ADVANCES IN MOLECULAR AND CELL BIOLOGY OXYRADICALS IN MEDICAL BIOLOGY Series Editor E. EDWARD BITTAR Department of Physiology University of Wisconsin Madison, Wisconsin Guest Editor JOE M. McCORD Webb-Waring Institute University of Colorado Health Sciences Center Denver, Colorado VOLUME 25 1998 @,A1 PRESS INC. Greenwich, Connecticut London, England Copyright 0 1998 )A/ PMSS INC. 55 Old Post Road No. 2 Greenwich, Connecticut 06836 ]A/ PRESS LTD. 38 Tavistock Street Covent Garden London WC2E 7PB England All rights reserved. No part of this publication may be reproduced, stored on a retrieval system, or transmitted in any way, or by any means, electronic, mechanical, photocopying, recording, filming or otherwise without prior permission in writing from the publisher. ISBN: 0-7623-0379-4 Manufactured in the United States of America LIST OF CONTRIBUTORS Patrick A. Baeuerle Tularik Inc. San Francisco. California Bruce A. Freeman Department of Anesthesiology The University of Alabama at Birmingham Birmingham, Alabama Sonia C. Flores Webb-Waring Institute University of Colorado Health Sciences Center Denver, Colorado Irwin Fridovich Department of Biochemistry Duke University Medical Center Durham, North Carolina D. Neil Granger Department of Physiology LSUMC School of Medicine Shreveport, Louisiana Norman R. Harris Department of Physiology LSUMC School of Medicine Shreveport, Louisiana Brooks M. Hybertson Webb-Waring Institute University of Colorado Health Sciences Center Denver, Colorado lonathan A. Leff Merck and Co., Inc. Rahway, New Jersey joe M. McCord Webb-Waring Institute University of Colorado Health Sciences Center Denver, Colorado vi i viii LIST OF CONTRIBUTORS Sally K. Nelson Webb-Waring Institute University of Colorado Health Sciences Center Denver, Colorado Larry W. Oberley Radiation Research Laboratory University of Iowa Iowa City, Iowa Terry D. Oberley Pathology Service William S. Middleton Memorial Veterans Hospital Madison, Wisconsin Klaus Schulze-Osthoff Department of Internal Medicine I University of Tubingen Tubingen, Germany David Patterson Eleanor Roosevelt Institute Denver, Colorado john E. Repine Webb-Waring Institute University of Colorado Health Sciences Center Denver, Colorado Homero Rubbo Department of Anesthesiology The University of Alabama at Birmingham Birmingham, Alabama PREFACE While we scientists would like to think that our view of the world is purely objec- tive, it is in fact colored by preconceived notions, false assumptions, and prejudices that are not so different from those that distort perceptions for the rest of human- kind. (Perhaps we can claim that we are at least more objective than they are.) In any case, the rapid expansion of the area of free radical biology in the last 25 years has occurred within a framework of assumptions and preconceived notions that has at times directed the course of this movement. The most dominant of these notions has been the view that free radical production is without exception a bad thing, and that the more efficient our elimination of these toxic substances, the better off we will be. The very important observation by Bernard Babior and colleagues in 1973 that activated phagocytes produce superoxidei n order to kill microorganisms,s erved to illustratet hat constructive roles are possible for free radicals. For many in the field, however, this merely underscored the deadly nature of oxygen-derived radicals, both from the microbe’s point of view and from the host’s as well. (Phagocyte- produced superoxide is responsible in part for the tissue injury manifested as in- flammation. See Harris and Granger, Chapter 5, and Leff, Hybertson and Repine, Chapter 6.) Mother Nature, however, has a penchant for being able to make a silk purse from a sow’s ear. If one is dealt a bad hand, one must simply make the best of it. After two decades of focusing on the destructive side of free radicals, the last few years have begun to reveal a new and finer perspective on free radical metabolism-a role in regulation of cellular function (see Schulze-Osthoffa nd Baeuerle, Chapter 2). Evi- ix X PREFACE dence from a number of sources suggests that an increase in the oxidative status of a cell encourages that cell to grow and divide. Increasing the expression of manga- nese superoxide dismutase can suppress the malignant phenotype of melanoma cells (see Oberley and Oberley, Chapter 3). Oxidative stress beyond a certain point seems to trigger a process in normal cells known as programmed cell death or upop- tosis (from the Greek, literally “to fall apart”). Is this suicide response an evolution- ary fail-safe device to curtail tumorogenesis? Does oxidative stress-induced apoptosis account for the loss of immune cells in AIDS (see Flores and McCord, Chaper 4)? This volume attempts to present the spectrum of roles, both good and bad, played by active oxygen species as understood at this point in the evolution of the field of free radical biology. Admittedly and obviously, our knowledge is incom- plete, and still rapidly expanding. Joe M. McCord Guest Editor REFERENCE Babior, B.M., Kipnes, R.S.,& Cumutte, J.T. (1973). Biological defense mechanisms. The production by leukocytes of superoxide, a potential hactericidal agent. J. Clin. Invest., 52,741-744. AN OVERVIEW OF OXYRADICALS IN MEDICAL BIOLOGY Irwin Fridovich I . Introduction-Oxyradicals in Living Cells? ........................... 2 II. Molecular Oxygen and the Spin Restriction. .......................... 2 Ill. O,.- How Much?. ............................................. 3 IV. 0;Some Properties. .......................................... 4 V . 0;-Macromolecular Targets ..................................... 5 VI . HO.fromO; + H,O, .......................................... 6 VII . Paraquat: An 0,. Dependent Mischief Maker ......................... 7 VIII . Nitric Oxide: Another Radical ..................................... 8 IX. Superoxide Dismutases: The First Line of Defense ..................... 8 X . The Consequences of Doing Without .............................. 10 XI . Lactobacilh Plantarurn and a Functional Replacement for SOD ..........1 1 XI1 . Catalases: Members of the Defensive Team ......................... 11 XIII . Summary ................................................... 12 Acknowledgments ............................................ 12 Advances in Molecular and Cellular Biology . Volume 25. pages 1.14 . Copyright Q 1998 by JAI Press Inc . All right of reproduction in any form reserved ISBN: 0-7623-0379-4 1 2 IRWIN FRlDOVlCH 1. INTRODUCTION-OWRADICALS IN LIVING CELLS? Knowledge of the normal, or physiological, provides an excellent foundation for building an understanding of the abnormal, or pathological. It is therefore appropri- ate that a book on Oxyradicals in Medical Biology begin with an overview of the normal biology of oxygen-derived free radicals. That there is a normal biology of oxyradicals would have seemed absurd to an earlier generation of scientists to whom oxyradicals were products of radiolysis and of gas phase reactions, with no relevance to ordinary biology. This view has been completely changed by explorations conducted during the past three decades. We now know that oxyradicals, such as superoxide anion, O;, are routinely produced within and upon living cells and that this occurs during both spontaneous and enzyme-catalyzedr eductions of molecular oxygen. We also know that 0,-i s itself capable of damaging essential biomolecules and that it can, moreo- ver, engender species more reactive than itself, among which are HO,., Fe(II)O, and HO.. This witches’ brew of oxygen-derivedf ree radicals would make aerobic life vir- tually impossible were it not for a multilayered system of defenses. Among these defenses are enzymes which selectively eliminate O;, H,O,, and alkyl hydroperox- ides; low molecular weight antioxidants, both water soluble for action in the cyto- sol and lipid soluble for action within membranes, which limit the extent of free radical chain reactions; enzymes which repair oxidatively damaged DNA and which recycle oxidatively damaged proteins; and finally oxidation-resistanti soen- zymes which, under conditions of oxidative stress, replace their oxidation-sensitive analogues. Let us now examine the biology of oxygen-derivedf ree radicals which underlies the toxicity of molecular oxygen, the oxygen-dependent toxicities of many com- pounds, and numerous pathologies. Then, having looked at the problems that ac- company the aerobic lifestyle, we will enumerate the defenses that allow us to live in the oxygenated biosphere bequeathed to us by the photosynthetic organisms. All of this, the problems posed by oxyradicals, and the defenses, will serve as an entrBe to the chapters that follow. 11. MOLECULAR OXYGEN AND THE SPIN RESTRICTION 0, in the ground state is paramagnetic,w hich denotes unpaired electronic spins. In- deed, 0, contains two unpaired electrons with parallel spin states. This electronic structure imposes a kinetic barrier towards divalent reduction. This hindrance is due to the relative slowness of the inversion of electronic spins and to the need for such inversion of spin when an electron pair is inserted into 0;. This can be made clear by using vertical arrows to depict electronic spin states, as in reaction 1 that depicts the divalent reduction of 0, to H,O, : Overview of Oxyradicals 3 ?? + ?$ +2H+ ?$ ?& -b w w + (1) 0, e pair H,Q We see three up-pointing arrows among the reactants on the left and only two in the product on the right. Obviously one electronic spin must be inverted during this di- valent reduction and that imposes the kinetic bottleneck that is often called the spin restriction. The spin restriction is a formidable barrier to reaction because electron transfer must occur during the moment of collision and because the time required for inver- sion of electronic spin is much longer than the lifetime of a collisional complex. Since there is relatively much more time between collisions, it is easier to reduce 0, by a univalent pathway, in which electrons are donated to it one at a time. This al- lows for spin inversion to occur during the long times between collisions, rather than requiring that it occur during the fleeting lifetime of the collisionalc omplex. The spin restriction thus makes 0, less reactive than it would otherwise be and at the same time favors the univalent pathway. The first of these consequences is for- tunate in that it prevents the spontaneous oxidation of many organic compounds. The second forces living things to deal with the oxyradicals that are intermediates on the univalent pathway, and this is surely the lesser of two evils. That it is so is demonstrated by photodynamic effects in which a dye mediates excitation of 0, by light. Excitation of 0, by -23 kcal inverts one electronic spin and the resultant singlet oxygen can rapidly attack many compounds including the unsaturated fatty acid residues found in biological membranes. Houseflies that have ingested meth- ylene blue-laced sugar water are rapidly killed by light, due to such a photodynamic effect. 111. 02--HOW MUCH? The univalent pathway involves three intermediates between 0, and H,O and these are 0,-,H ,O, and HO.. The first of these, Oy, is easily made both in vima nd in vivo. This is not to say that 0,- is ordinarily a major product of biological oxygen reduc- tion. To the contrary, most reduction of 0, in respiring cells is catalyzed by cyto- chrome oxidase which manages an overall tetravalent reduction of 0, to 2H,O, without the release of any intermediates. Respiring cells try to minimize 0,- pro- duction and do so quite successfully. 0,- production is consequently only a small percentage of 0, reduction. How much 0; is made in respiring cells? Escherichiu coli, growing in a rich aerobic medium at 37OOC, has been examined (May and Fridovich, 1991).T his cell uses 6.2 x lo6m olecules of 0, per second and produces 8 x lo3 0,- in the same time. Were 0; perfectly stable, its concentration within an E. coli cell would increase at a rate of 4.2 @per sIe cond! Since a hepato- 4 IRWIN FRlDOVlCH cyte and E. coli possess comparable defenses against OF, it seems reasonable to sup- pose that 0,- is made at comparable rates in these very different cells. IV. 02--SOME PROPERTIES 0,- can act either as a reductant or as an oxidant. Frequently used assays have been based on both of these properties. There are thus assays in which 0,- reduces ni- troblue tetrazolium (Beauchamp and Fridovich, 1971), or cytochrome c (McCord and Fridovich, 1969), and others in which it oxidizes epinephrine (McCord and Fri- dovich, 1969), 6-hydroxydopamine (Heikkila and Cabbat, 1976), hydroxylamine (Elstner and Heupel, 1976),o r pyrogallol (Marklund and Marklund, 1974). Indeed, in the dismutation reaction, b, 0,- acts both as a reductant and as an oxidant. This is the reaction catalyzed by superoxide dismutases (SODS): b. 0,- + 0,- + 2H++ H20, + 0, 0; is the conjugate base of a weak acid, the hydroperoxyl radical, which has a pK, of -4.8. Protonation of 0,- greatly increases its ability to act as an oxidant. This can be illustrated by the effect of pH on the spontaneous dismutation reaction, which has a second order rate constant at 25" C of -10' M-1S -' in acid solution. The rate constant increases to -lo8 M-' S-I at pH = 4.8, and then decreases by a power of ten + for each unit increase in pH above 4.8. In essence, the HO,. HO,. dismutation is + + + + fast and the HO,. 0,- H+r ate is even faster, while the 0,- 0,- 2H+d ismutation does not occur in the absence of catalysis. Another indication of the enhancement of oxidative capacity of 0,- by protonation is the rate of reaction with linoleic acid. Thus, 0,- does not perceptibly oxidize linoleate but HOz,d oes so at a rate of 1.2 x 103 M-l S-' (Bielski et al., 1983).P rotonation is not the only way to enhance the oxi- dative propensity of Oy. Association with other cationic centers, such as Mn(I1) (McPhail et al., 1976; Bielski and Chan, 1978) or V(V) (Liochev and Fridovich, 1986) exerts a similar effect. In the biological milieu, Oi causes mischief primarily by acting as an oxidant. Among the low molecular compounds known to be oxidized by 0; are glutathione, ascorbate, tetrahydropterins, sulfite, leukoflavins, catecholamines and the enedio- late tautomers of sugars and of sugar derivatives. Since 0; is a univalent oxidant its action can initiate self-propagating chain reactions which amplify the conse- quences of the initiating event. The net effect of such 0,--initiated oxidations is de- pletion of cellular reductants with increased production of H,O,. SOD, by removing O;, decreases such oxidations and spares cellular reductants while di- minishing the production of H,O,. Some investigators, who have observed deleterious consequences of overpro- duction of SOD, have argued that SOD increases the production of H,O, (Yarom et al., 1988; Scott et al., 1987). Were 0,- to be produced in isolation and were it quite