to be important i n the pathogenesis of oxidative damage in ischemic t i s s u e [13,194]. The r e s u l t s of the present study demonstrating the i n h i b i t o r y actions of a l l o p u r i n o l and i t s metabolite oxypurinol i n the t r a n s i t i o n metal ion-catalysed oxidation of ascorbic acid and oxidation of erythrocyte membrane l i p i d s suggest that the metal chelating actions of a l l o p u r i n o l and oxypurinol may be relevant to t h e i r protective actions against I/R injury. Transition metal ions catalyse the non-enzymatic oxidation of ascorbate through the intermediacy of redox reactions i n which ascorbate oxidaton occurs i n one-electron steps [225]. Since the mechanism of ascorbate oxidation l i k e l y involves formation of an ascorbate-transition metal ion complex preceding intramolecular electron transfer 178 [225], t r a n s i t i o n metal chelators would be expected to i n t e r f e r e with t h i s i nteraction, thereby decreasing the reaction rate. A l l o p u r i n o l and oxypurinol, as well as the metal chelator EDTA are capable of suppressing the basal oxidation of ascorbate (Fig. 19a), presumably caused by t r a n s i t i o n metal ion contaminants present in the phosphates and d o u b l e - d i s t i l l e d water . The stimulatory e f f e c t s of exogenously added cupric ions on the oxidation of ascorbate were also i n h i b i t e d by a l l o p u r i n o l i n a concentration-dependent manner (Fig. 19b) The use of EDTA to minimize basal oxidation of ascorbate permitted measurement of the r e l a t i v e l y modest increase in the rate of ascorbate oxidation induced by f e r r i c ions at micromolar concentrations. Under the assay conditions employed, cupric ions were found to be two-fold more e f f e c t i v e than f e r r i c ions i n catalysing ascorbate oxidation (Fig. 20). The cupric ion-catalysed reaction was also more susceptible to the i n h i b i t o r y actions of a l l o p u r i n o l and oxypurinol than that catalysed by f e r r i c ions. A s i m i l a r trend was observed for u r i c acid, a compound cl o s e l y related s t r u c t u r a l l y to a l l o p u r i n o l and oxypurinol. UV spectral analysis suggested the formation of an allopurinol-ascorbate-copper complex which i s unstable in the presence of EDTA (Fig. 21, Table XIV) . A s i m i l a r complexation process has previously been implicated in the i n h i b i t i o n by u r i c acid of the cupric ion-catalysed oxidation of ascorbate, as reported by Lam et. a l . [50]. 179 The absence of s i m i l a r spectral changes i n the a l l o p u r i n o l -ascorbate-ferric chloride mixture presumably indicates that complex formation i n t h i s system i s much less highly favoured. This i s consistent with our observation that f e r r i c chloride i s less e f f e c t i v e as a ca t a l y s t of ascorbate oxidation and i t s pro-oxidant action i s less susceptible to a l l o p u r i n o l i n h i b i t i o n than that of cupric chloride. Although antioxidant enzymes, such as catalase and superoxide dismutase, have been shown to i n h i b i t the cupric ion-catalysed oxidation of ascorbate, t h e i r protective e f f e c t s are more l i k e l y a t t r i b u t a b l e to protein binding of cupric ions rather than to scavenging of reactive oxygen-derived r a d i c a l s [280]. In addition, neither the t r a n s i t i o n metal ion-catalysed oxidation of ascorbate nor the formation of the allopurinol-ascorbate-copper ion complex i s affected by butylated hydroxytoluene (BHT), a l i p o p h i l i c antioxidant whose r a d i c a l scavenging a c t i v i t y i s also detectable in some aqueous systems, such as the hydroxyl radical-mediated degradation of deoxyribose (data not shown). Moreover, a l l o p u r i n o l and oxypurinol also i n h i b i t the cupric ion-TBHP-catalysed peroxidation of membrane l i p i d s (Fig. 25), presumably by chelating cupric ions, thereby preventing t h e i r reaction with hydroperoxides which can generate reactive oxy-radicals [281]. The lack of i n h i b i t o r y e f f e c t on l i p i d peroxidation induced by TBHP alone ( i . e . , in the absence of exogenously added t r a n s i t i o n metal ions) suggests that the actions of a l l o p u r i n o l or oxypurinol are not 1 8 0 mediated by d i r e c t free r a d i c a l scavenging. This i s further strengthened by the observation that increasing concentrations of a l l o p u r i n o l or oxypurinol did not cause a greater degree of i n h i b i t i o n of cupric ion-TBHP-induced l i p i d peroxidation (data not shown) . A l l these seem to suggest that the i n h i b i t i o n by a l l o p u r i n o l of the t r a n s i t i o n metal ion-mediated processes i s more l i k e l y a t t r i b u t a b l e to i t s metal chelating properties rather than to an oxy-radical scavenging action, despite the fact that a l l o p u r i n o l has been shown to be capable of i n a c t i v a t i n g exogenously and endogenously generated oxy-radicals [157,165]. I t i s now well established that l i p i d peroxidation in a var i e t y of systems requires the presence of iron in various c a t a l y t i c a l l y active forms [222,282-285]. With regard to the mechanism of iron-catalysed l i p i d peroxidation, the nature of the oxidant species responsible for i n i t i a t i n g the reaction i s s t i l l unresolved [282,286,287]. Minotti and Aust [286,287] have proposed that a s p e c i f i c F e 2 +-02~Fe 3 + complex, or at least a 1:1 r a t i o of F e 2 + to F e 3 + , acts as i n i t i a t o r of peroxidation i n liposomal and microsomal systems. Other studies involving the peroxidation of rat brain synaptosomes [222] or rat l i v e r microsomes [283] also support t h i s hypothesis. On the other hand, Aruoma et. a l . [287] have recently presented evidence arguing against the es s e n t i a l requirement for a Fe -Fe complex or a s p e c i f i c 3+ o+ . . . . . . r a t i o of Fe to Fe i n the i n i t i a t i o n of l i p i d peroxidation. They have shown that the Fe 2 +-dependent 181 peroxidation of liposomes made from ox-brain phospholipids was stimulated by A l 3 + and P b 2 + in a non-additive manner and these e f f e c t s were greater than those of F e 3 + . Nevertheless, the present study suggests the importance of a t t a i n i n g an optimal F e 3 + / F e 2 + r a t i o i n i n i t i a t i n g the Fe 3 + -induced l i p i d peroxidation. Our r e s u l t s indicate that the lag phase preceding the Fe 3 +-stimulated oxidation of erythrocyte membrane l i p i d s (Fig. 23) and the observed decrease i n the formation of TBARS at high concentrations of Fe (Fig. 22) may both be manifestations of a suboptimal F e 3 + / F e 2 + r a t i o . When only F e 3 + i s present i n the reaction mixture, reductive processes leading to the attainment of a favourable F e 3 + / F e 2 + r a t i o would be c r u c i a l in f a c i l i t a t i n g the i n i t i a t i o n of l i p i d peroxidation reactions. In t h i s regard, Minotti and Aust [282] have shown that the increase of Fe autoxidation produced by a l t e r i n g the c i t r a t e to 2 + . . . Fe r a t i o could eliminate the lag phase of the c i t r a t e -2 + • Fe -dependent peroxidation of microsomal phospholipids, presumably by f a c i l i t a t i n g the redox c y c l i n g of iron. In addition, the marked stimulatory e f f e c t s of ascorbic acid 3 + and GSH on Fe -induced l i p i d peroxidation, as described in the present study (Fig. 23), also agree with a recent finding suggesting a f a c i l i t a t i n g e f f e c t of iron reduction 3 + on the enhancement of Fe -dependent peroxidation of l i v e r microsomal l i p i d s [288]. Superoxide anion r a d i c a l s derived from GSH autoxidation [289] may lead to the generation of hydroxyl r a d i c a l s through the iron-catalysed Haber-Weiss 182 reaction [290], r e s u l t i n g i n a stimulatory e f f e c t on l i p i d peroxidation. Moreover, the increase i n the maximal l e v e l of TBARS attained i n ascorbate-stimulated peroxidation may indicate the enhancement of reactions i n the propagating phase caused by ascorbate r a d i c a l s generated from the in t e r a c t i o n of Fe and ascorbic acid [225]. A l l o p u r i n o l and oxypurinol, as well as other i r o n -chelators such as EDTA and u r i c acid, i n h i b i t the f e r r i c ion-induced oxidation of erythrocyte membrane l i p i d s . Based on the aforementioned hypothesis that an optimal r a t i o of 3+ o+ . . . . Fe to Fe i s important for the i n i t i a t i o n of i r o n -dependent l i p i d peroxidation [222,282], iron chelators may act by changing the redox potential of iron. A higher a f f m i t y of Fe for l i p i d regions of membranes than for a l l o p u r i n o l or oxypurinol might explain the weak i n h i b i t o r y e f f e c t s of a l l o p u r i n o l and oxypurinol on the Fe -induced l i p i d peroxidation in erythrocyte membranes. However, the l i p i d - s o l u b l e antioxidant BHT strongly suppressed t h i s l i p i d peroxidation. The finding that the i n h i b i t o r y actions of a l l o p u r i n o l and oxypurinol i n the Fe -induced oxidation of erythrocyte membrane l i p i d s were surmountable by increasing the concentration of Fe ions, s i m i l a r to the res u l t s obtained with EDTA (Fig. 24) , further suggests the involvement of metal chelation i n the actions of a l l o p u r i n o l . Although the i n h i b i t o r y e f f e c t of a low concentration of BHT can be p a r t l y overcome by increasing the concentration of Fe ions, t h i s i s probably due to the 183 complete depletion of exogenously added BHT as a r e s u l t of the increasing production of oxidants [291], which eventually leads to a greater extent of l i p i d peroxidation. Moreover, the a b i l i t y of BHT to reduce the extent of peroxidation induced by the Cu -TBHP mixture to a l e v e l lower than that produced by TBHP alone further suggests a free r a d i c a l mode of action. Under physiological conditions, most of the c e l l u l a r iron i s stored i n an inactive ( f e r r i c ) form in f e r r i t i n [94], Superoxide r a d i c a l s derived from activated neutrophils [308] or generated by xanthine oxidase [309] are able to release iron from f e r r i t i n which can catalyse the peroxidation of l i p i d s [292]. These processes could be important i n the mobilization of iron i n ischemic tissues in which oxy-radical production i s greatly enhanced. On the other hand, c e l l u l a r copper i s mostly incorporated into caeruloplasmin or amino acid complexes [310]. Large quantities of these copper complexes are released from l i v e r as a component of the acute-phase response to a number of disease states, such as infections, a r t h r i t i s and neoplasias [310]. I t i s therefore possible that t h i s response also occurs during acute myocardial i n f a r c t i o n . Although the copper ion-catalysed generation of hydroxyl r a d i c a l s i s only confined to the s i t e of binding of the copper ions [311], the r e s u l t i n g damage to the binding protein may lead to the release of free copper ions. Thus, during the course of tis s u e ischemia and reperfusion, the a v a i l a b i l i t y of 184 c a t a l y t i c a l l y active t r a n s i t i o n metal ions might be increased by a process of decompartmentalization, i . e . , mobilization from c e l l u l a r s i t e s [292,293]. This, in turn, could lead to an enhancement of reactive oxygen r a d i c a l generation and subsequent amplification of t i s s u e damage. The e f f e c t i v e concentrations of a l l o p u r i n o l or oxypurinol shown in the present study to i n h i b i t the t r a n s i t i o n metal ion-catalysed reactions are much higher than the plasma concentrations attained following a l l o p u r i n o l treatment i n animals during the course of ischemia/reperfusion studies [270]. However, the fact that optimal protection by a l l o p u r i n o l against I/R injury i s usually observed only following multiple drug dosing, as reported previously [155,156] and i n the present study, may r e f l e c t the progressive attainment of tissue a l l o p u r i n o l or oxypurinol l e v e l s s u f f i c i e n t to i n h i b i t the t r a n s i t i o n metal ion-catalysed generation of reactive oxidant species. Our data, therefore, support the hypothesis that the t r a n s i t i o n metal chelating actions of a l l o p u r i n o l and oxypurinol are relevant to t h e i r protective action against I/R injury. 4.10 The Effects of Allopurinol on Myoglobin-TBHP-Catalysed  Uric Acid Oxidation and Lipid Peroxidation The physiological function of myoglobin i s generally believed to be concerned with the i n t r a c e l l u l a r transport and storage of oxygen [197,294]. However, the existence of 185 a substantial i n t r a c e l l u l a r oxygen gradient in myocytes argues against an ess e n t i a l role of myoglobin i n the f a c i l i t a t i o n of oxygen d i f f u s i o n to mitochondria [295]. Recently, i t has been suggested that the redox c y c l i n g of myoglobin by ascorbate or GSH may serve as an electron "sink" for protecting against endogenously generated hydroperoxides i n muscle [54,55]. Our r e s u l t s , which have demonstrated the strong i n h i b i t o r y e f f e c t s of ascorbic acid and GSH on the myoglobin-TBHP-catlaysed oxidation of u r i c acid, are consistent with t h i s hypothesis. The c a t a l y t i c action of myoglobin i n the oxidation of u r i c acid induced by hydroperoxides has long been known [220], but the physi o l o g i c a l implications of t h i s peroxidation reaction are s t i l l unclear. The i n h i b i t i o n by u r i c acid of the myoglobin-TBHP-induced l i p i d peroxidation, as described in the present study, may suggest the involvement of u r i c acid in the defense against oxidative damage i n muscle. In t h i s regard, a recent study by Becker et. a l . [296] has demonstrated the b e n e f i c i a l actions of u r i c acid in isola t e d guinea pig hearts perfused with u r i c acid-containing buffer, suggesting that u r i c acid can serve as a physiological r a d i c a l scavenger and antioxidant, maintaining functional responsiveness of the coronary system and of the myocardium. Hydroperoxides oxidize myoglobin, r e s u l t i n g in the production of both alkoxyl r a d i c a l s and f e r r y l heme oxidants, which are powerful i n i t i a t o r s of l i p i d peroxidation reactions [202,229]. Our r e s u l t s have 186 demonstrated the stimulatory e f f e c t of a myoglobin-TBHP mixture on the peroxidation of erythrocyte membrane l i p i d s (Fig. 27), which was strongly suppressed by the l i p i d -soluble antioxidant BHT (Fig. 28), presumably by v i r t u e o f i t s free r a d i c a l chain terminating a c t i v i t y [291]. A l l o p u r i n o l also s l i g h t l y i n h i b i t e d t h i s myoglobin-TBHP-induced l i p i d peroxidation. The general finding that only those iron chelators with r a d i c a l scavenging properties i n h i b i t the hemoprotein-dependent l i p i d peroxidation [297] has suggested an esse n t i a l role of free r a d i c a l scavenging in the i n h i b i t i o n by a l l o p u r i n o l . The i n h i b i t i o n by a l l o p u r i n o l or oxypurinol of u r i c acid oxidation induced by myoglobin-derived oxidants, as shown i n the present study (Fig. 26), would be consistent with t h i s hypothesis. In t h i s regard, both a l l o p u r i n o l and oxypurinol have been shown to possess hydroxyl r a d i c a l scavenging properties in a system measuring the hydroxyl radical-induced degradation of deoxyribose [165]. On the other hand, a l l o p u r i n o l , by vi r t u e of i t s t r a n s i t i o n metal chelating properties (shown in the foregoing studies), could possibly i n t e r f e r e with the int e r a c t i o n between heme-iron and hydroperoxide by binding the iron-containing heme prosthetic groups in myoglobin, thereby suppressing the generation of f e r r y l heme oxidants. The important role of hydrogen peroxide in causing myocardial I/R injury has been suggested by various studies [124,298]. In t h i s regard, the pathogenic actions of hydrogen peroxide are often attributed to i t s a b i l i t y to 187 generate hydroxyl r a d i c a l s through t r a n s i t i o n metal ion-catalysed reactions [84,224,284]. However, hydrogen peroxide produced i n the ischemic myocardium would also react with myoglobin which i s abundant i n heart muscle [197]. During the course of myocardial ischemia and reperfusion, the increased production of hydrogen peroxide [124,298], which l i k e l y coincides with the depletion of t i s s u e ascorbic acid and GSH [170,229], could r e s u l t i n the uncontrolled production of f e r r y l heme oxidants, subsequently leading to tiss u e damage. Ascorbic acid and i t s l i p o p h i l i c d erivative 2-octadecylascorbic acid, e f f e c t i v e scavengers for myoglobin-derived oxidants [54,55], have been shown to protect against I/R injury in rat and dog hearts [127,131]. The r e s u l t s of the present study which demonstrated the i n h i b i t o r y e f f e c t s of a l l o p u r i n o l on myoglobin-hydroperoxide-induced u r i c acid oxidation and l i p i d peroxidation suggest that the a l l o p u r i n o l protection against I/R injury may, at least i n part, be related to i t s i n h i b i t o r y actions in hemoprotein-hydroperoxide oxidative reactions. 4.11 Summary and Conclusions (1) A marked impairment in myocardial antioxidant capacity developed in association with the onset of i r r e v e r s i b l e I/R injury i n rabbit hearts subjected to periods of 188 ischemia longer than 20 min followed by 60 min of reperfusion. (2) Reperfusion of the myocardium a f t e r a 40 min period of ischemia resulted in a progressive decrease in myocardial antioxidant capacity. The protracted time-course of myocardial a l t e r a t i o n s dissociated them from the early burst of r a d i c a l formation known to occur at the onset of post-ischemic reperfusion of the myocardium. (3) When the time-dependent changes i n functional indices of antioxidant status (TBHP-induced GSH depletion and formation of TBARS) of ischemic/reperfused myocardial tissues were analysed in r e l a t i o n to a c t i v i t i e s of antioxidant enzymes, evidence suggestive of fun c t i o n a l l y relevant impairment in Cu,Zn-SOD and GRD a c t i v i t i e s was found. These re s u l t s and the demonstration of s i g n i f i c a n t decreases in the a c t i v i t y of GSH-dependent antioxidant enzymes under a c i d o t i c conditions suggest that a transient impairment in the functioning of antioxidant enzymes may be involved in tr i g g e r i n g i r r e v e r s i b l e myocardial I/R injury. (4) Repetitive b r i e f episodes of ischemia and reperfusion did not produce any di s c e r n i b l e e f f e c t s on myocardial antioxidant capacity despite the substantial decrease in t i s s u e ATP l e v e l s . Moreover, ischemic preconditioning produced by r e p e t i t i v e b r i e f episodes of ischemia did not a f f e c t the severity of subsequently induced I/R injury. A l l these suggest that I/R-induced changes in myocardial antioxidant capacity and ATP l e v e l s can occur independently, although ATP depletion usually accompanies the development of i r r e v e r s i b l e I/R-induced injury. Isolated Langendorff-perfused rabbit hearts subjected to ischemia and reperfusion did not show any changes in myocardial antioxidant capacity, which contrasted with the marked impairment seen in blood-perfused in t a c t hearts. When inta c t hearts were subjected to ischemia in vivo and then a subsequent reperfusion i n v i t r o ( i . e . , Langendorff-perfusion), an impairment in myocardial antioxidant capacity became apparent. These suggest that blood elements may be a c r u c i a l factor involved in the development of I/R-induced oxidant injury. Chronic a l l o p u r i n o l pretreatment provided s i g n i f i c a n t protection against I/R-induced a l t e r a t i o n s in myocardial antioxidant capacity, but not the decrease in t i s s u e ATP l e v e l s . The chronic a l l o p u r i n o l regimen was found to enhance the a c t i v i t y of myocardial GRD, which may, at least i n part, account for the protection against I/R injury. 190 (7) A l l o p u r i n o l and oxypurinol i n h i b i t e d the t r a n s i t i o n metal ion-catalysed ascorbate oxidation and l i p i d peroxidation, l i k e l y as a consequence of t h e i r metal chelating actions, s i m i l a r l y , myoglobin-TBHP-catalysed u r i c acid oxidation and l i p i d peroxidation were also suppressed by a l l o p u r i n o l . Taken together, the res u l t s suggest that a l l o p u r i n o l may favourably a l t e r myocardial antioxidant capacity d i r e c t l y by v i r t u e of i t s t r a n s i t i o n metal chelating properties and i t s antioxidant action i n myoglobin-mediated oxidative processes. The finding that acute a l l o p u r i n o l or oxypurinol treatment did not protect against I/R injury suggests that the time-dependence of a l l o p u r i n o l -induced protection may r e f l e c t the gradual accumulation in tissues of a l l o p u r i n o l or i t s active metabolite oxypurinol to le v e l s s u f f i c i e n t to exert antioxidant e f f e c t s by t r a n s i t i o n metal chelation or other related antioxidant actions. (8) The acute administration of the 21-aminosteroid antioxidant U74006F under conditions comparable to those known to protect against trauma-induced damage in the central nervous system f a i l e d to reduce manifestations of oxidative injury in rabbit hearts subjected to ischemia and reperfusion. Although reactive free r a d i c a l s have been implicated in both types of tissue damage, the observed difference in 191 s u s c e p t i b i l i t y to protection by t h i s s t e r o i d a l antioxidant suggests that the molecular mechanisms involved are not i d e n t i c a l . Given the evidence suggesting that a multiple treatment regimen and heavy metal chelation are important in determining the antioxidant actions of a l l o p u r i n o l , future experiments exploring the possible e f f e c t s of chronic 21-aminosteroid pretreatment on myocardial s u s c e p t i b i l i t y to I/R injury should probably u t i l i z e a compound such as U74500A which i s more e f f e c t i v e than U74006F as a chelator of iron. (9) In the heart-lung transplantation study, erythrocytes from a l l o p u r i n o l - t r e a t e d pigs showed a time/dose-dependent enhancement in antioxidant capacity as r e f l e c t e d in the decrease i n MDA production following in v i t r o oxidative challenge. The extent of red c e l l protection i n both donor and re c i p i e n t animals correlated s i g n i f i c a n t l y with the functional v i a b i l i t y of the transplanted lung tissue, as assessed by tissue water content. I t i s , therefore, suggested that the antioxidant status of red c e l l s may provide a useful assessment of generalized a l t e r a t i o n s i n antioxidant status produced by pharmacological interventions. (10) F i n a l l y , the b e n e f i c i a l actions of a l l o p u r i n o l in various settings of I/R injury has suggested the c l i n i c a l use of a l l o p u r i n o l in protection of tissues 192 against I/R damage, given i t s r e l a t i v e l y low t o x i c i t y . 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