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Handbook of Oxidants and Antioxidants in Exercise Editors: Chandan K. Sen, Ph.D., FACSM Lawrence Berkeley National Laboratory University of California, Berkeley, USA and Department of Physiology University of Kuopio, Finland Lester Packer, Ph.D., FOS Department of Molecular and Cell Biology University of California, Berkeley, USA Osmo O.P. Hanninen, M.D., Ph.D. Department of Physiology University of Kuopio, Finland

2000 ELSEVIER Amsterdam - Lausanne - New York - Oxford - Shannon - Singapore - Tokyo

ELSEVIER SCIENCE BY. Sara Burgerhartstraat 25 P.O. Box 211, 1000 AE Amsterdam, The Netherlands © 2000 Elsevier Science B.V. All rights reserved. This work is protected under copyright by Elsevier Science, and the following terms and conditions apply to its use: Photocopying Single photocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery Special rates are available for educational institutions that wish to make photocopies for nonprofit educational classroom use. Permissions may be sought directly from Elsevier Science Rights & Permissions Department, PO Box 800, Oxford OX5 IDX, UK; phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. You may also contact Rights & Permissions directly through Elsevier's home page (http://www.elsevier.nl), selecting first 'Customer Support', then 'General Information', then 'Permissions Query Form'. In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone: (978) 7508400, fax: (978) 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London WIP OLR UK; phone: (+44) 171 631 5555; fax: (+44) 171 631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works Tables of contents may be reproduced for internal circulation, but permission of Elsevier Science is required for external resale or distribution of such material. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage Permission of the Publisher is required to store or use electronically any material contained in this work, including any chapter or part of a chapter. Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier Science Rights & Permissions Department, at the mail, fax and e-mail addresses noted above. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made.

First edition 2000 Library of Congress Cataloging in Publication Data A catalog record from the Library of Congress has been applied for.

ISBN: 0-444-82650-5 @ The paper used in this pubHcation meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper). Printed in the Netherlands

Preface Through the ages man has sought to harness the knowledge of exercise; interest in exercise science dates back at least to ancient Greece. Today, exercise is seen as not merely a leisure activity, but as an effective preventive and therapeutic tool in medicine. The study of exercise physiology and biochemistry has had a revolutionary overall impact on biomedical research, and exercise has been used as a model for studying the response of physiological regulatory mechanisms to stress. At the same time, advances in oxygen free radical biochemistry have lured exercise biochemists to study the effects of the increased oxygen consumption that accompanies exercise. The first reports in this field, published in the early 1970s, indicated that strenuous physical exercise might cause oxidative lipid damage in various tissues. Since then, a considerable body of research has accumulated concerning the effects of exercise, nutrition and training on indices of oxidative stress and antioxidant responses in various tissues. Most studies support the contention that during strenuous exercise, generation of reactive oxygen species (highly reactive, partially reduced metabolites of oxygen) is elevated to a level that overwhelms tissue antioxidant defence systems. The result is oxidative stress. The magnitude of the stress depends on the ability of the tissues to detoxify reactive oxygen species; i.e., antioxidant defences. Endurance training enhances such defence in various tissues, especially in skeletal muscle and heart. Antioxidants produced by the body act in concert with their exogenous (mainly dietary) counterparts to provide protection against the ravages of reactive oxygen as well as nitrogen species. Exercise and Oxygen Toxicity was first published in 1994. The purpose of this multiauthor volume — the first of its kind — was to examine different aspects of exercise-induced oxidative stress, its management, and how reactive oxygen may affect the functional capacity of various vital organs and tissues. Remarkable interest of the readers and favourable critical reviews published in leading journals provided the impetus to put together an enlarged second edition. We started to accomplish that goal. After two years of tireless effort of many, the current volume was ready to go the press. This volume was over double in size of the original edition. Key related issues such as analytical methods, environmental factors, nutrition, aging, organ function and several pathophysiological processes were thoroughly addressed. Leading experts provided unprecedented insight into the understanding of the role of reactive species and antioxidants. The combination of these properties makes this volume an authoritative treatise. During the course of review of the finalized manuscripts at the publishing house it was brought to our attention that the structure and contents of this volume more closely resembled a Handbook than just a second edition of Exer-

vi

Preface

cise and Oxygen Toxicity. This view was shared by many of our colleagues and peers, and thus we decided to name this volume as the Handbook of Oxidants and Antioxidants in Exercise. Since Exercise and Oxygen Toxicity was pubhshed, interest in exercise, reactive species and the possible role of endogenous and supplemented antioxidants has soared. Search of the PubMed database of the National Library of Medicine using a combination of the keywords "exercise" and "antioxidant" show that the number of research reports in 1997 is more than double of that in 1994. This handbook is therefore a timely publication. It is relevant to all those who have interest in biomedical sciences and is designed to be intelligible to a general scientific audience. We are dehghted to be involved in this project. The excellent editorial assistance of Dr. Savita Khanna and the outstanding contribution of authors are gratefully acknowledged. We hope that this volume will contribute to the further development of this important late-breaking field of research. Chandan K. Sen Lester Packer Osmo Hdnninen

Vll

Contents

Preface

v

Part I: Introduction to free radicals 1.

Free radical chemistry K.-D. Asmus and M. Bonifacic

3

Part II: Reactive species in tissues 2. 3.

Exercise and oxygen radical production by muscle MJ. Jackson

57

Exercise and xanthine oxidase in the vasculature: superoxide and nitric oxide interactions C.R. White, IE. Shelton, D. Moellering, H. Jo, R.R Patel and V Darley-Usmar

69

Part III: Oxidative stress: Mechanisms and manifestations 4. 5.

6. 7. 8. 9.

Chemical bases and biological relevance of protein oxidation O. Tirosh and A.Z. Reznick

89

Lipid peroxidation in healthy and diseased models: influence of different types of exercise KM. Alessio

115

Metal binding agents: possible role in exercise R.R. Jenkins and J. Beard

129

The role of xanthine oxidase in exercise Y. Hellsten

153

Acute phase immune responses in exercise JG. Cannon and JB. Blumberg

177

Oxidative DNA damage in exercise A. Hartmann and A.M. Mess

195

viii

Contents

Part IV: Antioxidant defenses 10. Physiological antioxidants and exercise training S.K. Powers and C.K. Sen

221

11. Superoxide dismutases in exercise and disease K. Suzuki, H. Ohno, S. Oh-ishi, T. Kizaki, T. Ookawara, J. Fujii, Z. Raddk and N. Taniguchi

243

12. Antioxidants and physical exercise C.K. Sen and AH. Goldfarb

297

Part V: Nutrition 13. Dietary sources and bioavailability of essential and nonessential antioxidants E.A. Decker and P.M. Clarkson

323

14. Vitamin E M.G. Traber

359

Part VI: Cellular and molecular mechanisms 15. Biological thiols and redox regulation of cellular signal transduction pathways C.K. Sen 16. Regulation and deregulation of vascular smooth muscle cells by reactive oxygen species and by a-tocopherol A. Azzi, D. Boscoboinik, N.K Ozer, R. Ricciarelli and E. Aratri . . . .

375

403

Part VII: Analytical methods 17. Oxidative stress indices: analytical aspects and significance D. Han, S. Loukianoff, and L. McLaughlin

433

18. Noninvasive measures of muscle metaboHsm T. Hamaoka, KK McCully, T. Katsumura, T. Shimomitsu and B. Chance

485

Part VIII: Environmental factors 19. Air pollution and oxidative stress D.M. MeacherandD.B. Menzel

513

Contents

ix

20. Risk of oxidative stress at high altitude and possible benefit of antioxidant supplementation I. M. Simon-Schnass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

555

2 1. Oxidants in skin pathophysiology S.Weber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

579

Part IX: Organ functions 22. Muscle fatigue: mechanisms and regulation M.B. Reid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

599

23. Oxidative stress in muscular atrophy H.Kondo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

63 1

24. Protection against free radical injury in the heart and cardiac performance D. K. Das and N Maulik . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

655

25. Exercise-induced oxidative stress in the heart L.L.Ji . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

689

26. Influence of exercise-induced oxidative stress on the central nervous system S. M. Somani and K. Husain . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

713

Part X: Aging 27. Oxidants and aging K. B. Beckman and B. N Ames . . . . . . . . . . . . . . . . . . . . . . . . . . . .

755

28. Caloric restriction, exercise and aging R.JM. McCarter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

797

29. Oxidative stress and the pathogenesis of sarcopenia M.E. Lopez, T A . Zainal, S.S. Chung, J M . Aiken and R.Weindruch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

83 1

30. Molecular mechanisms of oxidative stress in aging: free radicals, aging, antioxidants and disease M. Pollack and C. Leeuwenburgh . . . . . . . . . . . . . . . . . . . . . . . . . .

88 1

Part XI: Disease processes 31. Oxidative stress, antioxidants and cancer M.Gerber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

927

X

32. Alcohol and oxidative stress as. Lieber 33. Cigarette smoking as an inducer of oxidative stress in relation to disease pathogenesis G.G. Duthie, J.R. Arthur and S.J. Duthie

Contents

951

977

34. Regulation of inflammation and wound healing J.J. Maguire

995

35. Oxidative stress induced by the metabohsm of medical and nonmedical drugs M Paolini and G. Cantelli-Forti

1021

36. The paradoxical relationship of aerobic exercise and the oxidative theory of atherosclerosis R. Shern-Brewer, N. Santanam, C. Wetzstein, JE. White-Welkley, L. Price and S. Parthasarathy

1053

37. Claudication, exercise and antioxidants P.V.Tisi and C.P Shearman

1069

38. Exercise and oxidative stress in diabetes mellitus D.E. Laaksonen and C.K. Sen

1105

39. Exercise induces oxidative stress in healthy subjects and in chronic obstructive pulmonary disease patients J. Vina, E. Servera, M. Asensi, J. Sastre, F.V. Pallardo, A. Gimeno, L. Heunks, P.N.R. Dekhuijzen and J. A. Ferrero

1137

40. Hypoxia, oxidative stress and exercise in rheumatoid arthritis S. Jawed, S.E. Edmonds, V. Gilston and D.R. Blake

1147

Index of authors

1189

Subject index

1191

Parti Introduction to free radicals


©2000 Elsevier Science B.V. All rights reserved. Handbook of Oxidants and Antioxidants in Exercise. C.K. Sen, L. Packer and O. Hanninen, editors.

Part I • Chapter 1

Free radical chemistry Klaus-Dieter Asmus^ and Marija Bonifacic^ ^Radiation Laboratory, University of Notre Dame, Notre Dame, IN 46556, USA '^Ruder Bockovic Institute, Department of Physical Chemistry, RO. Box 1016, 10001 Zagreb, Croatia

1 INTRODUCTION 2 PROPERTIES AND DETECTION OF FREE RADICALS 2.1 What is a free radical? 2.2 How fast are free radical reactions? 2.3 Detection of free radicals by time-resolved optical spectroscopy 2.4 Detection of free radicals by electron spin resonance 2.5 Spin trapping 2.6 Product analysis 2.7 Reduction potentials 2.8 Where to find rate constants, ESR splittings, reduction potentials, etc. 3 OXYGEN RADICALS 3.1 The hydroxyl radical 3.2 Peroxyl radicals 3.3 Reversible oxygen addition 3.4 Alkoxyl radicals 3.5 Phenoxyl radicals 3.6 Semiquinone radicals 3.7 Superoxide (02*" / HOs*) 4 SULFUR-CENTERED FREE RADICALS 5 CONCLUSION 6 SUMMARY 7 ACKNOWLEDGEMENTS 8 ABBREVIATIONS 9 NOTES 10 REFERENCES

1

INTRODUCTION

It is well accepted these days in the scientific community that free radicals participate in many biological processes, often in a quite decisive manner [1—8]. This clearly emerges from the still rapidly increasing number of studies on this subject and the very important fact that such investigations are not restricted to the views of just one particular group of researchers. Free radical studies in biological systems embrace practically all fields from fundamental chemistry to applied clinical studies these days and have proved to be particularly successful when scientists of the various fields engage in collaborative work and extensive interdisciplinary discussions. In living systems, including humans, the presence of free radicals is something very natural. The decision of nature to provide the enzyme superoxide dismutase

4

Part I: Introduction to free radicals

(SOD), for example, clearly hints on the existence of the superoxide radical, O2* , - considered to be one of the most prominent radical species in oxygenated biological systems - and it also shows the obvious need of cellular systems to regulate its concentration [9—15]. While this serves as an example that a "too much" of a particular radical species is not in the interest of a biological system there are, on the other hand, also observations where a "too little" of radicals seems equally unwanted. For example, healthy cervix tissue has been reported to contain quite a measurable radical concentration whereas a cancerous tissue is almost completely devoid of radical species [16]. In general, however, it seems that the majority of malignant disturbances is associated with an increase in free radical concentration [17]. Free radicals are undoubtedly involved in many degenerative processes including aspects of aging, cancer, cardiovascular diseases, arteriosclerosis, neural disorders, skin irritations and inflammations. Reperfusion injuries, e.g., in the aftermath of surgery, are also typically characterized by an enhanced formation of free radicals [18]. Actually, any metabolism involving redox active centers is, per se, a possible source of continuous free radical production. This certainly allows the extrapolative conclusion that any stress situation, including exercise, carries the potential of excess free radical production associated with all the chemical consequences arising thereof Most of the radicals relevant in biological systems are either derived from or associated with the presence of molecular oxygen, in particular, the already mentioned superoxide anion (O2*) and peroxyl radicals (ROO*). Other oxygencentered and highly reactive radical species are hydroxyl (*OH) and oxyl radicals (RO*). Their biological significance is not in doubt but somewhat more in the debate than that of 02*^ and ROO*. Of course, it is not only oxygen-centered free radicals which are of importance and interest. Many others, generated both from endogenous as well as exogenous substrates are also of significance. A particularly relevant example is the thiyl radical, RS*, which is the one-electron redox intermediate between thiols and disulfides, two vital classes among the biologically abundant substrates [19,20]. The action of free radicals is generally determined by their chemical reactivity and the availability of a suitable reaction partner in the vicinity of their production site. In some cases such a radical-molecule interaction may directly lead to a biological damage in a few or even just one reaction step [7]. Thus, an •OH reaction with the sugar moiety of DNA can result in a strand break due to a radical specific phosphate cleavage reaction. Such a singular event does, however, not necessarily imply cell death as there are enzymatic and chemical, possibly even radical associated repair mechanisms. Accumulation of hazardous compounds as a result of free radical reactions is often the reason for long-term effects. In this case it may even be difficult to prove the free radical's responsibility It should also be pointed out that the appearance of free radicals does not automatically imply their direct participation in the disturbance of vital biological functions. In other words, free radical reactions are not necessarily the cause of disorders but may equally well just be a consequence thereof In this case free

Chapter 1: Free radical chemistry

5

radicals and their reactions may, however, serve as useful markers. Nature and modern medicine related science have provided us with mechanisms and substrates to cope with free radicals by "deactivation". Enzymes such as the above already mentioned SOD serve this function. Another important group of compounds in this respect are the so-called antioxidants [22,23]. Most prominent representatives in this respect are vitamin E (a-tocopherol) and vitamin C (ascorbate) which are most effective free radical scavengers in the lipid cell membrane and adjacent, more aqueous compartments, respectively. The same function is assigned to many other redox-active compounds such as, for example, thiols, carotenoids, quinones, etc. Whenever a free radical reacts with a molecular compound it loses its identity but at the same time it proliferates its general radical properties to a new radical formed in this reaction. In order to assess the action of free radicals it is, therefore, necessary to know not only the properties of the initiating but also of all subsequently generated species. The aim of the present article is, therefore, to elucidate on all these aspects and to provide an understanding for the chemical basis of free radical processes. In the first part of our essay we shall focus on the general properties of free radicals, their generation in chemical model systems, and provide some basic information on the most common methods of their detection. Later we shall focus on the identification and chemical properties of specific groups or individual free radical species which are considered to be of potential significance in biological processes. To find out whether the latter actually applies is then a challenge for the biochemist, biologist and medical researcher. It must, of course, always be remembered that a chemical "test tube" or in vitro experiment can only provide the basic information what a free radical can and will do chemically when meeting a suitable reaction partner. Such knowledge is an absolute prerequisite to understand a free radical's potential action. By no means can an in vitro experiment, however, substitute for the in vivo situation where many more parameters need to be taken into the consideration. 2

PROPERTIES AND DETECTION OF FREE RADICALS

2.1 What is a free radical? A free radical is, per definition, a molecule or just a single atom with an unpaired electron, conventionally symbolized by a radical dot "*". Examples, extremes from the molecular size point of view, would be a DNA radical (4' in sugar moiety) and a bromine atom. phosphate - O - CH2

base

7 O— phosphate ^^^'^

6


Both are species of interest in certain biological processes. The DNA radical is generated, for example, by reaction of an •QH radical with this vital macromolecule in any cellular system exposed to ionizing radiation and is a direct precursor for a potential strand lesion [7]. The bromine atom, on the other hand, is formed en route of reductive degradation of 1,2-dibromoethane, a well-known toxin [24-30]. What means "unpaired" electron? Typically, any electron seeks the association with another of its kind, so that they can pair up their spins in a system of lower energy A spin is the rotational motion of an electron which can attain two directions and, viewed in a simplified picture, sort of represent a right handed (or "up") and a left handed (or "down") version. Two radicals "shaking hands", i.e., teaming up two individually provided unpaired electrons of opposite spin, form a new bond with this process:

T

+

i

^

(1)

n

A chemical example would be the combination of two bromine atoms which results in the formation of the Br-Br a-bond: Br*^

+

Br*^

-^

Br - Br (with the spins t i in the bond) (2)

Generally, any even number of electrons in a molecule arranges in "up"-"down" spin coupled pairs, both in bonds or as nonbonding electron pairs. Usually this is an energetically quite favorable process. Sometimes, however, it may be more economic from the energy point of view that two electrons remain uncoupled, even within the same molecule. In this case the two unpaired electrons attain the same spin direction and the chemist talks about a "triplet state" or, if they are located at different atoms, a biradical. A most important example for this situation is the molecular oxygen which we generally view as a molecule with a double bond (a). The two electrons of its p-bond seem, however, to some extent separated and may thus act as if there were two centers providing one unpaired electron each (b): 0 = 0

(a)

^•O-O*^

(b)

The presence of unpaired electrons or unpaired spins has a most important consequence, namely the generally very high reactivity of such species.^ This, of course, reflects the genuine desire of the radical species to attain the most favorable energetic state by coupling its unpaired spin with that of an opposing sign in a new bond between two spin carrying atoms. Accordingly, most radicalradical and also many radical-molecule reactions occur as soon as the two reacting species meet each other. Such a reaction is, as we say, a diffusion controlled process. The time it actually takes for completion of any chemical reaction depends on two parameters, a reaction specific rate constant (which attains its


7

highest value for a diffusion controlled reaction) and the concentration of the reacting species. In biological systems the free radical concentration is usually very low and rarely acquires values high enough so that a radical-radical reaction could beat a reaction of a radical with a suitable molecular partner. The latter are typically present at much higher concentration in the near-by vicinity In order to illustrate this, let us discuss a simple example. Carbontretrachloride, CCI4, known as a potent liver toxin, is readily reduced by the enzymatic P-450 system of the endoplasmic reticulum [31]. In the presence of oxygen this results in the formation of CCI3OO*, i.e., trichloromethylperoxyl radicals as an important intermediate in the CCI4 degradation process (further mechanistic details on peroxyl radical decay will be presented later in this article). Its steady state concentration is, however, extremely small and only few research groups with highly sensitive equipment and great experience have been able to actually detect this radical in vivo [32]. It is probably even lower or, at most, comparable to the steady state radical concentration of ^ 1 pM (^10"^^ M) prevailing in systems which are subjected to y-irradiation in an average research ^^Co-source. In the absence of any suitable reaction partner the above peroxyl radicals have, indeed, no other chance than to react with each other: CClsOO* + CClsOO*

->

products

(2k = 2 x 10^ M ^ s"^) [33] (3)

The half-life for such a so-called second order process is mathematically given by an equation which includes the rate constant (for this kind of second order process defined as "2k") and the radical concentration (in molar units) [34]: ti/2 = 1 / {(2k) X [CCIBOOT } = 1 / {2 X 10^ X 10"^^} = 5 000 s

(I)

On the other hand, if a scavenger such as vitamin E (a-tocopherol) was around in just nanomolar (10^ M) concentration the following reaction would successfully take over: CClsOO* + Vit E

^

products

(k = 5 x 10^ M~^ s^) [35]

(4)

When the concentration of the molecular reaction partner (Vit E) significantly exceeds that of the radical (CCI3OO*) the reaction becomes of pseudo-first order (reflecting the fact, that the concentration of the excess component hardly changes during the course of the reaction). For such a condition, the mathematical equation attains a slightly different form, the most important difference being that it contains the concentration of the molecular reaction partner and not that of the radical: ti/2

=

In 2/ {k X [Vit E] }

=

1 / {5 x 10^ x 10 ^}

=

1.4 s

(II)

By comparison, these two half-lives show that the scavenging of the CCI3OO*

8


radical by Vit E is several orders of magnitude faster, i.e., more probable to occur than the mutual radical-radical deactivation. While this example, from the biological point of view, pertains more to the lipid phase (where Vit E is preferentially located) a similar system may be considered for the aqueous compartments. Thiyl radicals, RS*, although not oxygen-centered but nonetheless equally frequent and important as any oxygen-centered free radical in cellular systems, readily react with the water-soluble ascorbate (Vit C), as formulated in the following equation for the thiyl radical, GS*, from the probably most abundant thiol, glutathione (GSH): OS* + ascorbate (AH) -^ GS" + A*"

(5)

With a rate constant of k = 6 x 10^ M^^ s"^ [36] and an estimated Vit C concentration (e.g., in liver tissue) in the low millimolar range (10~^ M) the half-life of this process, calculated according to eq.(II), assumes a value of about 1 ms. For a second order thiyl radical recombination process (2k6 ^ 10^ M ^ s~^) [37] to compete, i.e., to occur with a comparable half-life, a GS* concentration of about 1 mM would be necessary (calculated with eq. (I)). Such a high steady-state radical concentration is, however, pretty unrealistic in any biological system. GS* + GS* -> GSSG

(6)

Therefore, any radical-radical reaction is extremely unlikely to occur and the discussion of radical reactions in biological systems should accordingly always be checked first with respect to possible radical-molecule processes. Even if the latter are slow they may still benefit from the usually considerably higher concentration of the molecular reaction partner as compared to the free radical concentration. 2.2 How fast are free radical reactions? As mentioned above most radical reactions are essentially controlled just by the diffusion of the reacting species. In liquid environment this typically results in rate constants within the order of 10^ M ^ s~^ with the individual value depending on the size and structure of the respective species, on solvation effects, and also on the viscosity of the environment. One intrinsic parameter lowering this value could be an internal stabilization of the free radical, meaning delocalization of spin and electron density into the existing regular bonds or to atoms of particularly high electron affinity. Often it is feasible to characterize such radicals by resonance structures, especially when aromatic TT-systems are involved. For example, phenoxyl type radicals (structurally similar to the chromanoxyl radical obtained upon oxidation of Vit E) mostly behave as oxygen-centered radicals (a) but certain reactions such as di- and oligomerizations can only be understood in terms of their carbon-centered resonance forms (b) and (c) [38]:


°^

°=0

(a)

(b)

(c)

Sometimes such resonance structures are so stable that the radical becomes more and more persistent and may, in extreme cases, even be bottled. At the same time the reactivity towards other radicals and molecular substrates becomes lower and lower, and rate constants accordingly diminish, possibly by many orders of magnitude. A second parameter which influences the rate of a radical reaction pertains to radical-molecule reactions: R*

+

A- B

-^

R - B

+

A*

(7)

In this case, the initial radical R* succeeds in pairing up its lonesome electron, a process certainly beneficial for a high rate constant. At the same time, however, it is necessary to break the molecular A - B bond, and this is a process which requires energy and is, therefore, of adverse influence on the overall rate of reaction. In fact, the complete assessment of the kinetics of such processes must also take into account the energy contents of the product species and, in particular, of the transition state in going from the educt to the product system. This transition state, in which (with reference to the above example) R* has associated already with A - B but A* has not left yet (i.e., [R...B...A]*, has, per definition, always the highest energy content along the reaction trajectory. The energy difference between the educts and the transition state is the so-called activation energy, and this is essentially the parameter which decides on the overall rate constant of a reaction. For a typical diffusion controlled process the activation energy is so small that it is essentially provided by the Brownian motion of the reacting partners. However, if this activation energy becomes higher statistically fewer and fewer of the reacting species provide the necessary internal energy to, literally spoken, push the reaction over the transition state mountain. Such reactions are not any more diffusion but activation energy controlled. An example, quite relevant and important from the biological point of view, would be an oxidative cleavage of a C - H bond by, e.g., a trichloromethyl peroxyl radical: I

-C-H

i

+

CC1300*

- ^

-C*

+

CCI3OOH

(8)

Abstraction of a bisallylic hydrogen atom from linoleic acid, for example, has been reported to occur with a rate constant on the order of 10^ M"^ s^ [39], i.e., three orders of magnitude below the diffusion limit. This means that, despite the strong activation these hydrogen atoms receive from the allylic double bonds of this PUFA, statistically only one in about a thousand encounters actually leads

10


to completion of the above reaction. An important consequence of these rate constant considerations is the following: radicals which are generated in the vicinity of substrates will definitely undergo fast and quantitative reaction with the latter whenever this reaction is a diffusion or near-diffusion controlled process. Such a situation does not allow any migration of the radicals over larger distances and must, therefore, not be considered for any direct action at distant sites or longer times after their generation. In this respect it may, in fact, be interesting to know how far a species can actually move until it reacts. Let us consider, for example, a hydroxyl radical *OH generated e.g., in a radiation exposed biological sample. These radicals are known to react with almost any available C - H bond with rate constants > 10^ Mr^ s"^ [40,41]. Assuming potential targets of this kind in a cellular fluid of at least millimolar concentration this translates into a half-life of the *OH radical of 1 V for the CHsOO* and CClsOO* systems, respectively [95]. One difficulty for an unambiguous determination of such potentials arises from the fact that peroxyl radicals may not only get involved in true electron transfer processes but often oxidize their reaction partners via an addition-elimination mechanism. This seems to apply, for example, for the peroxyl radical induced oxidation of organic sulfides (R2S, e.g., methionine) where the first step en route to a sulfide radical cation is an addition, followed by a substitution and equilibration of the transient three-electron bonded dimer radical cation to the molecular radical cation R2S*^ [96]. R2S

+

->

CCIBOO*

CCl300-*SR2 (R2S..SR2)^

+ ^

R2S R2S*^

CCl300-*SR2 -^

CCI3OO +

R2S

(34) +

(R2S.*.SR2)'^ (35) (36)

It will not be difficult to appreciate that such a complex mechanism cannot be linearly related to any redox potentials. Even more so, because the adduct radical, in competition with reaction 35, enters yet another pathway in which the sulfuranyl radical electron is transferred intramolecularly to the hydroperoxide moiety


29

(under participation of water) to yield sulfoxide and a reduced hydroperoxide (which decays into the oxyl radical): CCI3-O-O-SR2

•

(CC1300H)*"'

^ CClaO* + OH"

+ R2S(OHf

j

(37)

Yt + R2SO

This latter process constitutes a most interesting mechanism in the sense that it actually describes the second step of an overall two-electron transfer initiated by a peroxyl radical. The two-electron oxidation product is the sulfoxide and the corresponding two-electron reduction counter part is the oxyl radical. Formally, this peroxyl-radical-induced sulfoxide formation via Eqs. 34 and 37, may also be viewed as oxygen atom transfer, however, studies with labeled oxygen indicate that the sulfoxide oxygen is coming from the solvent water [96]. (This does not mean that the possibility of oxygen transfer has to be dismissed in all cases; it seems to be a real possibility in the peroxyl induced oxidation of certain organic tellurides, for example) [97]. The capability of peroxyl radicals to undergo both, one-electron and twoelectron processes pertains particularly for the sulfide-to-sulfoxide oxidation and would, therefore, be of special biological relevance for methionine and methionine-containing peptides and proteins. Generally, the sulfoxide yields, produced via the 2-e-mechanism, are higher than via the 1-e-mechanism [96,98] where, e.g., competing radical cation deprotonation may come into play Absolute rate constants for addition reactions of peroxyl radicals have hardly been measured so far but can reasonably be anticipated, at least for the above addition to sulfides, to be in the range of diffusion controlled values. Electron transfer reactions from a donor to the peroxyl show a distinct dependence on the electron density at the peroxyl radical site. The lower the latter the faster, in general, the reaction. This is nicely exemplified by a series of chloromethylperoxyl radical induced oxidations of ascorbate where the rate constant increases from 2.2 X 10^ to 2.0 X 10^ M"^ s"^ from CHaOO* to CClsOO*, respectively [99]. These early figures have, in the meantime, been corroborated by many more examples for 1-e-oxidation reactions of peroxyl radicals with different degrees of halogenation [69,92,95,99-102]. Besides electron transfer and addition reactions peroxyl radicals also undergo hydrogen atom abstraction reactions, and thus resemble quite similar reactivity features as *OH. The most important abstraction process is probably the chain carrying reaction in lipid peroxidation (with R - H = PUE\) and other autoxidation processes [1,7,13,15,17,103—106]. In principle, any carbon-centered radical, R*, may get involved in the following reaction sequence:

30

P^^t I' Introduction to free radicals

R*

+

ROO*

O2 +

^ R-H

ROO* ^

(38)

ROOH

+

R*

(39)

Reaction 39 is the slower of the two processes and thus rate controlUng. Not too many rate constants are known. Pubhshed values range from the 10"^ - 10"^ M ^ s"^ order of magnitude [66,69] and, to mention a biologically relevant example, k39 = 36 M"^ s^ has been reported for the peroxyl radical reaction in the autoxidation of linoleic acid [106]. It should further be mentioned that the chain length involving the above two processes significantly increases when going from homogeneous solutions to micellar aggregates [107,108] and, by extrapolation, lipids. A plausible explanation for this is the restricted diffusion of the radicals and, as a result thereof, a much lower probability for radical-radical termination processes. In all the discussion of peroxyl radical reaction kinetics, including those for the abstraction processes, it must be recognized that peroxyl radicals are highly polarizable [109] and its electron density distribution depends to a significant extent on the polarity of the solution. Therefore, absolute rate constants will vary with the nature of the environment [110,111] in which these reactions occur and caution is advised when trying to extrapolate aqueous or specific organic solvent in vitro figures to, for example, cellular conditions. In first approximation, it is probably safe though to operate within the given order of magnitude. In the absence of suitable molecular reaction partners, or if the corresponding reactions are too slow, peroxyl radicals may decay via unimolecular processes [7,21,112]. The most prominent and, from the biological point of view, most significant mechanism is a superoxide ehmination. Prerequisite for this process is a suff'iciently large electron delocalization into the peroxyl group. This may be achieved by simultaneous stabilization of a positive, i.e., electron deficient entity as it would be the case upon liberation of a proton. Since the latter is easily cleaved from hydroxyl groups, for example, any a-hydroxyl peroxyl radical is prone for such a process [113—117].

C

•

C = 0

+

H^

+

02*-

(40)

This elimination is considered to be assisted by a transient cyclic five-membered ring structure in which the hydroxyl proton interacts with the terminal peroxyl oxygen. Typical rate constants for this first-order decay are on the order of 10^ 10^ s ^ corresponding to half-lives in the lower microsecond region. The superoxide elimination becomes even faster in basic solutions [114,117] where the ahydroxyl group increasingly equilibrates to its conjugate anion (-OH ;^ - O ). The positive charge may also be stabilized within the remaining molecule. With


31

a diall^lamine substituent, for example, the peroxyl radical becomes even so unstable that it actually escapes detection [118]:

C "^

•

C=NR2

+

O2-

(41)

^NR2

Steric arguments can also be forwarded to rationalize intramolecular hydrogen abstraction or addition reactions by peroxyl groups. Five- and six-membered ring structures would thus facilitate hydroperoxide and epoxide formations (the latter via an endoperoxide radical) [21,119]. Respective examples are given in the following: >.

/

\

/

yO

yO

^ O — C / "

O*

C

/ \

O*

^ C = C

/

\ •

/

yO

C

/ \ .

^C

V

yO

/

\ o — C -

O

\

1/

C

OH

(42)

oI

— VQ . —

r\

/ \\ / ^ \ / C C

(43)

I

The actual rates of these processes depend very much on the electronic influence exerted by the substituents near the reactive sites. Finally, if there are neither suitable reaction partners nor the possibility for intramolecular conversion, the peroxyl radicals will suffer the ultimate free radical fate, namely, a bimolecular radical-radical deactivation. The actual mechanism of the generalized process ROO*

+

ROO*

^

[ROO-OOR]

^

products

(44)

may be quite manifold but is considered, in any case, to proceed via an intermediate tetroxide. At room temperature these tetroxides are usually not stable enough for analytical detection. Their transitory existence seems beyond doubt though as emerges from low temperature studies [120,122] as well as from the following mechanistic considerations. Generally, the bimolecular termination reactions (Eq. 44) are quite fast in terms of rate constants, and long lifetimes result mostly only because of the low steady-state concentrations of these radicals. This pertains, in particular for primary and secondary peroxyl radicals, i.e., those which still carry at least one hydrogen atom at the peroxyl carbon. Many measured bimolecular rate constants on the order of 10^ M ^ s^ (2k4o) indicate diffusion controlled processes


32

[66,69]. Deviations to lower values can mostly be rationalized in terms of steric hindrance. Tertiary peroxyl radicals, on the other hand, terminate with significantly lower rate constants, e.g., 2k = 2 x lO'* M~^ s~^ for /^r^butylperoxyl [123]. This probably does not only reflect steric constraints but also the smaller number of possible reaction channels available, as compared with primary and secondary peroxyl radicals. Tertiary peroxyl radicals, R3COO*, practically all decay via the tetroxide and by subsequent oxygen elimination to the respective oxyl radicals (the three substituents R do not need to be identical): 2 RsCOO*

[R3COOOOCR3]

2 RsCO*

+

O2

(45)

The further chemistry is then that of the oxyl radical which will be discussed in detail in a later chapter. This type of reaction may also apply to primary and secondary peroxyl radicals where at least one R=H. However, for these species additional routes become possible. The most preferred pathway is, in fact, a cyclic mechanism known as "Russell mechanism", the key element of which is considered to be a cyclic sixmembered transition structure of the tetroxide [124]. Three bond ruptures and a hydrogen transfer directly yields one molecule of oxygen, an alcohol and an aldehyde or ketone. From the redox point of view this constitutes a disproportionation. .0. O

R,H-

O

/V^^^'

R,H

I I

R,H / H

>C = 0

+

>CH-OH +

•R,H

(46)

An interesting aspect in this mechanism is the possible generation of single oxygen, ^02, which has been claimed to be formed in organic solutions [125,126]. In aqueous solutions, on the other hand, the natural triplet oxygen, ^02, seems to be cleaved which is then, however, accompanied by a triplet excited carbonyl compound [127]. Another decay mode of the tetroxide leads to two carbonyl molecules and, instead of oxygen, to hydrogenperoxide (R = H and ^ H): [R2HCOOOOCHR2]

2 R2C=0

+

H2O2

(47)

This alternative pathway is also considered to involve cyclic transition structures which, in aqueous solution, probably include water molecules [21,112,128]. In addition to these general features there are further specific characteristics to certain peroxides which are mainly attributable to individual structural and elec-


33

tronic parameters. This is not the place to present and discuss any of them in greater detail (for more information see [21,112]), and just one group-specific example shall be mentioned here. Thus, all peroxyl radicals generated as a result of oxidative or reductive radical attack at aromatics show a high tendency to embark on reaction routes which re-establish the aromaticity This clearly reflects thermodynamic stability criteria. A specific example is the •QH-induced oxidation of benzene in oxygenated solution for which phenol is formed as final product with about 60% efficiency via the following route [21]: OH H

OH H

O,

OH

r r S C ^r^•

-HO, (48)

Some further words also about halogenated peroxyl radicals: As mentioned above most bimolecular peroxyl termination processes lead to alcohols and aldehydes as stable molecular products. If any of the hydrogen substituents at the hydroxylcarrying or carbonyl carbon is a halogen atom then these compounds (a-haloalcohols, "oc-haloaldehydes" = acyl halides) assume, in fact, a higher oxidation state and, upon hydrolysis, directly convert into aldehydes/ketones and acids, respectively [65,129,130]: >CH-OH alcohol

>CX-OH a-haloalcohol

-CH=0 aldehyde

-CX=0 acyl halide

(H20)^

(H2O) -^

>C=0 aldehyde/ketone

(49)

-COOH acid

(50)

Any metabolic degradation of halogenated organic material, proceeding via peroxyl radicals as intermediates, would thus yield aldehydes and acids. The bad reputation of aldehydes as toxins is well known [131], and the organic acid (in combination of the inorganic acid H'^/X released in the hydrolysis processes) may exert a harmful influence due to pH changes. 3.3 Reversible oxygen addition Addition reactions, as organic chemistry has shown in many examples, inherently seem to include the possibility to be reversed. Free radical addition to a double bond and subsequent P-scission, in principle, also fall into this category although the cleaved radical may not be identical with the incoming one: R* + -C(Y) = C < ^

-C(R)(Y)-C*< ^

-C(R) = C < + V

(51)

The question whether a reaction is reversible or not is of principle significance.

34

Pcirt I: Introduction to free radicals

Any irreversible process is a once-and-forever event. A reversible reaction, however, always means an equilibrium which may have a preferred side but has not closed the door yet for the back reaction. And most important, any substrate which engages a "minority" component in an irreversible reaction pulls eventually the entire equilibrium back to the "wrong side". An instructive example is a thiyl radical mediated degradation of carbontetrachloride in the presence of isopropanol [132—135] where the equilibrium RS*

+

(CH3)2CHOH

^

RSH

+

(CH3)2C*OH

(52)

lies far on the left hand side. It may, nevertheless, completely be shifted to the right in the presence of CCI4 which engages the iso-propanol radical in a dissociative, i.e., irreversible electron transfer reaction: (CH3)2C*OH + CCI4

^

(CH3)2CO + H^ + CI

+ •CCI3

(53)

What about a possible reversibility of oxygen addition to free radicals? The answer is: Yes, it may well occur but it depends on the nature of the addition center and seems connected with the electrophilicity of the molecular oxygen [21]. Practically no addition has ever been observed, at least at room temperature, to organic oxygen-centered radicals, i.e., the possible reaction if occurring at all, must be reversible with the equilibrium way on the left hand side. -O*

+

0

2

^

-O-OO*

(54)

This pertains to all oxyl and peroxyl radicals. An interesting exception is the basic form of the hydroxyl radical, 0*~, which in the presence of oxygen has been reported to form the ozonide radical anion, O3* [136]. Carbon-centered free radicals are generally prone for irreversible oxygen addition. However, there are some examples also for a reversible process. One of them is the addition of O2 to the hydroxyhexadienyl radical formulated in Eq. 48. Here, the peroxyl radical is formed with a rate constant of 3.1 x 10^ M"^ s"^ (ti/2 ^ 8 ms in air saturated solution) and the back reaction, i.e., the re-elimination of oxygen occurs with 1.2 x lO'* s^^ (ti/2 ^ 57 ms [137]. A biologically most relevant example is the oxygen addition to those pentadienyltype PUFA radicals [138,139] which result from abstraction of the particularly labile bisallyllic hydrogens by practically any kind of reactive free radical, even RS* [86] and ROO* [69]. An interesting feature in this context is also that oxygen preferentially does not add to the bisallylic site but to a resonance form thereof with the result of a thermodynamically favorable conjugate double bond structure [139-141]:

(55)


35

The peroxyl radical, incidentally, exhibits the same UV absorption (234 nm) which is characteristic for conjugated dienes, in general. Another example for reversible oxygen addition, and also of particular biological relevance, is provided by thiyl free radicals. For quite some time and various reasons in dispute, there seems to be convincing evidence now for an equilibrium between thiyl and thioperoxyl radicals [142—145]: RS*

+

O2

^

RSOO*

(56)

Rate constants for the forward reaction and back reactions of about 2 x 10^ M"^ s^ and 6 X 10^ s ^ respectively, have been measured and they appear to be independent on the nature of R. Accordingly, an equilibrium constant of about 3 X 10^ M ^ applies. (Measured values refer to cysteine, glutathione and 2mercaptoethanol). Does such a reversible oxygen addition to a sulfur-centered free radical make sense? From the electronic point of view certainly yes. The electronegativity of sulfur lies in between that of oxygen and carbon, and, as we have seen before, oxygen does not add to oxygen-centered free radicals (i.e., an "addition" is highly reversible) while most of the respective processes with carbon-centered free radicals seem irreversible. The chemistry of thioperoxyl radicals has not yet completely been revealed and still leaves open questions particularly with respect to the redox properties of RSOO*. In part, this is probably due to the above equilibrium (Eq. 56). Further complicating factors with respect to establishing a reliable and quantitative picture arise from side reactions [145—150]. A most interesting one is a rearrangement by which the oxygen-centered is converted to a sulfur-centered sulfonyl free radical [146]. This process, depicted in Eq. 57, is of considerable significance because the sulfonyl radical can add another molecule of oxygen to yield a sulfonyl peroxyl radical [146,151] which itself is considered to be a precursor of sulfonic acids. O R-S-0-0*

^-^

•

R—S*

(RS02*)

(57)

O RS02*

+

O2

^

RS0200*

^ -^ ^

RSO3H

(58)

3.4 Alkoxyl radicals We have learned already that oxyl radicals may be formed en route of the peroxyl radical decay. One direct possibility to generate them is by hydrogen atom abstraction from a hydroxyl group, e.g., in the *OH reaction with an alcohol:

36

P(^^t I: Introduction to free radicals

R-OH

+

•OH

-^

R-O*

+

H2O

(59)

This reaction competes, of course, with the C - H cleavage in other parts of the molecule, particularly in a-position to the hydroxyl group, as discussed already in the chapter on *OH reactions. The yield of O - H cleavage in aliphatic compounds or, better, detectable alkoxyl radicals appears pretty small and attains, at most, ca 7% relative to the reacting 'OH in the case of methanol [88]. This figure might, however, only represent a lower limit. As we shall see later there is a fast intramolecular rearrangement (1,2-hydrogen shift) which may take place with certain oxyl radicals and thus obscure part of the initial yield. Oxyl radicals are also generated upon reductive degradation of peroxides and hydroperoxides. The key feature of the underlying reaction is that the incoming electron is accommodated in an antibonding a* orbital which causes an O - O bond lengthening and thus prepares it for easier rupture. R-O-O-R(H) + e

^

[R-0.\0-R(H)]

-^

RO* +

OR(H)

(60)

This mechanism is, incidentally the same, as for the reduction of H2O2 (resulting in •OH + OH" formation) and disulfides [75,149-153] (resulting in RS* + RS" formation) (for further details on this latter process and the three-electron bond " .*." notation, see section on sulfur-centered free radicals below). The intermediate ( R - O .*. 0-R)~ radical anion is generally much too short-lived as to become of direct chemical significance or to establish a measurable equilibrium with the oxyl radical. Reaction 60 reminds us of the well-documented Fenton-Haber-Weiss process. Whenever the reductive power of enzymatic systems may not be sufficient for an electron transfer to peroxides and hydroperoxides this function can be served by low oxidation state metal ions, particularly iron-(II) or copper-(I). The biologically most relevant reaction ROOH

+

Fe-(II)

->

RO*

+

OH

+

Fe-(III)

(61)

thus becomes understandable in view of the same electronic concept as outlined above for the general peroxide reduction mechanism. The most basic chemical property of alkoxyl radicals is that they are oxidizing species and may be detected through their reaction with an electron donor, e.g., iodide [88,154]: RO*

+

21

-^

l2*

+

RO

(62)

Alkoxyl radicals also engage in hydrogen atom abstraction reactions, a process which usually requires, however, considerable activation energy:

Chapter 1: Free radical chemistry RO*

+

R-H

37

->

ROH

+

R*

(63)

Although these oxidizing reactions are quite significant they probably do not constitute the most important characteristic of the alkoxyl radicals. Two features need particular attention, a 1,2-hydrogen shift and a p-scission process. Both occur at very short time scales and result in completely different chemical systems. The 1,2-hydrogen shift may be formulated as an intramolecular process but, at least in aqueous or protic environment, it has probably to be viewed as solvent assisted [155—157]: H -C-0*

^^ >

I

-C-OH

(64)

I

For our further discussion two aspects are important. First, the half-life of this rearrangement is typically on the order of a microsecond. In order to compete, even a diffusion controlled bimolecular reaction of the oxyl radical requires as much as 100 |iM or more of oxidizable substrate. As pointed out above, this fast rearrangement may be a likely cause for a-hydrogen substituted oxyl radicals to escape direct detection. The second important consequence of the 1,2-hydrogen shift is that it converts the oxidizing oxyl radical into a, usually strongly, reducing oc-hydroxyl radical. In other words, it dramatically changes the redox properties of the radical species. Whenever the oxyl carrying carbon is not substituted with hydrogen, as in the case of a tertiary oxyl radical, a P-scission process becomes a dominant pathway: R I

R-C-O*

•

R*

+

RoCO

I

R

(65)

This elimination process constitutes practically the reverse of a radical addition to a C=0 double bond and, therefore, reaction 65 should, in principle, be formulated as an equilibrium. Efficient back reaction would require R*, however, to be a good nucleophile since the site of addition is the carbon atom, i.e., the more positive of the two atoms in the carbonyl bond. But as the majority of radicals seem electrophilic in nature most p-scissions appear, indeed, fast and irreversible. The actual rates involved, both for the cleavage and possible readdition, depend, of course, on the identity of R*, possible resonance stabilizations, and steric parameters. Some biologically relevant consequences of this P-scission are probably be worth pointing out. Firstly, there is no reason to restrict this process only to tertiary oxyl radicals. The same may happen with hydrogen substituted species as well, e.g..

38

P(^^t J- Introduction to free radicals H

HCHO

I

R-C-0* H(R)

•

R*

+

(66) RCHO

It must only be recognized, however, that p-cleavage of a hydrogen atom itself, i.e., R2CHO* -> H* + R2CO does not take place. Whether or not reaction 66 occurs effectively is mainly a question of competition with the 1,2-hydrogen shift (Eq. 64). If it has a chance against the latter then it opens a route to aldehyde formation. Another potentially interesting aspect is associated with halogenated oxyl radicals. The radical cleaved by |3-scission may, in this case, be a halogen atom. For example, the carbon tetrachloride derived CCI3O* would liberate CI* atoms, leaving behind a molecule of phosgene, CCI2O: CClsO*

^

Cr

+

CCI2O

(67)

What about other halogen atoms? The following observation [158], made with oxyl radicals from halogenated acetic acids, reveals not only some significant (although, in view of bond strength considerations, probably not unexpected) difference between fluorine and the other halogens but it also stresses the need for individual product analysis before final conclusions on mechanisms and potential toxicity should be made. For example, in the following fluorinated model oxyl radical, •OCF2-CO2 , in principle two (3-cleavage processes may be envisaged, one involving C - F bond breakage with F* elimination (Eq. 68a) and the other involving C-C breakage with CO2* as remaining radical (Eq. 68b). y^—•

F*

+

CF(0)-C02"

(68a)

F F C02*-

+

CF2O

(68b)

The actual mechanism proceeds exclusively via C-C cleavage, as proven by the ultimate products CO2 and fluoride (involving Eqs. 69 and 70 in oxygenated solution). C02* CF2O

+ +

O2 H2O

^ ^

CO2 CO2

+ +

02* 2 F / 2 H^

(69) (70)

Corroborating support is provided by the complete lack of oxalate which has been shown to result upon hydrolysis of CF(0)-C02~ [159]. The bromine and chlorine analogues, on the other hand, decay quantitatively


39

via carbon-halogen bond cleavage, i.e., under formation of bromine and chlorine atoms. In this case all the halogens also end up as halide ions, Br" and CI , eventually.

Br (CI)

Br

+

CBr(0)-C02"

CI*

+

CC1(0)-C02"

(71a)

•O - C - CO." I

"^

Br (CI)

/ /

7Z

,

CBroO CO,*2

+

(71b)

CCI2O

With respect to the fate of the carbon skeleton, however, yet another interesting and perhaps surprising aspect emerged. Both, the oxaloyl bromide and chloride do not hydrolyse to oxalic acid as the oxaloyl fluoride but suffer C-C cleavage to generate CO and CO2 at a 1:1 molecular ratio [159]. CBr(0)-C02 / CC1(0)-C02

{H2O) ^

CO + CO2 + Br / CI

(72)

The formation of carbon monoxide as well as of the halogen atoms is, of course, a most significant outcome from a toxicological point of view Finally, a general consequence of any C - C cleavage in the P-scission process in an oxyl radical should be recognized, namely, the shortening of the carbon chain by one carbon unit. The eliminated radical R* is, of course, again a potential target for oxygen addition, and after going through another possible peroxyl and oxyl radical chemistry cycle, it may be prone for a further (3-scission. This may carry on and on until eventually a long aliphatic chain has been completely broken down into Ci-units. 3.5 Phenoxyl radicals Most features displayed and discussed for the formation and properties of alkoxyl radicals in the previous chapter also apply for phenoxyl-type free radicals, i.e., for species where the aliphatic (R) has been replaced by an aromatic substituent (Ar). Thus formation of ArO* is easily achieved by oxidation of the phenolic group, either by hydrogen abstraction or one-electron oxidation of the phenolate. The latter is a much more feasible process than for the aliphatic substrates, particularly in view of its biological relevance, since the pK of phenols is much closer to physiological pH than that of alcohols. Mechanistically the free radical induced oxidation of a phenolic hydroxyl group is, however, often not a simple one-step process. Hydroxyl radicals, for example, typically add to the aromatic 71-system in the first instance and subsequently ehminate H2O in an acid-catalyzed second step:

40

P(i^t I: Introduction to free radicals

•

*6

(•—r-OH

(73)

This is important, for example, in view of the radical's reactivity towards molecular oxygen. Whilst the phenoxyl, being an oxygen-centered radical, is practically unreactive in this respect, the carbon-centered •QH-adduct radical is prone for peroxyl radical formation. And furthermore, as we have seen before, hydroxylated peroxyl radicals may cleave superoxide. In other words, any *OH-induced oxidation of a phenol in oxygenated environment bears the potential of superoxide formation [21,112]. Phenoxyl radicals are moderately good oxidants with reported reduction potentials for the ArO*/ArO couples in the 400—1,000 mV range for phenol derivatives at pH 7 [70]. The actual values differ considerably and depend strongly on the other substituents. Naturally, the possibility to delocalize electron density within aromatic structures affects the actual oxidative power of the radical oxygen site. An important reaction phenoxyl-type free radicals readily undergo is the oxidation of ascorbate (AH ,Vit C). This pertains, in particular, to the most prominent of them, the chromanoxyl radical derived from a-tocopherol (Vit E) [160]. The reaction VitE(-0*)

+

AH

^

VitE(-OH)

+

A*

(74)

presumably constitutes the most important repair mechanism of this vital antioxidant. Although the stoichiometry looks like a hydrogen transfer reaction 74 appears, nevertheless, to be based on an electron transfer. Finally, let us consider a very special problem which, however, touches on a general aspect of phenoxyl radicals, particularly those derived from Vit E and related substrates. Some recent studies with the water soluble Vit E analogue Trolox C (I) [161,162] have revealed (and in part substantiate earlier findings) [163] that certain quinoid and prequinoid products arise from the chromanoxyl radical which cannot be explained exclusively on the basis of an oxygen-centered radical (II) alone. Assuming, however, participation of carbon-centered resonance forms such as (III), a disproportionation process via a carbocation (IV) intermediate can be envisaged which leads directly to a lactone (V) and, after hydrolysis of the latter, to an open-chain substituted quinone (VI), both of which have been identified as products in the *OH-induced oxidation of Trolox C.

COO" (I)

T (II)

^

COO"

T *^

COO"

(in)

(75)


41

disproportionation *-

(III)

• OH

(^v) —

COO"

LA^

)^^\ C—C=0 (V)

(75b) (VI)

The general message is, not to neglect possible electronic resonance structures, particularly when the predominant form of the radical is relatively unreactive. 3.6 Semiquinone radicals Semiquinones may be viewed as just a special kind of phenoxyl radicals. They typically involve resonance structures under participation of another oxygen function at the aromatic ring. To some extent even the chromanoxyl radicals fall into this category. The classical semiquinone is a one-electron intermediate between two relatively stable compounds, hydroquinones and quinones.

ô OH

•e-f-H^

O

(76)

As many free radicals do, also the semiquinone establishes an acid-base equilibrium in which the remaining hydroxyl group deprotonates. The respective pKa values of Q(H)*

^

Q*

+

H^

(77)

are typically in the range around 4, i.e., the prevailing form under physiological conditions is the anionic species, Q* . A characteristic feature of the semiquinone radicals is that they are not as good oxidants as simple phenoxyl radicals, with pH 7 reduction potentials of the semiquinone/ hydroquinone couples being around 400 mV for various 1,4-dihydroxybenzenes [66e,70]. In fact, semiquinones are generally more of a reductant. This is reflected, for example, in their role of biological electron shuttle agents in the photosystem, and also in an electron transfer equilibrium with molecular oxygen and quinone (Q) [164]:

42

Ptti^t I: Introduction to free radicals

Q*

O2

+

^

Q

+

(78)

02*

Semiquinones may thus effectively contribute to the superoxide formation in biological systems. The actual reduction potentials of the Q/Q*~ couples depend, of course, on the structure of the individual quinone involved but they are generally close to that of the 62/02*" couple (see chapter 2.7 on reduction potentials, pp. 19-21). 3.7 Superoxide (O2-, H02*) Superoxide is probably the most discussed and investigated among the biologically relevant oxygen radicals. Chemically, it is, however, a rather simple species and, in addition what has been said already in previous chapters, there is little more to add (see also two informative review articles [165,166]). As mentioned, superoxide is formed either by direct electron attachment to molecular oxygen, through some electron transfer reactions from other free radicals (e.g., disulfide radical anions or CO2*"), or via elimination reactions from certain peroxyl radicals. It exists in an acid-base equilibrium with a pKa of 4.8 [67], i.e., under physiological conditions the anionic form clearly prevails: H02*

^

02*

+

H^

(79)

It must be recognized though that any 0^~ which might find access to a less polar environment may protonate due to a shift in pKa, and thus assume the properties of the undissociated HO2*. 02*~ itself is a moderate reductant and transfers an electron to, for example, cytochrome-c-(III) [167], and is involved in electron exchange equilibria with quinones, as mentioned above. HO2*, on the other hand, is not as good a reductant but rather engages in oxidation reactions [67] and it would not be surprising if some of the reported oxidations by 02*~ of, e.g., hydroquinones were, in fact, due to the conjugate acid radical. The decay of superoxide, unless enzymatically assisted by SOD [9], proceeds fastest via the mutual H02*/02*~ reaction (with a rate constant of 10^ M ^ s"^ [67]) H02*

+

02*

^

HO2 (H2O2)

+

O2

(80)

while both the HO2* + HO2* and O2* + O2*" reactions occur much more slowly [168]. This is particularly true for the superoxide anion decay. The 02*~ is, in fact, a very stable species in the absence of a suitable reaction partner, a property which translates, of course, into relatively long pathways over which it may be transported. One other reaction of superoxide must, however, also be mentioned in this con-


43

text and that is its possible cross-termination with peroxyl radicals. Although only few data exist so far [169—171], it appears that this process, namely, ROO*

+

02*

^ -> ->

products

(81)

kinetically dominates over the competing mutual self-terminations (described in Eqs. 44-47, and 80). In a recent extensive study involving a halogenated model peroxyl radical, *CF2C02 , it was, in fact, quantitatively demonstrated that cross-termination under any circumstances accounted for at least 80%, often 100% of the peroxyl or superoxide decay [171]. The difficulty of these studies is that the products from reaction 81 and the peroxyl self-termination are often identical, thus preventing a distinction. Unambiguous quantification may, however, become possible through the hydrogen peroxide yields since crosstermination typically does not result in H2O2 formation and thus significantly lowers the yield of this product otherwise derived from the superoxide selftermination. 4

SULFUR-CENTERED FREE RADICALS

In several chapters we have come across free radicals with the unpaired electron located at the higher homologue atom to oxygen, namely, sulfur. In view of the very interesting redox chemistry of sulfur-centered free radicals and the recently increased focus on redox regulation of molecular processes in the bio sciences it is probably quite appropriate not only to summarize once more these various aspects under this separate headline but also to elaborate a little further on the role such radicals play in the context of thiol and disulfide chemistry. The most important of the sulfur-centered intermediates is probably the thiyl radical, RS* [172,173]. It may be generated via oxidation of thiols as well as by reduction of the corresponding disulfides. The former typically occurs as hydrogen atom abstraction by, in principle, almost any radical whenever the thiol prevails in its neutral form. Examples have been formulated in reaction 24 and in the back reaction of Eq. 52. Oxidation of thiolate, the conjugate base of the thiol, is likely to occur via an electron transfer, as formulated in the following equation with a general oxidizing radical X*: RS

+

X*

-^

RS*

+

X"

(82)

The possibility of this reaction is basically controlled by the respective redox potentials which for the RS*/RS couple typically are of the order of 0.7-0.8 V (vs. NHE) [70,173a]. Accordingly, any oxidant more potent than RS* may, in principle, engage in this process. This has been verified, for example, for the Br2* reactions with CysS [174] and PenS [175], respectively, the (CH3SSCH3)*^-induced oxidation of CysS , CyaS and PenS [175] and, possibly even more relevant from the biological point of view, for the reaction of the

44


lipoate radical cation Lip(SS)*"^ (E° = 1.10 V) with these conjugate bases of cysteine, cysteamine and penicillamine [175]. Rate constants, ranging from (1.13.7) X 10^ M"^ s~^ for the above-mentioned reactions, indicate that the electron transfer occurs almost diffusion controlled. With respect to the oxidation of thiolate (e.g., RS of cysteine, cysteamine, Aâcetyl-L-cysteine) it is noted that the rate constants for the reaction of •QH + RS are practically the same as for the reaction of *OH with RSH [176,177] ( ^ 3 x 10^ M"^ s^^) although the respective mechanisms are presumably quite different. While the reaction with the thiols clearly has to be viewed as a hydrogen atom abstraction, the corresponding reaction with the thiolate is likely to be an electron transfer. The positive redox potential of the hydroxyl radical, E°[*OH/ O H ] = 1.9 V [70], provides the necessary driving force. The other mechanism by which thiyl radicals are readily formed is by reduction of disulfides as formulated in the following general reaction: RSSR

+

e

-^

RS*

+

RS

(83)

This process is generally very fast and efficient, i.e., controlled only by the diffusion of the reaction partners. The electron itself may be, for example, a hydrated (solvated) electron, an electron transferred from a reducing radical, electrode, semiconductor, or an enzymatically provided one. For a full understanding of the disulfide reduction it must be recognized that Eq. 83 gives only a very simpHfied overall summary of this process [153,172]. The detailed mechanism is much more complex and interesting. Please note that the incoming electron finds two electronically already rather satisfied sulfur atoms (total of eight electrons around each sulfur with four in bonding and four in nonbonding mode). So, where does it go? Sulfur-d-orbitals may be one consideration but this is energetically not feasible, nor are the alkyl groups suitable sites for electron attachment. The answer is an antibonding a*-orbital of the existing S-S bond, so that this bond after the reduction contains two bonding aelectrons and one bond-weakening a*-electron. This 2-center-3-electron bond is generally denoted with a " .*." symbol. The effect of the antibonding a*-electron, in fact, drives the two sulfur atoms apart and provides the basis for the establishment of a reversible dissociation equilibrium, i.e.: RS-SR

+

e

^

[RS.'.SR]

^

RS*

+

RS

(84)

One immediately emerging consequence is that the transient disulfide radical cation is not only formed via disulfide reduction but also by association of thiyl radicals with thiolate. Furthermore, the lifetime of [RS.*. SR] is practically controlled by the thiolate concentration which, in turn, in thiol containing systems is a function of both, the total thiol concentration and the pH. In thiol-free solutions, half-lives of as short as a few hundred nanoseconds have been measured for the above equilibrium (K ^ 10"^ M), involving the reduction of simple ali-


45

phatic disulfides. Here, the RS* and RS components may freely diffuse apart once the sulfur-sulfur bond has been broken. Much longer lifetimes (up to milliseconds) are, however, obtained with increasing thiolate concentration or, and this is particularly interesting, if RS* and RS are prevented from diffusing apart through backbone linkage. The latter is the case in, for example, lipoic acid but may also be anticipated in proteins and many other macromolecular structures with either disulfide linkage or closely positioned thiol groups. The [RS.'.SR]/RS* equilibrium is of utmost importance because it strongly affects the overall redox behavior of the environment in which these two species coexist. While the thiyl radical is a moderately good oxidant [178], as outlined above in connection with Eq. 82, the disulfide radical anion is a reductant (E° ^ -1.6 V) [154], capable of reducing, for example, molecular oxygen [179—181]. [RS.-.SR]

+

O2

^

RSSR

+

02*

(85)

Reaction 85 leads us directly into the chemistry (and potential problems) of superoxide. The variety of other reactions the thiyl radical RS* may undergo need not be repeated here since they have been mentioned and discussed already in connection with Eqs. 52, 55 and 56. In summary, it is, however, noteworthy that thiyl radicals may engage in quite a number of equilibria and reactions involving molecular oxygen and oxygen-centered free radicals. 5

CONCLUSION

In this survey we have discussed the majority of the oxygen-centered and some sulfur-centered radicals with particular emphasis on general features of free radical chemistry. Nevertheless, this article can still not claim to provide a comprehensive coverage of all detailed information available up to now, nor was it intended to do. The exciting chemistry of singlet oxygen [1,6,182—185] or ozone [13,184], and species derived therefrom certainly warrants equal attention. Or, to go beyond the oxygen-centered species, many more sulfur-centered radicals [19], or the nitrogen-centered NOX (particularly NO [186—188]) free radicals are undoubtedly of similar interest and biological significance. Much has been documented in books, reviews and summarizing conference reports, and more information will unquestionably emerge from present days and future research. All of this will reflect, however, the fundamental chemical characteristics of radicals, and it is hoped that this article has provided some useful basis for the chemical understanding of those biological phenomena which can be related to these highly reactive species.

46

6

P^^t I- Introduction to free radicals

SUMMARY

This chapter on Free Radical Chemistry comprises of two main sections. In the first part some important general properties of free radicals are discussed, (i) A free radical is introduced as a species with an unpaired electron, (ii) As a consequence thereof, free radicals are usually highly reactive species, (iii) The knowledge of their chemical properties and absolute rate constants for their reactions is an indispensable requirement to assess any possible activity of free radicals, (iv) This information is best obtained by application of time-resolved detection techniques such as the radiation chemical method of pulse radiolysis and, complementary to this, laser flash photolysis, (v) Structural information on free radicals is mainly achieved by application of electron spin resonance (ESR); in biological systems spin trapping techniques are of particular value, (vi) Further useful information on the reactivity of free radicals may be obtained from redox potential measurements, and can also be deduced from stable products of free radical reactions. In the second part of this article the most important oxygen-centered and thiyl free radicals are discussed in some detail as there are: (1) the hydroxyl radical *OH, (2) peroxyl radicals ROO*, (3) alkoxyl radicals RO*, (4) phenoxyl radicals ArO*, (5) semiquinone radicals "O-Ar-O*, (6) superoxide 02*~, and (7) for comparison the redox-active species in disulfide/thiol systems. The examples chosen try to illustrate characteristic features of general interest such as: (a) acidbase equilibria of certain groups of free radicals, (b) the change of redox properties within a free radical reaction mechanism, (c) the possible involvement of electronic resonance forms of free radicals, and (d) the consideration of free radical lifetimes with respect to possible reactions and migration of these reactive species in solution or biological systems. In conclusion, it is hoped that the biologically or medically oriented reader appreciates the wealth of fundamental information free radical chemistry can provide for the understanding of probably many biochemical mechanisms. 7

ACKNOWLEDGEMENTS

Some of our own work referred to in this article has been supported by the International Association for Cancer Research (AICR) and the Office of Basic Energy Sciences of the U.S. Department of Energy. This is gratefully acknowledged (this is publication No NDRL-4045 of the Notre Dame Radiation Laboratory). 8

ABBREVIATIONS

b: y-rays: Ax: f.

Bohr magneton gamma-rays ( > 0.1 MeV electromagnetic radiation) mean diffusion distance frequency

Chapter 1: Free radical chemistry TT:

a: a*: $: AH /A* : Ar: CyaS: CysS : D: DNA: E: E°: ENDOR: ESR: e: âq •

eV: F: G: G°: GSH: Gy: g: H: h: hu: IR: lUPAC: J: K: K: k: 2k: Lip(SS): MeV: NAD: NHE: NMR: NOX: n: OD: Pens : PUFA: PZ:

molecular pi orbital molecular sigma orbital molecular antibonding sigma*orbital quantum yield ascorbate anion / ascorbate radical anion aromatic substituent (aryl) cysteamine thiolate cysteine thiolate diffusion coefficient deoxyribonucleic acid reduction potential standard reduction potential electron nuclear double resonance electron spin resonance electron hydrated electron electron-volt Faraday constant radiation chemical yield (species per 100 eV absorbed energy) standard free enthalpy glutathione Gray gyromagnetic factor magnetic field intensity Planck's constant photon, quantum of electromagnetic radiation infrared light range International Union of Pure and Applied Chemistry Joule equilibrium constant (as temperature unit) Kelvin rate constant radical-radical termination rate constant lipoic acid, thioctic acid mega electron-volt nicotinamide adenine dinucleotide standard (normal) hydrogen electrode nuclear magnetic resonance nitric oxide-derived reactive oxygen species number of electrons transferred in a redox process optical density penicillamine thiolate polyunsaturated fatty acid phenothiazine

Al

48


pH: pK: Q: R: R: SCE: SOD: T: ti/2 : UV: VIS: Vit C: Vit E: •

9 1. 2.

3.

4.

5.

negative decadic logarithm of proton concentration negative decadic logarithm of acid dissociation constant quinone (in AG° equation) gas constant aliphatic substituent saturated calomel electrode superoxide dismutase absolute temperature (in K) half-life ultraviolet light range visible light range vitamin C, ascorbic acid vitamin E, a-tocopherol unpaired electron three- electron-bond

NOTES There are exceptions, however, molecular oxygen, for example, is in fact relatively unreactive towards nonradical molecules. In radiation chemistry the yield of chemically changed species is usually expressed in terms of G which denotes the number of species per 100 eV absorbed energy This is approximately equivalent to ixmoles per 10 J, or fiM concentration per 10 J/kg (1 J/kg = 1 Gy); (Gy = Gray). In N2O saturated aqueous solution G(*OH) ?^ 6. In photochemistry the efficiency of a photo-induced process is generally expressed in terms of the quantum yield 0, i.e., the number of product species formed per quantum of absorbed light. If necessary the effect of H* atom reactions can be studied separately, and H2O2 and H2 do usually not interfere at the early pulse radiolysis time scale. Any effect by protons is usually controlled by the solution pH rather than the small amount generated as the result of the irradiation. Free radical studies may also be conducted by y-radiolysis. They can equally be trusted as the pulse radiolysis studies since it involves the same chemistry. In y-radiolysis the initial energy carrier is a high energy photon (y-ray) which on interaction with matter generates high energy electrons (up to MeV) in a Compton process. From any chemical point of view the situation is, therefore, the same as in the pulse radiolysis. Formally, a reduction process is not restricted to a full and isolated electron transfer. Thus, the oxidation state of a particular atom is also changed if a free radical adds to it. For example, the oxidation number of sulfur in an organic sulfide like methionine, CH3-S-CH2CH2— CH(NH2)C00H is -2. Addition of a hydroxyl radical to the sulfur, CH3-S*(OH)-CH2...., changes it to -1 and thus constitutes an oxidation.

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65. Asmus K-D, Bahnemann D, Krischer K, Lai M, Monig J. Life Chem Rep 1985;3:1 —15. 66. Landolt-Bornstein. Numerical Data and Functional Relationships in Science and Technology, New Series, Hellwege K-H, Madelung O (eds) Group IL Atomic and Molecular Physics, vol 13.: Radical Reaction Rates in Liquids. Fischer H. ed. Berlin: Springer: Part a: Beckwith ALJ, Griller D, Lorand JP. Carbon-Centered Radicals I, 1984. Partb: Asmus K-D, Bonifacic M. Carbon-Centered Radicals II, 1984. Part c: Ingold KU, Roberts BR Radicals Centered on N, S, P and other Heteroatoms. Nitroxyls, 1983. Part d: Howard JA, Scaiano JC Oxyl, Peroxyl and Related Radicals, 1984. Part e: Dohrmann JK, Scaiano JC, Steenken S. Proton and Electron Transfer. Biradicals, 1985 and Supplement additions, 1995/96. 67. Bielski BHJ, Cabelli DE, Arudi RL. Reactivity of H02*/02*~ radicals in aqueous solution. J. Phys Chem Ref Data 1985;14:1041-1100. 68. Neta P, Huie RE, Ross AB. Rate constants for reactions of inorganic radicals in aqueous solution. J Phys Chem Ref Data 1988;17:1027-1284. 69. Neta P, Huie RE, Ross AB. Rate constants for reactions of peroxyl radicals in fluid solutions. J Phys Chem Ref Data 1990;19:413-513. 70. Wardman P. Reduction potentials of one-electron couples involving free radicals in aqueous solution. J Phys Chem Ref Data 1989;18:1637-1723. 71. O'Neill P, Steenken S, Schulte-Frohlinde D. Angew Chem 1975;87:417-418. 72. Bonifacic M, Asmus K-D. J Chem Soc Dalton 1976;2074-2076. 73. Sanaullah, Hungerbiihler H, Schoneich Ch, Morton M,Vander Velde DO,Wilson OS, Asmus KD, Glass RS. J Am Chem Soc 1997;119:2134-2145. 74. Bonifacic M, Mockel H, Bahnemann D, Asmus K-D J Chem Soc Perkin Trans 2 1975; 675-685. 75. Asmus K-D. Ace Chem Res 1979;12:436-442. 76. Bobrowski K, Schoneich Ch. J Chem Soc Chem Commun 1993;795-797. 77. Hiller K-O, Masloch B, Gobi M, Asmus K-D. J Am Chem Soc 1981;103:2734-2743. 78. Monig J, Gobi M, Asmus K-D. J Chem Soc Perkin Trans 2 1985;647-651. 79. Asmus K-D, Gobi M, Hiller K-O, Mahling S, Monig J. J Chem Soc Perkin Trans 2 1985; 641-646. 80. Hiller K-O, Asmus K-D. J Phys Chem 1983;87:3682-3688. 81. Bobrowski K, Holcman J. Int J Radiat Biol 1987;52:139-144. 82. Bobrowski K, Schoneich Ch, Holcman J, Asmus K-D. J Chem Soc Perkin Trans 2 1991; 353-362. 83. Bobrowski K, Pogocki D, Schoneich Ch. J Phys Chem 1993;97:13677-13684. 84. Fujita S, Steenken S. J Am Chem Soc 1981;103:2540-2545. 85. McMillen DF, Golden DM. Ann Rev Phys Chem 1982;33:493-532. 86. Schoneich Ch, Asmus K-D, Dillinger U, Bruchhausen F Biochem Biophys Res Commun 1989; 161:113-120. 87. Schoneich Ch, Dillinger U, Bruchhausen F, Asmus K-D. Arch Biochem Biophys 1992;292: 456-467. 88. Asmus K-D, Mockel H, Henglein A. J Phys Chem 1973;77:1218-1221. 89. Bonifacic M, Schafer K, Asmus K-D. J Phys Chem 1975;79:1496-1502. 90. Asmus K-D, Henglein A,Wigger A, Beck G Ber Bunsenges Phys Chem 1966;70:756-758. 91. Mertens R, von Sonntag C Angew Chem Int Ed Engl 1994;33:1262-1264. 92. Lai M, Schoneich Ch, Monig J, Asmus K-D Int J Radiat Biol 1988;54:773-785. 93. Bahnemann D, Asmus K-D,Willson RL. J Chem Soc Perkin Trans 2 1983;1661-1668. 94. Bahnemann D, Asmus K-D,Willson RL. J Chem Soc Perkin Trans 2 1981;890-895. 95. Huie RE, Neta P Int J Chem Kinet 1986;18:1185-1191. 96. Schoneich Ch, Aced A, Asmus K-D. J Am Chem Soc 1991;113:375-376. 97. Engman L, Persson J, Merenyi, Lind J. Organometallics 1995;14:3641—3648. 98. Aced A. PhD Thesis, Technical University Berlin, Germany D 83, 1991. 99. Packer JE,Willson RL, Bahnemann D, Asmus K-D. J Chem Soc Perkin Trans 2 1980:296-299.

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100. Huie RE, Brault D, Neta P. Chem Biol Interact 1987;62:227-235. 101. Neta P, Huie RE, Mosseri S, Shastri LV, Mittal JP, Mamthamuthu P, Steenken S. J Phys Chem 1989;93:4099-4104. 102. Monig J, Asmus K-D. In: Bors W, Saran M,Tait D (eds) Oxygen Radicals in Chemistry and Biology Berlin: Walter de Gruyter, 1984;57-63. 103. Martin RA, Richard C, Rousseau-Richard C. In: Vigo-Pelfrey C (ed) Membrane Lipid Oxidation. Boca Raton, FL: CRC Press, 1989;63-100. 104. Vigo-Pelfrey C (ed). Membrane Lipid Oxidation. Boca Raton, FL: CRC Press, 1989. 105. Korcek S, Chenier JHB, Howard JA, Ingold KU. Can J Chem 1972;50:2285-2297. 106. Gebicki JM, Bielski BHJ. J Am Chem Soc 1981;103:7020-7022. 107. Gebicki JM, Allen AO. J Phys Chem 1969;73 2443-2445. 108. Patterson LK, Redpath JL. In: Mittal KL (ed) Micellization, Solubilization, and Micro-emulsions, vol 2. New York: Plenum Press, 1977;589-601. 109. Fessenden RW, Carton PM, Shimamori H, Scaiano JC. J Phys Chem 1982;86:3803-3811. 110. Neta P, Huie RE, Maruthamuthu P, Steenken S. J Phys Chem 1989;93:7654-7659. 111. Alfassi ZB, Huie RE, Neta P J Phys Chem 1993;97:7253-7257. 112. von Sonntag C, Schuchmann H-P Angew Chem 1991;103:1255-1279. 113. Rabani J, Klug-Roth D, Henglein A. J Phys Chem 1974;78:2089-2093. 114. IlanY, Rabani J, Henglein A. J Phys Chem 1976;80:1558-1565. 115. Bothe E, Behrens G, Schulte-Frohlinde D. Z Naturforsch 1977;32b:886-889. 116. Bothe E, Schulte-Frohlinde D, von Sonntag C. J Chem Soc Perkin Trans 2 1978;416-420. 117. Bothe E, Schuchmann MN, Schulte-Frohlinde D, von Sonntag C. Photochem Photobiol 1978; 28:639-644. 118. Das S, Schuchmann MN, Schuchmann H-P, von Sonntag C Chem Ber 1987;120:319-323. 119. Bloodworth AJ, Courtneidge JL, Davies AG. J Chem Soc Perkin Trans 2 1984;523-527. 120. Adamic K, Howard JA, Ingold KU. Can J Chem 1969;47:3803-3808. 121. Howard JA. ACS Symposium Ser 1978;69:413-432. 122. Furimsky E, Howard JA, Selwyn J. Can J Chem 1980;58:677-680. 123. Bennett JE. J Chem Soc Faraday Trans 1990;86:3247-3252. 124. Russell GA. J Am Chem Soc 1957;79:3871-3877. 125.Nakano M, Takayama K, Shimizu Y, Tsuji Y, Inaba H, Migita T J Am Chem Soc 1976;98: 1974-1975. 126. Niu Q, Mendenhall GD. J Am Chem Soc 1990;112:1656-1657. 127. Lee S-H, Mendenhall GD. J Am Chem Soc 1988;110:4318-4323. 128. Bothe E, Schulte-Frohlinde D. Z Naturforsch 1978;33b:786-788. 129. Asmus K-D. In: Fielden EM, Fowler JF, Hendry JH, Scott D (eds) Radiation Research, Proceeding 8th International Congress of Radiation Research, vol 2. London: Taylor and Francis, 1987;48-53. 130. Lai M, Monig J, Asmus K-D. J. Chem Soc Perkin Trans 2 1987;1639-1644. 131. Esterbauer H, Zollner H, Schaur RJ. In: Vigo-Pelfrey C (ed) Membrane Lipid Oxidation. Boca Raton, FL: CRC Press, 1989;239-268. 132. Schoneich Ch, Bonifacic M, Asmus K-D. Free Radical Res Communs 1989;6:393-405. 133. Schoneich Ch, Asmus K-D. Radiat Environ Biophys 1990;29:263-271. 134. Schoneich Ch, Bonifacic M, Asmus K-D. J Chem Soc Faraday Trans 1995;91:1923-1930. 135. Schoneich Ch, Bonifacic M, Dillinger U, Asmus K-D. In: Chatgilialoglu C, Asmus K-D (eds) Sulfur-Centered Reactive Intermediates in Chemistry and Biology NATO ASI Series, A: Life Sciences, vol 197. New York: Plenum Press, 1990;367-376. 136. Czapski G, Dorfman LM. J Phys Chem 1964;68:1169-1177. 137. Pan X-M, von Sonntag C. Z Naturforsch 1990;45b:1337-1340. 138. Porter NA, Lehman LS, Weber BA, Smith KJ. J Am Chem Soc 1981;103:6447-6455. 139. Porter NA,Wujek D G In: Quintanilha A (ed) Reactive Oxygen Species in Chemistry, Biology, and Medicine. NATO ASI Series A, vol 146. New York: Plenum Press, 1988;55-79.


53

140. Frankel EN. In: Simic MG, Taylor KA,Ward JF, von Sonntag C (eds) Oxygen Radicals in Biology and Medicine. New York: Plenum Press, 1988;265—282. 141. Barclay LRC In: Minisci F (ed) Free Radicals in Synthesis and Biology Dordrecht: Kluwer, 1989;391-406. 142. Monig J, Asmus K-D, Forni LG,Willson RL. Int J Radiat Biol 1987;52:589-602. 143. Tamba M, Simone G, Quintihani M. Int J Radiat Biol 1986;50:595-600. 144. Wardman P. In: Chatgilialoglu C, Asmus K-D (eds) Sulfur-Centered Reactive Intermediates in Chemistry and Biology NATO ASI Series, A: Life Sciences, vol 197. New York: Plenum Press, 1990:415-427. 145. Zhang X, Zhang N. Schuchmann H-P, von Sonntag C. J Phys Chem 1994;98:6541-6547. 146. Swarts SG, Becker D, DeBold S, Sevilla MD. J Phys Chem 1989;93:155-161. 147. Chatgilialoglu C, Guerra M. In: Chatgilialoglu C, Asmus K-D (eds) Sulfur-Centered Reactive Intermediates in Chemistry and Biology NATO ASI Series, A: Life Sciences, vol 197. New York: Plenum Press, 1990;31-36. 148. Chatgilialoglu C In: Patai S (ed) The Chemistry of Sulfenic Acids and Their Derivatives. New York: Wiley 1990;549-569. 149. Asmus K-D, Schoneich Ch. Oxidative Damage & Repair. In: Davies KEJ (ed) Chemical, Biological and Medical Aspects. New York: Pergamon Press, 1991 ;226—233. 150. Asmus K-D. In: Yagi K (ed) Active Oxygen, Lipid Peroxides, and Antioxidants. Tokyo: Japan Sci Press; Boca Raton: CRC Press, 1993;57-67. 151. Chatgilialoglu C, Schoneich Ch, Asmus K-D. Unpublished results. 152. Asmus K-D. In: Nygaard OF, Simic MG (eds) Radioprotectors and Anticarcinogens. New York: Academic Press, 1983;23-42. 153. Asmus K-D. In: Chatgilialoglu C, Asmus K-D (eds) Sulfur-Centered Reactive Intermediates in Chemistry and Biology NATO ASI Series, A: Life Sciences, vol 197. New York: Plenum Press, 1990;155-172. 154. Dainton FS, Janovsl^ IV, Salmon GA. Proc Roy Soc Ser A 1972;327:305-316. 155. Berdnikov VM, Bazhin NM, Fedorov VK, Polyakov OV. Kinet Catal 1972;13:986-987. 156. Gilbert BC, Holmes RGG, Laue HAH, Norman ROC. J Chem Soc Perkin Trans 2 1976; 1047-1052. 157. Schuchmann H-P, von Sonntag C. J Photochem 1981;16:289-295. 158. Makogon O, Fliount R, Asmus K-D. J Adv Oxid Techn 1998;3:11-21. 159. Fliount R, Makogon O, Asmus K-D. To be published. 160. Packer JE, Slater TF, Willson RL. Nature 1979;278:737-738. 161. Willnow A. PhD Thesis, Technical University Berlin, D83, 1994. 162. Willnow A, Liebler DC, Asmus K-D To be published. 163. Thomas MJ, Bielski BHJ. J Am Chem Soc 1989;111:3315-3319. 164. Patel KB, Willson RL. J Chem Soc Faraday Trans I 1973;69:814-825. 165. von Sonntag C, Schuchmann H-P Angew Chem Int Ed Engl 1991;30:1229-1253. 166. Bielski BHJ, Cabelh DE. Int J Radiat Biol 1991;59:291-319. 167. Bielski BHJ, Richter HW J Am Chem Soc 1977;99:3019-3023. 168. Bielski BHJ. Photochem Photobiol 1978;28:645-649. 169. Shastri LY, Shrinavasan K, Rama Rao KVS Proc Nucl & Radiat Chem Symp, University of Pune. Poona, India 1967;49 ff. 170. Schuchmann MN, von Sonntag C. J Am Chem Soc 1988;110:5698-5701. 171. Fliount R, Magogon O, Asmus K-D J Phys Chem 1997;101:3547-3553. 172. Asmus K-D. In: Packer L, Glazer AN (eds) Methods of Enzymology, vol 186. San Diego: Academic Press 1990;168-180. 173. see following articles in ref 19: a) Armstrong DA: 121 — 134 and 341—352; b) von Sonntag C: 359-366; c) Dunster C, Willson RL: 377-388; d) Mason RP, Rao DNR: 401-408; e) von Sonntag C, Schuchmann HP: 409-414;f) Wardman P: 415-428;Quintihani M: 435-443. 174. Adams GE, Aldrich JE, Bisby RH, Cundall RB, Redpath JL, Willson RL. Radiat Res 1972;49:

54

Pci^t I' Introduction to free radicals

278-289. 175. Bonifacic M, Asmus K-D. Int J Radiat Biol 1984;46:35-45. 176. Mezyk SP. Radiat Res 1996;145:102-106. 177. Mezyk SR J Phys Chem 1996;100:8295-8301. 178. Surdhar PS, Armstrong DA. J Phys Chem 1987;91:6532-6537. 179. Chan PC, Bielski BHJ. J Am Chem Soc 1973;95:5504-5508. 180. Willson RL. Chem Commun 1970;1425-1426. 181. Micic OI, Nenadovic MT, Carapellucci PA. J Am Chem Soc 1978;100:2209-2212. 182. Frimer AA (ed). Singlet O2. Boca Raton, FL: CRC Press, 1985. 183. Wilkinson F, Brummer JG. Rate constants for the decay and reactions of the lowest electronically excited singlet state of molecular oxygen in solution. J Phys Chem Ref Data 1981; 10: 809-1000. 184. HippeH S, Elstner EF. In: Sies H (ed) Oxidative Stress, Oxidants and Antioxidants. London: Academic Press, 1991 ;3—55. 185. Foote CS. In: Quintanilha A (ed) Reactive Oxygen Species in Chemistry, Biology, and Medicine. NATO ASI Series A, vol 146. New York: Plenum Press, 1988;107-116. 186. Saran M, Michel C, Bors W Free Rad Res Commun 1990;10:221-226. 187.Noack E, Murphy M. In: Sies H (ed) Oxidative Stress, Oxidants and Antioxidants. London: Academic Press, 1991;445-489. 188. Snyder SH, Bredt DS. Sci Am 1992;May:28-35.

Part II Reactive species in tissues


r 2000 Elsevier Science B.V. All rights reserved. Handbook of Oxidants and Antioxidants in Exercise. C.K. Sen, L. Packer and O. Hanninen, editors.

^^ *^ '

Part II • Chapter 2

Exercise and oxygen radical production by muscle Malcolm J. Jackson Muscle Research Centre, Department of Medicine, University of Liverpool, P.O. Box 147, Liverpool, L69 3GA, UK

1 2 3

4 5 6 7

1

INTRODUCTION EXERCISE POTENTIAL SOURCES OF FREE RADICAL GENERATION IN EXERCISING MUSCLE 3.1 Primary sources of free radicals 3.1.1 Mitochondrial generation of free radical species 3.1.2 Xanthine oxidase 3.1.3 Prostanoid metabolism 3.1.4 Catecholamines 3.1.5 NAD(P)H oxidase 3.2 Secondary sources of free radicals 3.2.1 Radical generation by phagocytic white cells 3.2.2 Generation of radicals secondary to muscle calcium accumulation 3.2.3 Free radical formation due to disruption of iron-containing proteins CONSEQUENCES OF EXERCISE-INDUCED FREE RADICAL PRODUCTION CONCLUSIONS ABBREVIATIONS REFERENCES

INTRODUCTION

The idea that oxygen radicals might be produced in excess during physical exercise and that these substances are responsible for some of the deleterious effects of excessive or unaccustomed exercise has been widely discussed for about 20 years [1]. In the initial stages it became clear that oxygen radicals could be produced in excess under certain exercise regimens [2], but this was not an inevitable consequence of all forms of exercise and did not always initiate cellular damage or degeneration (see Jackson et al. [3] for a review). Much of the confusion in this area can probably be attributed to the different types of exercise examined in various studies and the predominant manner in which muscle is used in different protocols. Prior to a discussion of the potential sites, and effects, of oxygen radical generation during exercise this review will therefore briefly consider these different forms of exercise with a view to a clearer recognition of the implications for oxygen radical production.

58

2

P^î II' Reactive species in tissues

EXERCISE

The "umbrella" title of exercise is used to describe a wide range of physical activity ranging from a small number of repeat contractions of single or small groups of muscles (e.g., "hand exercises") to extensive, rapid and exhaustive contractions of many of the major muscle groups of the body (such as occurs in many competitive sports events). It would therefore be reasonable in discussion of free radical generation during exercise to refine the question from "is exercise associated with increased free radical generation?" to more specific ones such as "are free radicals generated during muscle contractions?" or "are free radicals generated by increased oxygen consumption by muscle?" However, it is clear that this logical approach has not been widely followed and most studies have examined whole body exercise in man or animals. Such exercise is very complex and any observed changes in indicators of increased free radical activity may be theoretically derived from many tissues (e.g., lungs, heart, liver) in addition to skeletal muscle or may be associated with a variety of factors such as mechanical stresses, increased mitochondrial activity or increased supply of blood borne substrates in addition to muscular contraction. A number of such compounding factors therefore potentially influence free radical generation during exercise and it is also clear that the intensity and duration of the exercise will play a major role in the process. Thus high intensity, short term exercise is essentially anaerobic while longer term and endurance exercise is aerobic and dependant on substantial increases in mitochondrial oxygen consumption. Various theories for the production of free radicals in these two extreme areas have been proposed (e.g., see Sjodin et al. [4] for a review), but clear comparisons of the production of free radicals in these two extreme areas do not appear to have been undertaken. A further complication concerning the study of potential damaging processes during exercise is the predominant manner in which the muscle is used. Muscle may contract in three ways: where the active muscle is allowed to shorten (concentric contractions), remains at fixed length (isometric contractions) or is lengthened (eccentric contractions). A number of comparisons between experimental concentric and eccentric contractions have demonstrated that repetitive eccentric contractions are much more damaging that concentric contractions [5—9], although the time course of onset of the pain and damage is much slower than seen following excessive concentric contractions. Most standard exercise protocols (e.g., running, cycling, etc.) involve a combination of concentric and eccentric contractions with some muscles being used primarily in a concentric and some in an eccentric manner. Occasional forms of exercise, such as predominantly downhill running involve extensive use of a number of muscles in an eccentric manner and, are therefore very damaging to muscle tissue. Further characteristics of these different forms of muscle activity are presented in Table 1. It is therefore clear that any consideration of the potential role of free radicals in exercise-induced muscle injury should carefully control the nature of the con-

Chapter 2: Exercise and oxygen radical production by muscle

59

Table 1. Characteristics of different types of contractile activity. Eccentric

Isometric

Concentric

Movement of muscle Mechanical forces Electrical activity Metabolic cost Oxygen consumption

Lengthening High Low Low Low

Static Low High High High

Shortening Low High High High

Tendency to induce: Damage Fatigue Pain

High High High

Low Low Low

Low Low Low

tractile activity undertaken and the intensity and duration of that activity The different types of exercise discussed have such different energy requirements, oxygen consumption, and mechanical stresses on the tissue that it is unlikely that similar mechanisms for generation of damaging free radicals are active in each situation. It is entirely feasible that different mechanisms in each model predispose to the excessive generation of free radicals, since many potential mechanisms for generation of free radical species in exercising tissue exist. These are discussed in the next section. 3

POTENTIAL SOURCES OF FREE RADICAL GENERATION IN EXERCISING MUSCLE

There are a number of potential sites for the generation of free radical species within exercising muscle tissue and further possible mechanisms by which free radical species may be increased secondary to damage induced by other mechanisms. One of the major problems with assessment of the role of free radicals in any situation where tissue damage occurs is to differentiate between free radicals mediating the cell damage and occurring secondarily to it. Halliwell and Gutteridge [10] have pointed out that free radicals are produced by degenerating tissue in a number of situations and this possibility must always be considered. 3.1 Primary sources of free radicals 3.1 A Mitochondrial generation of free radical species Oxidative metabolism in mammals involves the reduction of molecular oxygen in mitochondria. Where oxygen is not limiting, this highly regulated system provides a means for the continuous generation of "high energy" phosphates (ATP) for muscular contraction. The ability of muscle to undertake co-ordinated increases in mitochondrial oxygen consumption is substantial. As part of the process for

60

P^^t II- Reactive species in tissues

delivery of energy supplies molecular oxygen generally undergoes four electron reduction catalysed by cytochrome oxidise. This process has been claimed to account for 95—98% of the total oxygen consumption of tissues, but the remainder (i.e., 2—5% of the total) may undergo one electron reduction with the production of the superoxide radical [O2']. Further one electron reduction of superoxide produces hydrogen peroxide which has been shown to be released by isolated mitochondria [11,12]. The site of this apparent "loss" of electrons to oxygen has been proposed to be coenzyme Q [4]. Thus it is envisaged that the quinones which constitute coenzyme Q are reduced to semiquinones by electrons from NADH and that these lipophilic compounds are able to readily diffuse to come into contact with oxygen with the generation of superoxide radial [11]. this hypothesis agrees well with what is known about the radical species which are visible by electron spin resonance studies of exercising muscle [2,13]. The only free radical signal observed in intact normal muscle has a g-value of approximately 2.004 and has been claimed to derive mainly from the mitochondria of cells [14]. The actual nature of the radical species has been disputed [15] although it is likely that it primarily derives from semiquinones [16]. It is therefore apparent that substantial aerobic exercise could theoretically lead to an increased production of superoxide radicals because of the vastly increased electron flux through the mitochondrial electron transport chain. However, it should also be noted that the mitochondrion has well-developed systems for protection against oxygen radical mediated damage with the presence of a specific mitochondrial superoxide dismutase to prevent local superoxide-mediated degeneration. There is increasing evidence that aerobic exercise leads to release of mitochondria-derived superoxide radicals from the muscle cell. Reid and co-workers [17,18] found that superoxide produced in contracting myocytes was released into the interstitial fluid and O'Neill and co-workers have recently reported clear evidence of increased hydroxyl radical production by contracting skeletal muscle [19]. In this elegant study the authors used L-phenylalanine as a probe for hydroxyl radicals and examined stimulated intermittent static contractions of the triceps surae muscle in anaesthetised cats. Contractile activity induced a rapid and substantial rise in the content of m-, and (9-tyrosines (formed by reaction of phenylalanine with hydroxyl radicals) indicating production of hydroxyl radicals during intermittent contractions (Table 2). This production appeared to be related to the tension developed by the muscle. Furthermore these authors pretreated animals with desferrioxamine and were able to show a reduction in hydroxyl radical production (Table 2). The overall conclusion of these disparate studies appears to be that superoxide is generated within mitochondria during contractile activity. Superoxide (and probably hydrogen peroxide) are subsequently lost from the muscle cell into the interstitial fluid. Hydroxyl radicals are then generated from the released superoxide/hydrogen peroxide by an iron-catalysed system (the lack of cell penetration of desferrioxamine indicate that this latter process must occur extracellularly).

Chapter 2: Exercise and oxygen radical production by muscle

61

Table 2. Hydroxyl radical production during intermittent static contractions of triceps surae muscles in cats.

1 min precontractions After 1 min contractions After 3 min contractions After 4.5 min contractions 1 min postcontractions

Hydroxyl radical production

Hydroxyl radical production with desferrioxamine treatment

0.30 ± 0.08 7.98 ± 2.30 10.70 ± 3.50 11.65 ± 2.03 5.09 ± 1.90

0.26 ±0.15 4.10 ± 1.80 5.16 ± 1.56'' 6.71 ± 1.37^ 1.65 ± 0.78''

Hydroxyl radical production will calculated from the increase in isomeric tyrosines formed from L phenylalanine. Blood was sampled from the venous output (popliteal vein) of triceps surae muscles stimulated to contract via the sciatic nerve. Muscle received 15 s of stimulation followed by 15 s recovery for the period shown. An alternative group of animals received desferrioxamine (10 mg/kg iv) 15 min prior to commencement of stimulation. ''pTrp> His=Tyr>Phe [63]. The loss of Histidine matched to the formation of aspartate or of a derivative that is a precursor of aspartic acid. Ozone induced loss of His residues in BSA is 1.5—2 times faster than in glutamine synthetase indicating

Chapter 4: Chemical bases and biological relevance of protein oxidation

101

that the susceptibiUty to oxidation is dependent on the primary, secondary, tertiary, and quaternary structure of proteins. Oxidative modification of residues may be mediated by physiologic and nonphysiologic systems including oxidases, ozone, hydrogen peroxide, superoxide, and ionizing irradiation. Methionine residues are remarkable for their high susceptibility toward oxidation by most of these systems [64] with the product generally being methionine sulfoxide. Cysteine, when oxidized at room temperature by 1 or 2 equivalents of hypochlorous acid produced cysteic acid as the only product [31]. 1.8 Lipid peroxidation derived aldehydes and protein damage Lipid peroxidation processes that occurs in biological membranes results in membrane instability Peroxidation processes has been connected to changes in both chemical and physical properties of the biological membrane, and to impairment of activity of enzymes located in the membrane environment [65]. Aldehydes are stable end products of lipid peroxidation. Unlike reactive free radicals, aldehydes are rather long lived and can therefore diffused from the site of their origin to reach distant targets. Aldehydes are formed during the peroxidation process mainly in the presence of transition metals which catalyzed the breakdown of the acyl chains and the release of reactive aldehydes [1]. Aldehydes can react with -SH and -NH2 groups on proteins to form both intramolecular cross-links, and also cross-links between different proteins or between proteins and lipids such as in phosphatidylethanolamine (PE) and phosphatidylserine (PS). Among the aldehydes that originate from the peroxidation of cellular membrane lipids, 4-hydroxy-2-nonenal (HNE) is believed to be a product which responsible for pathological effects observed during oxidative stress [2,66]. HNE reacts with sulfhydryl groups leading to the formation of thioether adducts that further undergo cyclization to form hemiacetales. HNE also reacts with histidine and lysine residue of proteins to form stable Michael addition type adducts [67,68]. Reaction of SH containing amino acids, peptides or proteins with HNE involves a classical Michael addition and the formation of a stable adduct [2]. This 1,4 addition as described in Fig. 6 requires the formation of a sulfhydryl ion by dissociation of the thiol. The ionized thiol can then attack the aldehydes on the fourth carbon which possesses a relatively high partial positive charge due to derealization of the double bond electrons of the aldehydes. The enol which is then formed is rearranged in keto-enol tautomerism to the corresponding saturated aldehyde. In reactions with 2-alkenals without the hydroxy group this is the final adducts. Cyclization occurs in alkenals containing hydroxy residue at a carbon-4 position. Since all the individual steps in this pathway are reversible, the overall reaction is also reversible. 4-Hydroxyalkenals may react with various amino acid residues of a protein. For example, the binding to lysine is reversible and in order to stabilize the bond the

Part III: Oxidative stress: mechanisms and manifestations

102 or lysine (NH2) RSH

^

RS-

1,4 Michael addition

R—C—C=C—CHO

R—0—C—C=C—O"

I

I

+ Glycine

H

OH

OH

HO

\

N

R—S

\

I

H2C—COOH

R—C—C—C—C=0 I H2 OH

R—SR-

OH

Fig. 6. Reaction mechanism for the formation of protein-aldehyde conjugates.

formed Schiff base has to be reduced to an secondary amine. HNE is highly reactive against amino acids such as Pro, Gly, His, Lys, and Ser; and the lowest reactivity is seen with Ala, Phe, He, Arg, Asp, Leu, Asn, Val, Gin and Glu [2]. HNE was found to react with the simplest amino acid glycine to form a pyridinium, derivative, 1 -(1 -carboxymethyl)-3-(2-hydroxypropyl)-pyridinium betaine [69] (Fig. 6). Uchida and Stadtman proposed [67] that HNE reacts with lysine also by a Michael addition mechanism as showed in Fig. 6. 2.

BIOLOGICAL RELEVANCE OF PROTEIN OXIDATION

2.1 Introduction There are several major areas which are the subject of this review concerning protein oxidation and its biological relevance: — Oxidative stress, protein oxidation, and aging. — Protein oxidation in pathological conditions and various diseases. — The role of protein oxidation in the process of protein inactivation and degradation. — Exercise, immobilization, and protein oxidation. — Plasma protein oxidation and modification due to exposure to cigarette smoke.


103

2.2 Oxidative stress, protein oxidation and aging The aging phenomenon is a gradual, multifactorial process that has been an enigma for humanity since time immemorial. Many theories have been put forward in this century in order to attempt to explain the causes and processes of aging. However, one can divide the important theories into two main categories, as seen in Fig. 7: the molecular and cellular theories and the systemic and physiological theories. The molecular and cellular theories can be further subdivided into genetic and nongenetic theories (see Fig. 7). Among this multitude number of theories, the free radical theory of aging has gained tremendous support and become the most popular hypothesis to explain the ubiquitous process of aging. The reason for this stems from the fact that this is the only theory for which extensive experimental evidence today supports the involvement of free radical damage in aging and age-related diseases. It was Harman [70] who first suggested, in 1956, that free radicals produced during respiration and cellular metabolism might cause oxidative damage to biological macromolecules. In addition, external factors such as toxic agents, irradiation, and excessive exercise may also raise the level of oxidative stress, and its cumulative effects over the years will lead to cellular damage, tissue and organ malfunctioning, and subsequently aging and death. This can be seen in Fig. 8. Several interesting findings in the last few decades have provided support and credibility to the free radical theory of aging. It was the discovery of the enzyme superoxide dismutase (SOD) [71] that showed that evolution has provided the living organism with antioxidant capacity to neutralize oxygen free radicals such as superoxide. Later, Cutler [72] observed that the metabolic rate of organisms is inversely related to the life span and aging rate in many animals. The higher the metabolic rate and, thus, the greater the increase in mitochondrial respiration and production of oxygen free radicals, the shorter the life span observed in aging

Theories of Aging molecular and cellular theories \

systemic and physiological theories

^

genetic theories

nongenetic theories

-the somatic mutation theory -the genetic control theory -the error catastrophe theory

-the immunological theory -the CNS-hormonal theory -the extracellular cross-linking theory

-the free radical theory of aging -the wear and tear theory -the membrane theory of aging -the intracellular molecular cross linking theory

Fig. 7. Theories of aging.


104

/

ROS can be formed by: -metabolism -irradiation -toxic effects

PUFA

I \

DNA

enzymes and proteins

I

\ aldehydic products of lipid peroxidation

altered DNA

\ I

altered proteins and enzymes

/

membrane and cellular damage

aging and age-related diseases Fig. 8. Effects of reactive oxygen species on biological systems.

mammals with some number of exceptions. Finally, very compelling evidence was presented several years ago which demonstrated that the increase of expression of the two antioxidant enzymes catalase and SOD retarded the age-related oxidative damage of Drosophila melanogaster [73] and increased the life span of these flies [74]. As seen in Fig. 8, the free radical theory of aging predicts that one should be able to observe elevated oxidative damage to proteins and enzymes with advanced age. Using the carbonyl assay for protein oxidation [75], the content of protein carbonyls has been found to increase considerably in several aging systems [3,73,75—81]. Protein carbonyls refer to aldehydes or ketone groups which maybe introduced into proteins by either metal-catalyzed oxidation reactions, the reaction of proteins with carbohydrates or lipids, or by Michael addition of aldehydes to proteins. Accordingly, it has been found that protein carbonyls increase by almost 50% in muscles of old versus young rats [75]. Similarly, protein oxidative damage estimated by the carbonyl assay was associated with the life expectancy of houseflies [73]. The use of estimation of carbonyl content as a measure for protein oxidation in biological tissues and in aging was questioned by Cao and Cutler [76,77] when they encountered some technical difficulties in measuring protein carbonyls. Thus, they could not detect any changes in the level of protein carbonyls in gerbil liver and brain with age or with a-phenyl-A^-tertbutyl nitrone (PBN)-treated animals. This is in contrast to the work of Carney et al. [78] who could detect such changes in protein oxidation in aging gerbils and with PBN treatment. However, Dubey et al. [79] later repeated the work of Carney et al. [78] and confirmed the original finding that PBN treatment causes a

Chapter 4: Chemical bases and biological relevance ofprotein oxidation

105

decrease of age-related elevation of protein carbonyls in gerbil brain cortex. In other studies it was observed that the carbonyl content of proteins dramatically increased in oxidized proteins during the final third of the life span of several species such as human, rat, and the housefly D. melanogaster [82]. Stadtman [3] estimated that oxidized protein content of old tissues represents at least 30—50% of the total protein content. This observation was compared to the finding that many enzymes such as aldolase, superoxide dismutase and inolase have lower catalytic activity in aging animals in the range of 25—50% [82]. A very interesting work was reported that showed that accumulation of protein carbonyls in mitochondrial proteins increased considerably with age [81]. This study showed that protein oxidation during aging was not a random process, it was show that high molecular weight mitochondrial proteins were relatively more oxidatively damaged than small molecular weight proteins during aging [81]. In addition to carbonyl accumulation of protein oxidation were measured by the loss of the membrane protein-sulfhydryl (-SH) group, Agarwal and Sohal [80] could show a decrease of membrane protein-SH content in old male houseflies with a concomitant decrease in the activity of alcohol dehydrogenase and glucose-6-phosphate dehydrogenase using the quantitation of QO'-dityrosine and O-tyrosine in proteins as a measure of protein oxidation. Recent work showed a marked increase of dityrosine with age in cardiac and skeletal muscles, but not in liver and brain, of aging mice [83]. O-tyrosine did not change with age in any of the above-mentioned tissues studied. However, caloric restriction to 60% of an ad libitum diet was capable of attenuating the dityrosine increase in cardiac and skeletal muscle [83]. It is interesting to note that elevation of protein oxidation with age is a relatively new area of research in the biology of aging compared to the previous observation of increased lipid peroxidation in aging tissues. Nonetheless, oxidative damage to biological molecules and their role in the aging process are not clear at this time, and more research is needed to elucidate this point. 2.3 Protein oxidation in pathological conditions and various diseases 2.3,1 Diabetes and the modification and oxidation of proteins In diabetes, long-term elevation of glucose has been shown to cause the phenomenon of non-enzymatic glycosylation or "glycation" of proteins. Indeed, an increased level of glycosylated hemoglobin has been observed in vivo in diabetic patients [84], and the extent of hemoglobin glycation has been used in the clinic as an index of progression of glycemia [85]. The molecular mechanism of protein modification due to reaction of glucose with proteins leads to the formation of Schiflf base reaction between the protein amines and the aldehydic moiety of sugars (see Fig. 9). Upon rearrangement, these Schiflf base compounds formed what has been called Amadori adducts containing a single group of ketosugars that can be further degraded into deoxyglucosomes containing dicarbonyl deriva-

106

Pcirt III: Oxidative stress: mechanisms and manifestations reducing sugars

_[_

primary amines of proteins

{} schiff base formation

^ amadori products

^ deoxyglucosomes H-protein-NHj

{} advanced maillard products (advanced glycation endproducts-AGE) Fig. 9. Nonenzymatic glycation and formation of AGE.

lives. These latter compounds are very reactive and cross-react with proteins to form adducts known as Maillard reaction products or advanced glycation end products [86]. The physiological consequences of this phenomenon has been the modification and alteration of many proteins like collagen [87] and serum albumin [88] and the aggregation of lens proteins in glycosilation associated cataracts [89]. Indeed, the stiffness of joints and arteries of diabetic patients has been attributed to increased cross-linking of collagen, possibly due to non-enzymatic glycosylation of collagen [87]. 2.3,2 Protein oxidation in atherosclerosis In the process of formation of atherosclerotic plaques, oxidized low-density lipoprotein (LDL) plays a major role. In a review article, the major oxidative changes to the protein component of LDL were summarized [90]. These include the appearance of tyrosyl radical reactive nitrogen intermediates and the formation of hypochlorous acid. In contrast, protein oxidation products that are characteristics of metal-catalyzed oxidation like carbonyls were not elevated in vitro models of LDL oxidation [90]. More specifically, 3-chlorotyrosine, which is a specific marker for myeloperoxidase-catalyzed oxidation, was found to be markedly elevated in LDL isolated from human atherosclerotic plaques [37]. Moreover, careful work has validated that hypochlorite-oxidized proteins including oxidized apoHpoprotein B, are present in atherosclerotic plaques [91]. Nitrotyrosine, which is used as a marker for peroxynitrite formation in diseased tissues, could not be detected in atherosclerotic lesions [92]. However, 3-nitrotyrosine was elevated [93]. In a recent publication, a significant increase of dityrosine and not 0-tyro-

Chapter 4: Chemical bases and biological relevance ofprotein oxidation

\ 07

sine was observed in fatty streaks of atherosclerotic plaques [94]. This finding supported the notion that tyrosyl radical is involved in the oxidative damage occurring in human atherosclerosis [94] and also suggest that MCO plays a minor role in this process. 2.3.3 Protein oxidation in cancer The involvement of free radicals and oxidative stress has been documented in a number of malignancies. The proposed mechanisms have been postulated to range from free radicals causing DNA double-strand breaks to the possibility of free-radical activation of oncogenes [95]. However, evidence for direct changes in the level of protein oxidation in cancer cells has been quite meager. In vitro studies of red blood cells from cancer patients compared to controls that were exposed to lipid or protein oxidation showed that methemoglobin formation was significantly higher in cancer patients [96]. Free radicals can cause oxyhemoglobin oxidative stress, which will result in an increase of methemoglobin [96]. The conclusion of the above authors was that free radicals in cancer alter erythrocyte permeability, which results in higher susceptibility to oxidative stress. A recent report on the capacity of benzo[a]pyrine of carcinogenesis initiation relates this chemical to the formation of a so-called micronucleus [97]. Evidence was presented that micronucleus formation in rat skin fibroblasts is connected to the elevation of oxidation of protein (carbonyl formation) and oxidation of DNA. The main conclusion of the above work was that carcinogenic initiation requires enzymatic bioactivation and it is peroxidase dependence and probably mediated by oxidation of proteins and DNA [97]. 2.3.4 Protein oxidation and neurological diseases Up to the present day, the etiology of many neurological and neurodegenerative diseases remains unclear. An interesting hypothesis was put forward by Beal [98] in which a defect in energy metabolism will lead to neuronal depolarization and an increase in intracellular calcium, which will, in turn, result in increased free radical generation [98]. A similar hypothesis was recently advanced by Markesbery [99] in which the etiology of Alzheimer disease (AD) was attributed to increased oxidative stress observed in the brain of AD patients. A corollary of the above hypothesis is that one should be able to detect increased oxidation to protein and lipids in neurodegenerative diseases. Indeed, studies in human have demonstrated an increase in oxidized proteins in the brains of patients with different neurological diseases (Alzheimer, Parkinson, and Huntington disease) [100]. A more detailed study of the level of oxidative damage to proteins, lipids, and DNA in seven different brain areas of AD patients was reported [101]. Overall, only a slight increase of protein carbonyls was found in areas such as frontal, occipital, and temporal lobes. In the parietal lobes the increase was quite significant. In addition, measuring some oxidized

108

P^^t III: Oxidative stress: mechanisms and manifestations

DNA bases showed that there was a significant elevation of DNA oxidation in a number of brain regions [101]. These findings partially support the notion that oxidative damage may play a role in the pathogenesis of AD, at least in some brain regions. On the other hand measuring protein carbonyls in motor cortex of sporadic motor neurons disease could not detect a significant elevation of protein oxidation compare to control patients [102]. 2.3.5 Protein oxidation in other pathologies Protein oxidation as measured by protein thiol loss and protein carbonyls has been detected in chronic ethanol-induced liver pathology [103], in inflammation [104], and in synovial fluid of patients with rheumatoid arthritis [105]. Elevated protein carbonyls and oxidation of protein thiols has also been observed in muscles of chickens with inherited muscular dystrophy [106]. A more recent report of measuring protein carbonyls in six human patients afflicted with Duchenne muscular dystrophy indicated that in quadriceps femoris there was an increase of 211% in protein oxidation compared to normal control subjects [107]. The latter finding was related to the fact that in Duchenne muscular dystrophy the protein dystrophin was missing, along with neuronal-type nitric oxide synthase. This was postulated to alter calcium homeostasis and to increase intracellular calcium levels, resulting in activation of the xanthine dehydrogenase/xanthine oxidase system, which causes elevation of superoxide radicals [107]. 2.4 The role of protein oxidation in the process of protein inactivation and degradation Early studies on glutamine synthetase from E. colt showed that oxidative damage renders the enzyme inactive. Oxidation of the enzyme could be generated by the cytochrome P-450 system or by ascorbic acid and oxygen [51]. After the enzyme was oxidized and inactivated, it was susceptible to proteolytic degradation systems of bacterial extracts [51]. In later studies in the mid-1980s, Davis and coworkers [55,108—110] showed that oxidation of proteins by hydroxyl radicals caused rapid protein denaturation and increased hydrophobicity, which was followed by protein degradation. The above process was dependent on the extent of denaturation and hydrophobicity, which seem to be the main signals for initiation of the proteolytic process [111]. Mild oxidative modification of protein caused a linear increase of denaturation and hydrophobicity However, more severe protein oxidation (usually in the absence of oxygen) resulted in stabilization of the proteins, due to both intra- and intermolecular cross-links. This process prevented further denaturation, which caused the proteins to become less susceptible to proteolytic degradation [111]. The latter finding may explain the phenomenon observed in aging when a number of proteins and enzymes are found to have longer half-lives and degrade more slowly [112,113]. This seemingly contradicts the observation that mildly oxidized proteins are degraded more rapidly.


109

There have been at least two explanations for the above finding. One is that oxidized proteins in aging may result in cross-linked in some proteins that render them more resistant to proteolytic degradation [114]. Indeed, cross-linked extracellular proteins like collagen and elastin are known to accumulate in aging tissues. The second explanation is that with aging there is a gradual reduction and loss of activity in the proteolytic system responsible for the removal of faulty proteins in aging [115]. In a later work, the increase in activity of neutral proteases was suggested to be responsible for the accumulation of oxidized proteins in aging [116]. Support for the above contention was found when an increased relationship was observed between the amount of abnormal protein accumulated in aging hepatocytes and the level of activity of neutral proteases found in these cells [47]. In the last decade there have been several attempts to identify the specific proteolytic system responsible for the degradation of oxidized proteins. Several works have suggested that it is the 19s multicatalytic cytosolic protease, part of the intracellular proteolytic system, known as proteosome, that is mainly responsible for degradation of oxidized proteins [117,118]. 2.5 Exercise, immobilization, and protein oxidation It is suggested that with strenuous exercise there is a marked elevation of oxidative stress to muscles. The first report to demonstrate that rats subjected to exhaustive exercise accumulate higher level of protein carbonyls was published in 1992 [119]. However, several weeks of pre-exercise feeding with high levels of vitamin E could ameliorate the accumulation of protein carbonyls [120]. Rats exercised for long-term training of 12 weeks also exhibited an increase in the level of protein carbonyls from 2.6 to 5.4 nmol/mg protein. However, after 2 weeks of cessation from exercise this value of protein oxidation was reduced to 3.6 nmol/mg protein [120]. More recent reports have also demonstrated an increase of protein carbonyls in muscle of rats subjected to high-altitude training [121]. In that study, however, a concomitant increase of lipid peroxidation products was not observed. In another recent study, long-term exercise for 8 weeks increased protein carbonyls and lipid peroxidation measured by thiobarbituric acid-reactive substances. Moreover, 8 weeks of vitamin E supplementation to animals supplemented with fish and soy oils markedly reduced the level of protein oxidation and lipid peroxidation [122]. Table 1 summarizes reports in which an increase protein oxidation was observed in exercised animals. Immobilization of hind-limb muscles of rats was shown to be associated with an increase of lipid peroxidation that could be reduced to some extent when immobilized animals were administered vitamin E [123]. A xanthine oxidase dependent molecular mechanism for this oxidative stress observed in immobilization has been proposed [124]. More recently, the increase of protein carbonyls in immobilized muscles of aging rats was also reported [125,126]. Administration of rat growth hormone to the immobilized animals could retard some of the protein oxidation and lipid peroxidation as well as other parameters of immobilization-

110


Table 1. Summary of protein carbonyl levels in exercise and immobilization. Type of tissue

Experimental conditions

Results

Reference

Red and white muscle, quadriceps muscle

Running until exhaustion

t

Reznicketal., 1992 [119]

Total hind-limb muscle

8 weeks of running

|

Wittetal., 1992 [120]

Quadriceps femoris muscle

4 weeks of high-altitude running 4 weeks of running at sea level

|

Radaketal., 1997 [121]

Muscles and liver

8 weeks of running

|

Gastrocnemius muscles

4 weeks of immobilization ]

CarmeUetal., 1993 [125]

Plantaris muscle

4 weeks of immobilization

|

Fares etal., 1996 [126]

Various brain areas

Immobilization

|

Liuetal., 1996 [127]

^

Senetal., 1997 [122]

associated damage [125,126]. Another paper [127] reported that immobilization causes increased levels of protein oxidation as well as lipid and DNA oxidative damage in several areas of the brain, supporting the notion that general stress such as immobilization may cause oxidative damage to brain and contribute to degenerative diseases observed in aging [127]. It is noteworthy that both severe modes of exercise and immobilization result in elevated lipid and protein oxidation. Nevertheless, the mechanism of these two phenomena may not be the same. 2.6 Plasma protein oxidation and cigarette smoke In early studies it was shown that exposure of human plasma, in vitro, to gasphase CS resulted in oxidative damage to lipids. Concomitant with the increase of lipid hydroperoxide due to CS exposure, there was a marked depletion of plasma and antioxidants such as rapid decrease of ascorbic and uric acids and slower disappearance of plasma a-tocopherol [128]. In subsequent studies, the effect of exposure of plasma to CS on protein carbonyl levels was investigated. Interestingly, exposure of humane plasma to nine puflFs of CS over a period of 3 hours produced a 500% increase in plasma protein carbonyls that could not be inhibited by the addition of glucose, ascorbate, or desferrioxamine. Only small thiols containing molecules like glutathione or dihydrolipoic acid could ameliorate the time-dependent accumulation of protein carbonyls [129]. In a later study, an attempt was made to elucidate the mechanism of these protein modifications. It was found that with the increase of protein carbonyl ac-


\\\

cumulation due to CS exposure, there was a concomitant decrease in protein-SH groups. Using purified unsaturated volatile aldehydes, such as acrolein and crotonaldehyde, known to be present in CS, it was possible to show that increase of protein carbonyl due to exposure to aldehydes was accompanied by a 1:1 molar ratio decrease of protein-SH groups. This explained quite clearly that aldehydes in CS interact with the SH group of proteins in a Michael addition reaction, leading to accumulation of carbonyls contributed by the aldehydic groups acrolein and crotonaldehyde [130]. 3

SUMMARY

A wide variety of chemical protein modifications do occur during protein oxidation. The modification of a protein by either a direct oxidation of a specific amino acid or cleavage of the protein backbone might results in impaired biological activity and changes in the secondary and tertiary structure of the protein. In this chapter we reviewed the chemistry and the patho-physiological relevancy of biologically irreversible protein oxidation. The physiological relevancy of the fine tuning of redox regulation on proteins activity was not included. Indeed oxidative stress in proteins results in faulty proteins accumulation in the organism's cells, tissues and body fluid. The numerous publications that describes the accumulation of oxidized proteins in the aging process and in various pathological disorders including diabetes, atherosclerosis, and brain disorders clearly demonstrate the relevancy of oxidative stress in these pathologies. 4

ACKNOWLEDGEMENT

Many thanks to Dr. Christiaan Leeuwenburgh for critically reading this work and providing comments. Part of this work was supported by the Krol Foundation, Lakewood, N.J., USA, to AZR 5

ABBREVIATIONS

ROS: MCQ: NFK: LDL: SOD: EDTA: BOC: BSA: MPO: HNE: AD:

reactive oxygen species metal-catalyzed oxidation N' -formylkynurenine Low-density lipoprotein superoxide dismutase ethylenediaminetetraacetic acid êr^butyloxycarbonyl bovine serum albumin myeloperoxidase 4-hydroxy-2-nonenal Alzheimer disease

112


PBN: CS: 6 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.

oc-phenyl-A^-rer^butyl nitrone cigarette smoke

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74-80. 84. Trivelli LA, Ranney HM, Lai HT N Engl J Med 1971;284(7):353-357. 85. Kennedy L, Baynes JW Diabetologia 1984;26(2):93-98. 86. Wolff SP, Jiang Z y Hunt JV. Free Radio Biol Med 1991;10(5):339-352. 87. Bailey AJ, Kent KJC In: Baynes JW, Monnier VM (eds) The Maillard Reaction in Aging, Diabetes and Nutrition. New York: Alan R. Liss, 1989. 88. Shaklai N, Garlick RL, Bunn HE J Biol Chem 1984;259(6):3812-3817. 89. Monnier VM, Stevens VJ, Cerami A. J Exp Med 1979;150(5):1098-1107. 90. Heinecke JW Curr Opin Lipidol 1997;8(5):268-274. 91. Hazell LJ, Arnold L, Flowers D,Waeg G et al. J Clin Invest 1996;97(6):1535-1544. 92. Evans P, Kaur H, Mitchinson MJ, Halliwell B. Biochem Biophys Res Commun 1996;226(2): 346-351. 93. Leeuwenburgh C, Hardy MM, Hazen SL,Wagner P et al. J Biol Chem 1997;272(3):1433-1436. 94. Leeuwenburgh C, Rasmussen JE, Hsu FF, Mueller DM et al. J Biol Chem 1997;272(6): 3520-3526. 95. Reizenstein P Med Oncol Tumor Pharmacother 1991;8:229-233. 96. Delia Rovere F, Granata A, Broccio M, Zirilli A et al. Anticancer Res 1995;15(5B):2089-2095. 97. Kim PM, DeBoni U, Wells PG Free Radic Biol Med 1997;23(4):579-596. 98. Beal ME Ann Neurol 1995;38(3):357-366. 99. Markesbery WR. Free Rad Biol Med 1997;23(1):134-147. 100. Carney JM, Carney AM. Life Sci 1994;55(25-26):2097-2103. 101. Lyras L, Cairns NJ, Jenner A, Jenner P et al. J Neurochem 1997;68(5):2061-2069. 102. Lyras L, Evans PJ, Shaw PJ, Ince PG et al. Free Rad Res 1996;24(5):397-406. 103. Rouach H, FataccioH V, Gentil M, French SWet al. Hepatology 1997;25(2):351-355. 104. Krsek-Staples JA, Webster RO. Free Radic Biol Med 1993;14(2):115-125. 105. Chapman ML, Rubin BR, Gracy RW J Rheumatol 1989;16(1):15-18. 106. Murphy ME, Kehrer JR Biochem J 1989;260(2):359-364. 107. Haycock JW, MacNeil S, Jones P, Harris JB et al. Neuroreport 1996;8(1):357-361. 108. Davies KJ, Delsignore ME. J Biol Chem 1987;262(20):9908-9913. 109. Davies KJ. J Biol Chem 1987;262(20):9895-9901. 110. Davies KJ, Lin SW, Pacifici RE. J Biol Chem 1987;262(20):9914-9920. 111. Davis KJA. Biochem SocTrans 1993;21:346-352. 112. Reznick AZ, Gershon D. Mech Ageing Dev 1979;ll(5-6):403-415. 113. Reznick AZ, Lavie L, Gershon HE, Gershon D FEBS Lett 1981;128(2):221-224. 114. Grant AJ, JessupW, Dean RT. Biochim Biophys Acta 1992;1134(3):203-209. 115. Lavie L, Reznick AZ, Gershon D. Biochem J 1982;202(1):47~51. 116. Starke-Reed PE, Oliver CN. Arch Biochem Biophys 1989;275:559-567. 117. Rivett AJ. Curr Top Cell Regul 1986;28:291-337. 118. Pacifici RE, Salo DC, Davies KJ. Free Rad Biol Med 1989;7(5):521-536. 119. Reznick AZ, Witt E, Matsumoto M, Packer L. Biochem Biophys Res Commun 1992;189: 801-806. 120. Witt EH, Reznick AZ,Viguie CA, Starke-Reed P et al. J Nutr 1992;122(3 Suppl):766-773. 121. Radak Z, Asano K, Lee KC, Ohno H et al. Free Radic Biol Med 1997;22(6):1109-1114. 122. Sen CK, Atalay M, Agren J, Laaksonen DE et al. J Appl Physiol 1997;83(1):189-195. 123. Kondo H, Miura M, ItokawaY. Acta Physiol Scand 1991;142(4):527-528. 124. Kondo H, Nakagaki I, Sasaki S, Hori S et al. Am J Physiol 1993;265(6, Pt 1):E839-E844. 125. Carmeh E, Hochberg Z, Livne E, Lichtenstein I et al. J Appl Physiol 1993;75(4):1529-1535. 126. Fares FA, Gruener N, Carmeh E, Reznick AZ. Ann N Y Acad Sci 1996;786:430-443. 127. Liu J, Wang X, Shigenaga MK,Yeo HC et al. Faseb J 1996;10(13):1532-1538. 128. Frei B, ForteTM, Ames BN, Cross CE. Biochem J 1991;277(Pt 1)):133-138. 129. Reznick AJ, Cross CE, Hu M-L, Suzuki YJ. Biochem J 1982;286:607~611. 130. O'Neill CA, Halliwell B, van der Vliet A, Davis PA et al. J Lab Clin Med 1994;124(3):359-370.

(t'2000 Elsevier Science B.V. All rights reserved. Handbook of Oxidants and Antioxidants in Exercise. C.K. Sen, L. Packer and O. Hanninen, editors.

11^ 11 J

Part III • Chapter 5

Lipid peroxidation in healthy and diseased models: influence of different types of exercise Helaine M. Alessio Miami University, Oxford, Ohio 45056, USA. Tel.: +1-513-529-2707. E-mail: [email protected]

1

2

3 4 5 6 7

1

INTRODUCTION 1.1 Lipid peroxidation: What is it and why is it important? 1.2 How is lipid peroxidation measured? LIPID PEROXIDATION AND EXERCISE 2.1 Healthy models 2.1.1 Acute exercise 2.1.2 Regular exercise 2.1.3 Aerobic and nonaerobic exercise 2.1.4 Antioxidant supplementation 2.2 Diseased models 2.2.1 Cardiovascular disease 2.2.2 Diabetes SUMMARY PERSPECTIVES ACKNOWLEDGEMENTS ABBREVIATIONS REFERENCES

INTRODUCTION

Free radicals and lipid peroxides play an important role in the oxidative stress balance between prooxidant and antioxidant activities. If the balance tilts towards prooxidants, the functional health of an organism is jeopardized. The major site for oxidative metabolism is the mitochondria. Energy-releasing reactions of oxidation-reduction for transporting electrons cause a proton (H"^) gradient across the inner mitochondrial membrane. Energy stored in the proton gradient combined with the inner mitochondrial membrane potential, provide the electrochemical basis for coupling electron transport with ADP phosphorylation. This is how large amounts of ATP are produced. Uncoupling may occur if an electron escapes the inner mitochondrial membrane, leaving the final electron receiver, oxygen, with one less electron in its possession. Uncoupling of oxidative phosphorylation results in the generation of oxygen radicals and lipid peroxides. Many of these radicals are derived from normal mitochondrial oxygen consumption and electron transport flux or movement across the inner mitochondrial membrane. Examples of free radicals formed in part by oxygen consumption and electron transport flux include superoxide radical (O2* ), hydroxyl radical

116

Pcirt III: Oxidative stress: mechanisms and manifestations

(*OH), perhydroxyl radical (HO2*), and conjugated peroxyl radical (LOO*), all of which may induce lipid peroxidation reactions [1—3]. 1.1 Lipid peroxidation: what is it and why is it important? Lipid peroxidation reactions involve free radical attack on polyunsaturated fatty acids. The availability of polyunsaturated fatty acid substrates and the antioxidant protection in lipid-rich cell membranes, control the pathophysiology of lipid peroxidation chain reactions that can produce free radicals and lipid peroxides. The way in which lipid peroxides may ultimately harm an organism is when the fatty acid under radical attack becomes a lipid radical with allylic double bonds. These double bonds are relatively weak and can combine with oxygen to produce lipid peroxy radicals and ultimately lipid peroxides. Lipid peroxides usually decompose to form aldehydes (e.g., malonaldehyde) which can cross-link or change the structures of proteins, lipids, carbohydrates, and DNA. Lipids are generally hydrophobic, and act as fuel for metabolism, support for membrane structure, and selective cell membrane transport. Unsaturated fatty acids are vulnerable to free radical attack, especially by the hydroxyl radical. When a hydrogen is removed from a hydrocarbon chain and a lipid peroxide is formed, the unsaturated fatty acid becomes more hydrophilic than usual. This new characteristic of fatty acids being attracted to water, alters the ability of lipids to selectively transfer metabolites through the cell membrane. As a result, excess water may enter the cell and inflammation may occur. Inflammation may trigger more O^ release, thus contributing to a positive feedback system that facilitates or propagates lipid peroxidation reactions, and produces more aldehydes, moving the cell towards destruction. Active oxygen species, including 02*~, *OH, HO2*, and LOO* can initiate and propagate lipid peroxidation reactions (Fig. 1). Free radical mediated lipid peroxidation generally requires radicals and weakly bonded polyunsaturated fatty acids for the initial steps. Oxygen and a lipid radical are necessary for the intermediate or propagation steps which ultimately produce lipid peroxidation byproducts. 02* OH* R-CH=CH-CH2-R • loss of an electron

R-CH=CH-C-«H-R (a lipid radical)

HO; LOG* Fig. 1. Summary of initial step for free radical initiation of lipid peroxidation reactions. 02*: superoxide radical; OH«: hydroxyl radical; H02«: perhydroxyl radical; LOO«: conjugated peroxyl radical; R: alkyl group; R-CH=CH-CH2-R: unsaturated lipid; -: single bond; =: double bond; R-CH=CHC-»H-R: lipid radical.

Chapter 5: Lipid peroxidation in healthy and diseased models

117

1.2 How is lipid peroxidation measured? Malonaldehyde (MDA), thiobarbituric acid reactive substances (TEARS), lipid hydroperoxides (LH), and 4-hydroxyalkenals (4-HNE) are examples of lipid peroxidation by-products that have been used as barometers of lipid peroxidation reaction rates. By-products of lipid peroxidation reactions have been investigated in many studies that have focused on exercise using both healthy and diseased models. Increases in MDA, TEARS, LH, or 4-HNE are directly linked to increased lipid peroxidation rates, and indirectly related to electron transport disturbances, uncoupling of oxidative phosphorylation, increased mitochondrial respiration, and/or elevated oxygen uptake (VO2). These lipid peroxidation byproducts are, themselves, toxic. Aldehydes such as MDA may be harmful because of their carcinogenic [4,5], mutagenic [6], and protein cross-linking [7] properties. The basis of the TEARS method is the measurement of MDA, which is one of the secondary products formed during the oxidation of polyunsaturated fatty acids. There are, however, other endogenous sources of MDA in tissues, which come from a variety of reactions such as side products of prostaglandin and thromboxane synthesis [8,9]. LH are the first separate products of oxidation, and interaction between LH and proteins or any other compounds, can damage membranes and enzymes [10]. 4HNE is a well characterized oxidation product of polyunsaturated fatty acids and increases in direct proportion to both LDL oxidation and lipid peroxidation reactions [11]. Steady-state MDA, TEARS, LH, and 4-HNE content of a tissue is the net result of the endogenous rate of lipid peroxidation chain reactions, the metabohc removal of lipid peroxidation by-products, and the antioxidant status of the tissue. It is generally agreed that oxygen toxicity is often accompanied by lipid peroxidation by-products However, it is not clear whether lipid peroxidation byproducts themselves are directly harmful to cells or if lipid peroxidation reaction rates coincidentally increase when oxygen radicals overwhelm antioxidants. An interesting side to the incubus perception of lipid peroxidation is that lipid peroxidation has been shown to be essential for normal cell development. Sevanian and Hochstein [12] suggested that only when lipid peroxidation takes place in uncontrolled free radical chain reactions that overwhelm antioxidant cell protection does lipid peroxidation cause cell damage. The implication that lipid peroxidation is necessary for normal growth and development is intriguing. Nevertheless, much evidence has accumulated implicating lipid peroxidation to a number of diseases including diabetes mellitus, atherosclerosis, rheumatoid arthritis, ischemia-reperfusion [11], hemolytic anemias, and pulmonary dysfunction [12]. Thus, an elevated lipid peroxidation rate as indicated by elevated MDA, TEARS, LH, or 4-HNE levels, implies that those cells that accumulate lipid peroxidation by-products, are less apt to function properly Lipid peroxidation can also be driven by agents other than oxygen or oxygen derived radicals, such as chelated iron, redux metal irons, ascorbate, enzymes

118


such as cyclooxygenase-dependent peroxidation of arachidonate to prostaglandin and thromboxane precursors or the NADPH-cytochrome P450 reductase dependent reactions in microsomes. The rate of hpid peroxidation can also be moderated by the amount and/or activity of certain antioxidants. That lipid peroxidation by-products can increase without evidence of higher oxygen consumption or oxygen derived radical activity, is of interest in studies on lipid peroxidation, disease, and research using different modes of exercise. 2

LIPID PEROXIDATION AND EXERCISE

Many studies have hypothesized that physical exercise can induce oxidative stress and accelerate lipid peroxidation reactions in humans [13—17]. Virtually all of these studies implicate aerobic exercise in producing either increased active oxygen species (e.g., 02*~, *OH, HO2*, and LOO*) that overwhelm antioxidant defenses to the point where free radicals and lipid peroxidation reactions run amuck. Most of the protocols used to study exercise-induced lipid peroxidation have used healthy models that participate in aerobic exercise such as running or swimming with durations of at least 20 min to exhaustion. 2.1 Healthy models Most studies on exercise and lipid peroxidation have used healthy subjects. Exercise-induced oxidative stress in healthy subjects has been shown to increase above baseline levels, but has not been blamed for serious health problems or compromised performance. Virtually all studies on aerobic exercise report that healthy and fit persons generally have a higher capacity to consume oxygen compared to age-matched unhealthy or unfit persons, and have less oxidative stress associated with aerobic exercise. 2,1,1 Acute exercise How dangerous can oxygen be if it is such a key component in aerobic exercise? Figure 2 gives a theoretical estimate of superoxide radicals produced by oxygen consumption at rest and during exercise. In this example, a 150 pound person is estimated to have a resting VO2 equivalent to about 1 MET or 3.5 ml- kg"^- min ^ This basal metabolic rate translates to about 353 1 of oxygen consumed in a day or 14.7 moles oxygen consumed in a day. Halliwell [18] has speculated that 1% of VO2 escapes univalent reduction in the electron transport system and 0.146 moles of superoxide radicals are formed. Over a year time, 0.146 moles of O2* would be equivalent to 1.72 kg of 02*~.

Chapter 5: Lipid peroxidation in healthy and diseased models

119

/ \

r / f \

Fig. 2. Estimation of VO2 and O2* production per year if a person does not exercise and if a person engages in daily moderate exercise. A: A 150 lb adult consumes 3.5 ml/kg/min, 352.8 1 02/day or 14.7 moles 02/day Assuming 1% of VO2 becomes O2* , then 0.146 moles O2* /day is produced adding 1.72 kg O2* each year. B: If a 150 lb adult consumes the same amount of oxygen at rest but exercises for 40 min each day at approximately 35 ml/kg/min, then this person consumes 441 1 02/day or 18.4 moles 02/day. Assuming 1% of VO2 becomes O2* , then 0.184 moles O2* /day is produced adding 0.43 more O2* for a total of 2.15 kg O2* each year.

2.1,2 Regular exercise The second scenario has this same 150 pound person raising their basal metaboHc rate 10-fold (with a V02= 35 ml- kg '• min^\ by exercising regularly for 40 min each day. Under the same assumption that 1% of the oxygen consumed will become O2* , this person, by adding a regular exercise regime, will produce 2.15 kg of O2* in a year. One can hypothesize that the effect on oxidative stress balance will vary quantitatively, with exercise time and intensity. It is likely that with no further antioxidant protection arising from either endogenous or exogenous sources, 2.15 kg of O2* compared to 1.72 kg of O2* would result in greater production of O2* , lipid peroxidation by-products, oxidative stress, and cell damage. 2.1,3 Aerobic and nonaerobic exercise Exhaustive exercise has been shown to incur the most signs and symptoms of oxidative stress. Radak et al. [19] reported that exhaustive running induced xanthine oxidase-derived oxidative damage in liver. Kidney tissue showed an increased lipid peroxidation after the run, which the authors suggested was due to a washout-dependent accumulation of peroxidized metabolites. When treated with antioxidants, a superoxide dismutase derivative, before exercising to exhaustion.

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P^^t III- Oxidative stress: mechanisms and manifestations

rats showed lower levels of TEARS and xanthine oxidase activity after the exercise [19]. Based on these results, oxidative stress associated with exhaustive exercise resulted in higher levels of lipid peroxidation by-products, namely MDA, and antioxidant supplement attenuated lipid peroxidation activity and MDA production. Swimming to exhaustion caused some forms of chemical change and oxidative damage to the membranes of erythrocytes, cardiac and skeletal muscle tissue of rats [20]. Following exhaustive exercise MDA increased, phospholipids decreased, protein oxidation increased and total cellular sulfyhdryls in blood plasma and skeletal muscle, increased. Sulfhydryl group oxidation was reported in another exercise study where seven moderately trained males completed a marathon race [13]. It is important to protect sulfhydryl groups, because a decrease in sulfhydryls resulting from their oxidation or a change in the disulfide/sulfhydryl ratio can inactivate certain enzymes and damage cells [21]. Although plasma TEARS did not change significantly, plasma protein-bound sulfhydryl group levels were 22, 12, and 13% lower than baseline immediately, 1 day, and 2 days, respectively, following the race [13]. These sulfhydryl levels may have been lower following exercise due to oxidization, and may tilt the disulfide/sulfhydryl ratio in a way that inactivates enzymes and causes cell damage. Few studies have focused on nonaerobic types of exercise that last only a minute or two. Radicals that initiate lipid peroxidation reactions are not always generated aerobically Superoxides have been observed during the autooxidation of catecholamines, hemoglobin, and thiols [22,23]. Ey-products of lipid peroxidation accumulate under conditions of both and low and high oxygen consumption. Alessio, Goldfarb and Cutler [24] reported increased lipid peroxidation following both aerobic and nonaerobic exercise in rats. One minute of high-intensity running (45 m-min^) resulted in 167 and 157% elevated TEARS in red slow-twitch and white fast-twitch muscle, respectively and in 34 and 31% elevated LH in red slow-twitch and white fast-twitch muscle, respectively. Since the high-intensity running only lasted for 1 min, it is likely that increased oxygen consumption may not be the exclusive rate limiting factor for lipid peroxidation. A pilot study by Alessio, Fulkerson and Wiley, [25] lends support to this suggestion. In this study, blood levels of prooxidant and antioxidant biomarkers were compared in six volunteers who exercised to exhaustion on a treadmill and a week later, exercised to exhaustion by squeezing a hand grip dynamometer at 50% of 1 repetition max (RM) at 45 s on/off intervals for the same amount of treadmill exercise time. Results showed that aerobic exercise resulted in a 14-fold increase in VO2 compared to a 2-fold increase in VO2 with isometric exercise (Fig. 3). MDA increased 5.6% above rest following aerobic exercise compared to 25.4% (p

O2

+

OH«

+

OH

(4)

It was subsequently shown that the original Haber-Weiss reaction was not likely to occur in vivo. However, if transition metals such as iron or copper are available then the hydroxyl radical may be formed. This has become known as the Fecatalyzed Haber-Weiss reaction which is shown in reaction [5,36]. O2

+ H2O2-Fe catalyst

->

O2 + OH# + OH

+ Fe ^^ (5)

Ryan and Aust [82] recently reviewed published work and discuss the important role of iron in determining rates of lipid peroxidation. They argue that the simultaneous presence of Fe^^ and Fe"^^ in a ratio of approximately 1 is the essential factor rather than the rate of formation of oxygen radicals. While in general, data seem to support that notion, it has not held for all experimental situations [90]. A number of workers in this area [36,82] point out that reactive species in addition to OH« may be generated from the interaction of iron chelates with H2O2. However, there is no clear evidence for the importance of these other species at physiological pH [91]. Tissue damage and destruction appear to lead to the release of iron ions and haem proteins in forms that can catalyze free radical damage [92]. It should be pointed out that transition metals are especially toxic to the nervous system, liver and kidney [93]. While the reason for the difference in susceptibility to oxidative stress is unclear, the mechanism involving transition metal induction seems consistent. The influence of iron and copper in the involvement of DNA damage has received considerable attention recently [94,95]. Iron [96], ferritin [97], and ironsulfur clusters [98] are present in the nucleus in certain circumstances. There is evidence that a metallothionein may serve to protect the nuclear environment against iron driven radical chemistry [99].

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P^^t JII- Oxidative stress: mechanisms and manifestations

Cabanatchik and colleagues [100] recently developed a fluorescence assay which should provide an important tool for measuring chelated intracellular iron. The technique has been applied successfully to the study of the labile (low molecular) iron pool of the cell [101]. 9

IRON AND PHYSICAL ACTIVITY

9.1 Nutrition, sport and anemia It is not difficult to understand the preoccupation of sports medicine investigators with iron deficiency since it is the most common single nutrient deficiency for about 15% of the world's population [102]. In developing countries, successful pregnancy outcomes and infant growth are highly dependent on nutrition interventions. In more developed countries, people make food choices for other reasons. The decision by many athletes to reduce food quantity and to reduce meat consumption might reasonably be expected to result in a marginal provision of iron. [103,104]. However, there is now an increasing amount of evidence indicating that the typical emphasis on correcting any appearance of low iron levels in athletes may be misdirected. For instance the body exerts a homeostatic adaptation to the stimulus of low iron stores by increasing iron absorption [105]. While habitual runners may be at an increased risk for iron deficiency, full-blown anemia is rare [106] and should be diagnosed carefully by assessing serum ferritin and hemoglobin markers. The relationship between these two markers in the clinical stages of iron-deficiency states are illustrated in Table 1. Although serum Table 1. States iron-deficiency Stage

Indicator = Low serum ferritin and:

Cutoff values

I. Storage Fe depletion

Normal Normal Normal Normal

250-500 îg/dl >15% > 80 |ig/dl >12g/dl

11. Fe deficient erythropoiesis

Increased total Fe binding capacity Decreased percent transferrin saturation Normal hemoglobin

350-650 îg/dl 12g/dl

Or Decreased serum Fe concentration Normal hemoglobin

< 60-80 jig/dl >12g/dl

Increased total Fe binding capacity Decreased percent transferrin saturation Decreased serum iron concentration Low hemoglobin

350-650 |ig/dl 55 years) when compared to young men ( < 30 years) during the 24-h period following 45 mins of downhill running; vitamin E supplementation significantly increased the postexercise rise in circulating neutrophils and CK activity in the older subjects to levels similar to those of the younger men. At the time of peak concentrations in the plasma, CK was significantly correlated with superoxide release from neutrophils. Urinary 3-methylhistidine excretion after the exercise Table 8. Aging, nutrition and the acute phase response. Acute phase reactant

Apparent aging effect

Nutritional effect

Complement Neutrophilia

No change Decreased

Elastase

Decreased

cytokine

No change

Not affected [33] Vitamin E supplementation had no effect on the stress-induced neutrophilia of young subjects, but "restored" responses of older subjects [41] Neutrophil degranulation, measured by plasma elastase levels, is related to plasma arachidonic acid/eicosapentaenoic acid (AA/ EPA) ratios changed by fish oil supplementation [31] Lipid peroxide and AA levels are production correlated with cytokine production; dietary factors that change lipid peroxide and AA levels change cytokine production [61,65]

Chapter 8: Acute phase immune response in exercise

\ 89

was correlated with mononuclear cell secretion of both IL-ip and PGE2. Moreover, increases in superoxide production by circulating neutrophils and in vitro endotoxin-induced IL-1 and TNF production by neutrophils were attenuated when subjects were supplemented with vitamin E. These data indicate that enhancing antioxidant status via vitamin E supplementation may affect the rate of repair of skeletal muscle following muscle damage and that these effects may be more pronounced in older subjects. The alterations observed in fatty acid composition, vitamin E, and lipid conjugated dienes in muscle and in urinary lipid peroxides after the eccentric exercise were consistent with the concept that vitamin E provides protection against exercise-induced oxidative injury [104]. The disruption of the contractile properties of human skeletal muscle following eccentric exercise-induced muscle damage also results in a substantial loss in maximal voluntary contraction and a selective loss of contractile force at low compared to high stimulation frequencies [105,106]. This selective low-frequency fatigue has been attributed to oxidative stress and free radical injury to the sarcoplasmic reticulum [107]. Thus, Jakeman and Maxwell [108] tested the effects of vitamins C and E supplementation on muscle function prior to an eccentric box-stepping exercise in physically active young people. They found a reduction in the loss of contractile function after the eccentric exercise and in the first 24 h of recovery in the group supplemented with vitamin C but not vitamin E; this protective effect was most evident with the low-frequency fatigue component (20/50 Hz tetanic tension). Interestingly, recent studies of a more severe form of tissue injury, blunt trauma, suggest that the major mechanism involved in neutrophil dysfunction in these patients is the triggering of the complement cascade and release of complementderived chemotactic factors, followed by inappropriate activation and subsequent deactivation of these cells [109]. These changes are associated with increased rates of auto-oxidation, declines in serum and neutrophil concentrations of vitamins C and E (but not glutathione), and loss of reducing capacity [110]. Employing a randomized cHnical trial, Maderazo et al. [Ill] found that intravenous infusion of vitamins C and E to victims of serious blunt trauma (from motor vehicle accidents) significantly reverses neutrophil locomotory dysfunction. 13 SUMMARY Heavy exercise, especially when muscle injury occurs, initiates an acute phase response that contributes to the breakdown and clearance of overloaded tissue. Following the exercise, the mobilization and activation of neutrophils appear to contribute to increased myocellular enzyme efflux. The acute phase reactions are unified by a common set of cytokine mediators including IL-ip, IL-6, and TNF which in turn influence some of their target tissues via induction of eicosanoids like PGE2. Importantly, these events are all modulated by peroxide tone and antioxidant defense status. The antioxidant vitamins C and E are consumed during acute phase immune reactions. A limited numuber of studies employing

190


interventions with these vitamins have shown a beneficial impact on some aspects of the acute phase immune response, including improvements in neutrophil function. 14 PERSPECTIVES The ability to modulate acute phase immune responses through nutritional means suggests new approaches to determining the fundamental role of the acute phase response in exercise. Despite several unsuccessful attempts to demonstrate a direct ergogenic effect of antioxidant nutrients, these compounds appear very important to the process of repair and recovery following intensive exercise. Thus, there may be a potential for increasing the intensity and/or frequency of physical training to improve performance following appropriate increases in antioxidant defenses as optimal physical performance capacity cannot be achieved without optimum cellular function. Further, as some data already suggests, information about the relationship between nutrition, exercise, and acute phase immune reactions may be directly applicable to the prevention and/or treatment of noninfectious inflammatory diseases such as rheumatoid arthritis and ankylosing spondylitis. 15 ABBREVIATIONS AA: bFGF: CL: EPA: IFN: IL-1: LPS: LT: PDGF: PG: PUFA: SOD: TGFP: TNF:

arachidonic acid basic fibroplast growth factor creatine kinase eicosapentaenoic acid interferon interleukin-1 lipopolysacchar ide leukotriene platelet-derived growth factor prostaglandin polyunsaturated fatty acids superoxide dimutase transforming growth factor (3 tumor necrosis factor

16 REFERENCES 1. Kluger MJ, Rothenburg BA. Science 1979;203: 374-376. 2. Rees RF, Gewurz H, Siegel JN, Coon J et al. Clin Immunol Immunopath 1988;48:95-107. 3. KlebanofF SJ. In: Gallin JI, Goldstein IM, Snyderman R (eds) Inflammation: Basic Principles and Clinical Correlates. New York: Raven Press, 1992;541-588. 4. Dinarello CA, Cannon JG, Wolff SM. Rev Infect Dis 1988;10:168-189. 5. Gelfand JA, Donelan MB, Burke JF. Ann Surg 1983;198:58-62.


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50. St Pierre BA, Tidball JG. J Appl Physiol 1994;77:290-297. 51. Robertson TA, Maley AL, Grounds MD, Papadimitriou JM. Exper Cell Res 1993;207: 321-331. 52. Nathan CF. J Clin Invest 1987;79:319-326. 53. Schmidt JA, Mizel SB, Cohen D, Green I. J Immunol 1982;28:2177-2182. 54. Krane SM, Dayer JM, Simon LS, Byrne S. Collagen Res 1985;5:99-117. 55. Cannon JG, Kluger MJ, Science 1983;220:617-619. 56. Haahr PM, Pedersen BK, Fomsgaard A,Tvede N et al. Int J Sports Med 1991;12:223-227. 57. Cannon JG, Fielding RA, Fiatarone MA, Orencole SFet al. Am J Physiol 1989;257:R451-455. 58. Sprenger H, Jacobs C, Nain M, Gressner AM et al. Clin Immunol Immunopathol 1992;63: 188-195. 59. Cannon JG, Tompkins RG, Gelfand JA, Michie HR et al. J Infect Dis 1990;161:79-84. 60. Viti A, Muscettola M, Paulesa L, Bocci Vet al. J Appl Physiol 1985;59:426-428. 61. Cannon JG, Meydani SN, Fielding RA, Fiatarone MA et al. Am J Physiol 1991;260: R1235-R1240. 62. Cannon JG, Evans WJ, Hughes VA, Meredith CNet al. J Appl Physiol 1986;61:1869-1874. 63. Pals KL, Change RT, Ryan AJ, Gisolfi CV. J Appl Physiol 1997;82:571-576. 64. Ku G, Thomas CE, Akeson AL, Jackson RL. J Biol Chem 1992;267:14183-14188. 65. Cannon JG, Fiatarone MA, Meydani M, Gong J-X et al. Am J Physiol (In press). 66. Packer L. J Sports Sci 1997;15:353-363. 67. Karlsson J World Rev Nutr Diet 1997;82:81-100. 68. Leaf DA, Kleinman MT, Hamilton M, Barstow TJ Med Sci Sports Exercise 1997;29:10361039. 69. Margaritis I,Tessier F, Richard MJ, Marconnet P Int J Sports Med 1997;18:186-190. 70. Magnusson B, Hallberg L, Rossander L, Swolin B. Acta Med Scand 1984;216:157-164. 71. Liesen H, Dufaux B, Hollmann W Eur J Appl Physiol 1977;37:243-254. 72. Anderson R,Theron AJ, Ras GJ Am Rev Resp Dis 1987;135:1027-1032. 73. Goldschmidt MC. Am J Clin Nutr 1991;54:1214S-1220S. 74. Bergsten P, Amitai G, Kehrl J, Dhariwal RK et al. J Biol Chem 1990;265:2584-2587. 75. Bendich A. Adv Exp Med Biol 1990;262:35-55. 76. Coquette A,Vray B,Vanderpas J Arch Int Physiol Biochem 1986;94:S29-S34. 77. Harris RE, Boxer LA, Baehner RL. Blood 1980;55:338-341. 78. Boxer LA. Adv Exp Med Biol 1990;262:19-33. 79. Baehner RL, Boxer LA, Allen JM, Davis J Blood 1977;50:327-331. 80. Engle WA, Yoder MC, Baurley JL, Yu PL. Pediatr Res 1988;23:245-248. 81. Anderson R,Theron AJ, Myer MS, Richards GA et al. J Nutr Immunol 1992;1:43-63. 82. Baehner RL, Boxer LA, Ingraham LM, Butterick et al. Ann NY Acad Sci 1982;293:237-250. 83. Azzi A, Boscoboinik, Szewczwyk A. Arch Biochem Biophys 1991;286:264—269. 84. El-Hag A, Clark RA. J Immunol 1987;139:2406-2413. 85. Anderson R, Smit MJ, Joone GK,Van Staden AM. Ann NY Acad Sci 1990;587:3448. 86. Esposito AL. Am Rev Resp Dis 1986;133:643-647. 87. Meydani SN, Barklund PM, Liu S, Meydani M et al. Am J Clin Nutr 1990;52:557-563. 88. Steinhilber D, Moser U, Roth HJ, Schmidt KH. Ann NY Acad Sci 1987;498:522-524. 89. Siegel BV, Leibovitz B. Int J Vit Nutr Res 1982;23(Suppl):9-22. 90. Johnston CS, Martin LJ, Cai X. Am Coll Nutr 1992;11:172-176. 91. Maderazo EG,Woronick CL, Albano SD, Breaux SP et al. J Infect Dis 1986;154:471-477. 92. Peters EM. Internatl J Sports Med 1997;18:S69-S77. 93. Mobarhan S, Bowen P, Andersen, Evans M et al. Nutr Cancer 1990;14:195-206. 94. Blumberg JB. Clin Appl Nutr 1991;1:53-61. 95. LeeTH, Hoover RI,Williams JD, Ravalese J et al. N Engl J Med 1985;312:1217-1224. 96. Payan DG,Wong M y Chernov-RoganT,Valone FH et al. J Clin Immunol 1986;6:402-410. 97. Endres S, Ghorbani BS, Kelley VE, Georgilis K et al. N Engl J Med 1989;320:265-271.


193

98. Meydani SN, Endres S,Woods MN, Goldin RD et al. J Nutr 1991;121:546-555. 99. Virella G, Kilpatrick JM, Rugeles MT, Hyman B et al. Clin Immun Immunopathol 1989;52: 257-270. 100. Kremer JR, Schoene N, Dougless LW, Judd JTet al. Am J Clin Nutr 1991;54:896-902. 101. Fielding RA, Meydani M. Aging 1997;9:12-18. 102. Zerba E, Komorowski TE, Faulkner JA. Am J Physiol 1990;258:C429-C435. 103. Oliver CN, Ahn B, Moerman EJ, Goldstein S et al. J Biol. Chem 1987;262:5488-5491. 104. Meydani M, Evans WJ, Handleman G, Biddle L et al. Am J Physiol 1993;264:R992-R998. 105. Newham DJ, Jones DA, Clarkson PM. J Appl Physiol 1987;63:1381-1386. 106. Evans WJ, Cannon JG. Exerc Sport Sci Rev 1991;19:99-125. 107. Byrd SK. Med Sci Sports Exerc 1992;24:531-536. 108. Jakeman P, Maxwell S. Eur J Appl Physiol 1993;67:426-430. 109. Maderazo EG,Woronick CL, Albano SD, Breaux SP et al. J Infect Dis 1986;154:471-477. 110. Maderazo EF, Woronick CL, Hickingbotham N, Mercier E et al. Crit Care Med 1990;18: 141-147. 111. Maderazo EG,Woronick CL, Hickingbotham N, Jacobs L et al. J Trauma 1991;31:1142.


( 2000 Elsevier Science B.V. All rights reserved. Handbook of Oxidants and Antioxidants in Exercise. C.K. Sen, L. Packer and O. Hanninen, editors.

^Q^ iyJ

Part III • Chapter 9

Oxidative DNA damage in exercise Andreas Hartmann^ and Andreas M. Niess^ Ândreas Hartmann, Novartis Pharma AG, Genetic Toxicology, WS2881.5.14 CH-4002 Basel, Switzerland Andreas M. Mess, Medical Clinic and Polyclinic, Department of Sports Medicine, University of Tuebingen, Hoelderlinstr 11, D-72074 Tuebingen, Germany. E-mail: [email protected]

1 INTRODUCTION 2 DNA: CARRIER OF GENETIC INFORMATION 3 OXIDATIVE DNA DAMAGE 4 DNA DAMAGE AFTER EXERCISE 4.1 Exercise-induced damage in cellular DNA 4.2 Analysis of oxidized bases in cellular DNA 4.3 Urinary excretion of oxidized DNA bases and nucleosides 4.4 Cytogenetic methods 4.5 Exercise-induced apoptosis: Secondary induction of DNA fragmentation 5 FREE RADICAL GENERATION IN EXERCISE: POTENTIAL MECHANISMS FOR DNA DAMAGE INDUCTION 5.1 Generation of radicals during exercise 5.2 Generation of radicals after exercise: inflammatory processes 6 BIOLOGICAL SIGNIFICANCE OF EXERCISE-INDUCED DNA DAMAGE: EPIDEMIOLOGICAL EVALUATION 7 SUMMARY 8 PERSPECTIVES 9 ACKNOWLEDGEMENTS 10 ABBREVIATIONS 11 REFERENCES

1

INTRODUCTION

Oxidative stress due to vigorous exercise is a well-described phenomenon. In living cells, reactive oxygen species are formed continuously and physical exercise leads to an increased generation of radicals. Free radicals are also generated as part of the reparative process following muscular injury. If not adequately removed by antioxidant defenses, free radicals react with membranes, proteins, nucleic acids, and other cellular components to initiate cellular damage or degeneration. Indicators of free radical damage, such as oxidized proteins and products of lipid peroxidation have been widely described. Recent reports provide evidence that certain regimens of physical exercise are also capable of inducing DNA modifications. Induction of DNA damage after excessive exercise was described, whereas moderate exercise was shown to have no effects on cellular DNA. Antioxidants, as well as regular training seem to have a protective effect against the occurrence of DNA damage after exercise and the involvement of oxidative stress as one underlying mechanism was proposed. Further evidence for

196


oxidative stress as one underlying mechanism was provided by demonstrating a concomitant increase of heat shock proteins in leukocytes. The induction of structural DNA alterations by exposure to reactive oxygen species is of great interest since such modifications seem to play a key role in human cancer development. However, the relationship between oxidative stress due to vigorous exercise and induction of DNA modifications is unknown and their long-term effects on health have yet to be elucidated. The aim of this chapter is to provide insight into the relatively new field of exercise-induced DNA damage, the techniques used to detect this specific damage, and the biological significance of these findings. 2

DNA: CARRIER OF GENETIC INFORMATION

All living organisms, prokaryotes and eukaryotes, are composed of cells. The genetic information of cells is encoded in their deoxyribonucleic acid (DNA). Nucleic acids are macromolecules assembled from nucleotides. The sequence in which the individual blocks are joined together is the critical factor that determines the property of the resulting macromolecule. The maintenance of the exact structure is important for both, cellular metabolism and passing the correct information from one generation to the next. The unit of inheritance is called a gene and each gene constitutes a specific sequence of DNA that carries the information representing a particular protein. Genes are carried on chromosomes and the set of genes of a particular organism is called a genome. Genetic material is subjected to a number of stresses including oxidative damage and damage due to environmental mutagens/carcinogens. These agents induce structural alterations in DNA such as modified bases or chromosome breaks. The alterations induced have a certain probability of remaining damaged or being misrepaired. When a cell divides, such damage may manifest in changes in the DNA sequence, a mutation. The sequence of bases in the polynucleotide chain is important not in a sense of structure per se, but because it codes for the sequence of amino acids that constitutes the corresponding protein. Therefore, mutations may result in dysfunctional proteins but may not affect the maintenance of DNA itself. Certain proteins are important for the regulation and control of the cell cycle and before a cell can become malignant, disturbance of homeostasis in a number of cellular systems is necessary. Neoplastic expansion of a mutated cell may result from activation of stimulatory mechanisms or the inactivation of inhibitory mechanisms. It is assumed that dynamic processes and several genetic changes are required before a normal cell can become tumorigenic [1]. However, DNA is continuously damaged by oxidative reactions and DNA damage induced by endogenous mechanisms is high [2,3]. Cells are able to respond to DNA damage with a variety of repair mechanisms to restore DNA structure and sequence. Oxidative DNA damage can be repaired by excising damage bases or nucleotides. Deficiencies in DNA repair systems may lead to the accumulation of genetic changes and can have deleterious effects on organisms. Several human hereditary diseases are known which are caused by

Chapter 9: Oxidative DNA damage in exercise

197

mutations in DNA repair genes or genes involved in eliminating reactive oxygen species [4]. The rare human disease Xeroderma Pigmentosum is caused by a defect in nucleotide excision repair and is characterized by photodermatoses including skin cancer at an early age [5]. The importance of an intact defense system against reactive oxygen species is further underscored by another rare genetic disease, Amyotrophic Lateral Sclerosis. Some of the familial forms are associated with mutations in Cu/Zn-superoxide dismutase which are accounted for the accumulation of reactive oxygen species with catastrophic effects on motor neurons [6]. 3

OXIDATIVE DNA DAMAGE

Oxygen is the terminal oxidant for respiration and other oxidative reactions in aerobic organisms. Some reactive oxygen species have important physiological functions, but also represent a source of endogenous damage to the genome. It is generally accepted that a certain amount of oxidative DNA damage occurs in animals and humans. Ames and colleagues [7] estimated that each liver cell from adult rats contain 10^ oxidative lesions and that 10^ new lesions are added daily. Cells possess mechanisms to minimize, but not necessarily ehminate, the effects of normal levels of oxidative damage. Oxidized DNA is abundant in human tissues and it is suggested that damaged DNA bases accumulate with age [7,8]. A relationship between oxidative modification of mitochondrial DNA and the process of aging has emerged [9]. Subjecting tissue to oxidative stress, and thereby overwhelming the antioxidative capacity of a cell can result in severe metabolic dysfunctions [2,10], including un- or misrepaired DNA lesions as demonstrated in Fig. 1. Different types of oxidative DNA damage are known DNA

exposure to reactive oxygen species ( H ^ , a 2 , O H , R O )

induction of primary DNA lesions • single- and double strand breaks • base/nucleotide modifications (e.g. 8-OHG/8-OHdG) H/^-^N-

• DNA-protein crosslinks

replication • gene mutations • chromosome mutations ^

unaltered genetic information

modulation of gene/protein activity initiation of carcinogenesis

Fig.l. DNA damage induced by reactive oxygen species and their biological consequences.

198

P(^^t III: Oxidative stress: mechanisms and manifestations

such as base lesions, sugar lesions, single- and double-strand breaks, abasic sites, and DNA-protein crosslinks. The oxidized desoxynucleoside 8-hydroxy-7,8dihydro2'-deoxyguanosine (8-OHdG) is of special interest. Due to its mispairing properties it has been suggested that it is directly involved in the process of carcinogenesis [11,12]. The cell is able to rapidly remove this DNA lesion from nuclear DNA and from the nucleotide pool by different repair mechanisms. This might explain, why elevated levels of activated endogenous oxygen species are rather inefficient at inducing gene mutations, but tend to generate gross chromosomal rearrangements [13]. The carcinogenic impact of oxygen radicals may be based on oncogene activation or suppressorgene inactivation through chromosomal rearrangements. Furthermore, oxygen radicals may induce genotoxic effects by indirect mechanisms leading to the formation of chromosome breaking agents (clastogenic factors) [14]. Besides its key role in human cancer development, oxidative DNA damage also seems to be of causal importance in various other disease processes. It has been suggested that damage to mitochondrial DNA is related to age-associated degenerative diseases. Mitochondrial DNA (mtDNA) is a major source and target site of reactive oxygen species. An accumulating rate of mtDNA mutations as a result of oxidative DNA damage will result in deficient mitochondrial respiratory function which, in turn, leads to cellular energy deficit [9,15]. Mounting evidence suggests that mitochondrial dysfunction and oxidative stress contribute to the pathogenesis in several neuro-degenerative diseases such as Parkinson disease. Amyotrophic Lateral Sclerosis, Alzheimer disease and Huntington disease [16]. An elevated content of 8-OHdG in mtDNA extracted from cerebrums and cerebellums [17], as well as increased levels of other oxidized DNA bases in different brain areas [18] have been shown in patients with Alzheimer disease. However, it remains unclear whether oxidative DNA damage is a primary or secondary event in this disease. Furthermore, other energy-requiring postmitotic tissues such as skeletal and heart muscle also seem to be sites in which mutations of mtDNA are associated with chronic degenerative diseases [15]. Measurements of an accumulated content of 8-OHdG in human heart mitochondria with age confirms the assumption that oxidative damage to mitochondrial DNA may be involved in failure of the aging heart [19,20]. Induction of DNA damage is measured using a variety of techniques. Primary DNA lesions induced by free radicals such as strand breaks, abasic sites, and DNA-protein crosslinks are determinated with biochemical methods. These methods allow the detection of DNA strand breaks and DNA adducts with high sensitivity. However, primary DNA lesions are usually repaired by repair enzymes with high efficiency and for the assessment of the biological significance of DNA damage, cytogenetic techniques and mutation assays are employed. These methods allow the detection of DNA lesions which remained unrepaired or misrepaired and were transformed into chromosome modifications or mutations during cell proliferation [21].


4

199

DNA DAMAGE AFTER EXERCISE

Only recently, an increasing number of studies demonstrated the induction of DNA modifications in human subjects as a consequence of exhaustive exercise. Oxidative stress has been discussed as a mechanism causing these effects. In principle, three different endpoints were used to investigate exercise-induced DNA damage: 1. Analysis of the steady-state level of DNA alterations as an indicator of the balance between damage (analysis of primary DNA lesions) and repair. This analysis was performed in DNA from peripheral leukocytes or lymphocytes which are well-established indicators of exposure to DNA damaging agents in human biomonitoring studies [21]. 2. An index of the total oxidative DNA damage which has been repaired. This is provided by analysis of DNA base damage products excreted in the urine. 3. Analysis of exercise-induced chromosome alterations. The following overview provides an insight into the techniques and endpoints used to assess exercise-induced DNA damage and discusses differences in the findings. Figure 2 summarizes the techniques used to measure DNA damage after exercise. 4.1 Exercise-induced damage of cellular DNA DNA lesions such as strand breaks, abasic sites, and DNA-protein crosslinks are measured with biochemical methods. In the majority of the studies reported so far, the comet assay (single-cell gel test) was used to demonstrate elevated DNA breaks in leukocytes of human subjects after exercise. The comet assay is a microgel electrophoresis technique which enables the detection of DNA damage and - DNA damage in leukocytes: primary DNA lesions

^chromosome alterations

- strand breaks: comet assay FADU

- micronuclei - sister chromatid exchanges

- oxidized DNA bases: comet assay + specific enzymes HPLC analysis

- oxidized DNA bases HPLC-analysis of urinary excretion of 8-OHdG and 8-OHG Fig.2. Techniques used to demonstrate exercise-induced DNA damage. FADU, fluorescence analysis of DNA unwinding; 8-HdG, 8-hydroxy guanosine; 8-OHG, 8-hydroxy guanine.


200

DNA repair in individual cells with high sensitivity [22]. This technique has widespread potential applications and is becoming an established tool in biomonitoring studies, as well as in genotoxicity testing [23]. Detailed protocols for the assay have been described [24]. In brief, a single-cell suspension (e.g., whole blood or isolated lymphocytes) is dispersed in agarose and spread on a microscope slide. Then, the cells are lysed with high salt and detergents, alkali-denatured and electrophoresed. After staining, the microscopic image of cells with increased DNA damage displays increased migration of chromosomal DNA out of the nucleus, resembling the shape of a comet. The amount of DNA migration indicates the amount of DNA breakage in the cell. Unlike other methods, results of the comet assay are believed not to be confounded by dead cells, which might have undergone programmed cell death (apoptosis, see below) or necrosis [25]. Using the comet assay, various exercise protocols have been studied, such as incremental runs to exhaustion on a treadmill [26—28], half marathons [29,30], triathlon competitions [31] or repeated bouts of anaerobic runs (Zankl, personal communication). The results with the comet assay unequivocally show the induction of DNA damage in human leukocytes independent of the exercise protocols used. In all studies, DNA damage was detected with a delay of several hours after the exercise. Interestingly, the exercise protocol used seems to have an influence on the induction of DNA damage. Figure 3 compares the effects of an single bout of exhaustive exercise on the treadmill with the occurrence of DNA damage after a triathlon competition. After a run on a treadmill, the highest increase in DNA damage was found 24 h postexercise, whereas after the triathlon, an additional and very strong increase in DNA damage was found 72 h after the race. The results indicate that the exercise protocol used has an important influence

before after

6h

24 h

48 h

72 h

incremental treadmill run

before after 24 h

48 h

72 h

J

triathlon competition

Fig. 3. DNA damage in leukocytes of humans measured as increased DNA migration, (mean and SEM of 50 cells per data point), in the comet assay, a: DNA damage after a run on a treadmill (n = 3); b: DNA damage after a triathlon competition (n = 6). Reprinted with the permission of Oxford University Press 26] and also with permission from Elsevier Sciences Inc [31].


201

on occurrence and persistence of DNA damage. It should be stressed that the treadmill run was performed by untrained individuals, whereas the triathlon was done by well-trained athletes. Another study described a difference in the extent of DNA damage induced in trained vs. untrained individuals. The results of a comparative study on trained and untrained individuals after a treadmill run are shown in Fig. 4. In most of the studies discussed above, either whole leukocytes or isolated lymphocytes were analyzed. One investigation studied DNA damage in ten trained individuals after a half marathon and a differential analysis was performed with leukocytes and separated mononuclear cells [30]. It was shown that the percentage of damaged cells 24 h after the race was higher in total leukocytes than in mononuclear cells (Fig. 5). The authors concluded that the exercise protocol used induces DNA damage primarily in granulocytes. This might also explain negative or equivocal findings using techniques with which only proliferating cells (lymphocytes) could be used for an analysis (see section 4.4, Cytogenetic methods). Using a fluorometric analysis of DNA unwinding (FADU), Sen and colleagues [32] detected an association between a single bout of exercise and the induction of DNA strand breaks in leukocytes. In this study, two submaximal exercises were carried out 7 days apart, each lasting 30 min. Blood samples were taken before and 2 min after each exercise. This study found an induction of DNA damage in 13 out of 18 samples. In contrast to the studies described above, DNA damage was detected directly after the runs suggesting that the technique used might be more sensitive. Unfortunately, no blood samples were analyzed at later time points after the runs.

Untrained

Trained

Fig.4. Effects of a run on a treadmill on DNA damage in leukocytes of trained (n = 6) and untrained (n = 5) individuals. The results are presented as percent changes of DNA migration in the comet assay from resting to 24 h postexercise values (A DNA migration) and displayed as boxplots, in which the horizontal line represents the median, the edges of the box the 25% and 75% quantiles and the end of the bars the extremes. Data modified from [28] with kind permission.

202


60-

leukocytes

mononuclear cells

50CD

o 40-

•D CD

D)

.

30-

CO

E 20CO

ii -> -^ ii -> ii ii -> T

Pereira et al. [93]

(Continued.)

swimming swimming swimming swimming swimming

swimming swimming swimming swimming swimming swimming swimming swimming swimming swimming

—)•

- > •

Chapter 11: Superoxide dismutases in exercise and disease

259

Table 5. Continued. Investigator

Subject

Tissue

Type of work

SOD

Change

Shimojo etal. [106]

Mice Mice Mice Mice Mice Mice Rats Rats Normotensive rats Normotensive rats Normotensive rats Normotensive rats Normotensive rats Hypertensive rats Hypertensive rats Hypertensive rats Hypertensive rats Hypertensive rats 2-month-old mice 2-month-old mice 26-month old mice 26-month-old mice Rats

Brain Lung Heart Liver Kidney Red blood cells Heart Heart Outer wall of 1. ventricle Longissimus dorsi m. Quadriceps femoris m. Liver Kidney Outer wall of 1. ventricle Longissimus dorsi m. Quadriceps femoris m. Liver Kidney Diaphragm Diaphragm Diaphragm Diaphragm Heart

Total Total Total Total Total Total Cu,Zn Mn Total Total Total Total Total Total Total Total Total Total Cu,Zn Mn Cu,Zn Mn Total

^ -^ -^ T -^ T T T -^ TT -^ i ^ -^ -^ T i -^ T -^ -^ -^ -^

Food-restricted rats

Heart

Total

"^

Rats, normal protein diet Rats, normal protein diet Rats, normal protein diet Rats, normal protein diet Rats, casein protein diet Rats, casein protein diet Rats, casein protein diet Rats, casein protein diet Rats, soy protein diet Rats, soy protein diet Rats, soy protein diet Rats, soy protein diet 77-week-old rats

Liver

9 weeks swimming 9 weeks swimming 9 weeks swimming 9 weeks swimming 9 weeks swimming 9 weeks swimming 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 6 weeks swimming 6 weeks swimming 6 weeks swimming 6 weeks swimming 18.5-month wheel running 18.5-month wheel running 12 weeks running

Cu,Zn

T

Liver

12 weeks running

Mn

-^

Gastrocnemius m.

12 weeks running

Cu,Zn

-^

Gastrocnemius m.

12 weeks running

Mn

-^

Liver

12 weeks running

Cu,Zn

-^

Liver

12 weeks running

Mn

-^

Gastrocnemius m.

12 weeks running

Cu,Zn

->

Gastrocnemius m.

12 weeks running

Mn

->

Liver

12 weeks running

Cu,Zn

^

Liver

12 weeks running

Mn

-^

Gastrocnemius m.

12 weeks running

Cu,Zn

-^

Gastrocnemius m.

12 weeks running

Mn

-^

Heart

9 weeks running

Total

r

Somani et al. [80] Hong et al. [94]

Oh-ishi etal. [117]

Kim et al. [107]

Song et al. [104]

Somani et al. [99] (Continued.)

Part IV: Antioxidant defenses

260 Table 5. Continued. Investigator

Subject

Tissue

Type of work

SOD

Change

Husain and Somani [100] Oh-ishi et al. [84] Oh-ishi et al. [85] Leeuwenburgh et al. [95]

Rats Ethanol-treated rats Rats Rats Rats Rats Rats Rats Rats Rats Rats Rats Rats Rats Rats Rats Rats Rats Rats

Heart Heart Soleus m. Soleus m. Diaphragm Diaphragm Liver Liver Liver Heart Heart Heart Deep vastus lateralis m. Deep vastus lateralis m. Deep vastus lateralis m. Soleus m. Soleus m. Soleus m. Heart

Total Total Cu,Zn Mn Cu,Zn Mn Cu,Zn Mn Total Cu,Zn Mn Total Cu,Zn Mn Total Cu,Zn Mn Total Total

T T T T T T -> -* -^ -^ ^ ^ T -> T -^ -^ —> -^

Rats

Heart

Total

~^

2-month-old mice 2-month-old mice 26-month-old mice 26-month-old mice

Kidney Kidney Kidney Kidney

6.5 weeks running 6.5 weeks running 9 weeks running 9 weeks running 9 weeks running 9 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 10 weeks running 6 weeks sprint running 6 weeks endurance running 6 weeks swimming 6 weeks swimming 6 weeks swimming 6 weeks swimming

Cu,Zn Mn Cu,Zn Mn

-^ -^ -^ T

Libonati et al.[108]

Toshinai etal. [122]

physical training, because the adaptive changes produced by swimming training were revealed more closely to resemble those produced by cold acclimation rather than those resulting from running exercise [111 — 115]. Actually, 4-week exposure to cold at 5°C definitely increased rat liver Mn-SOD level estimated by an ELISA [116]. On the other hand, the effect of physical training on diaphragmatic SOD activity appeared to be constant, namely, positive [85,92,117]. These data indicate that endurance training can cause tissue-specific adaptation of SOD. Meanwhile, zinc has been reported to affect the free radical production in sedentary mice [118]. In the work of Cao and Chen [110], zinc deficiency eliminated the training-induced increases in blood and hepatic SOD activities which existed in zinc-adequate mice. However, a high amount of dietary zinc had even a harmful effect on both sedentary and exercised zinc-deficient animals. Physical training also increased SOD activity in blood [105,106,110], being in accord with human studies [55,60,119]; however, SOD activity in bone marrow was not increased after 4-week running training [77]. Finally, training effect on cardiac SOD activity was apparently reduced by aging [96].

Chapter 11: Superoxide

dismutases

in exercise and disease

261

Table 6. Acute exercise performance and SOD level in humans. Investigator

Subject

Tissue

Type of work

SOD

Change

Ohno et al. [57] Ohno et al. [49]

Untrained students Volleyball players Volleyball players Soccer players Soccer players Soccer players Soccer players Soldiers Soliders Untrained students Trained students

Red blood cells Plasma Plasma Serum Serum Urine Urine Serum Serum Plasma Plasma

30 min bicycle, 75% max 15 min bicycle, 75% max 15 min bicycle, 75% max 2 h soccer 2 h soccer 2 h soccer 2 h soccer 93 h ranger training 93 h ranger training Vo, max test, bicycle V(), max test, bicycle

Cu, Cu, Mn Cu, Mn Cu, Mn Cu, Mn EC EC

^ -^ -^ ii -^ TT ^''

Ohno etal. [123]

Ohnoetal. [124] Haga et al. (unpublished data)

Zn Zn Zn Zn Zn

- » •

TT -^ T

^No immunoreactive Mn-SOD level in urine was detected before and after 2 h soccer training.

3.3 Effect of exercise on SOD level in humans 3.3,1 Acute exercise Sedentary students were studied, using a bicycle ergometer, for 30 min at about 75% Vo2max [57]. As a result, red blood cell Cu,Zn-SOD level (which was measured by a single radial immunodiffusion technique) did not change substantially (Table 6). On the other hand, Cu,Zn-SOD level (estimated by an ELISA) in plasma decreased markedly at 15 min and 24 h after 15-min cycle ergometer exercise, whereas the Mn-SOD level did not vary significantly (Table 7) [49]. The reduced plasma level of Cu,Zn-SOD, the molecular weight of which is 32,000 (smaller than that of Mn-SOD) (Table 1), could be the result of an increased glomerular permeability, although the mechanism remained obscure. Cu,Zn-SOD actually emerged into urine, in connection with the reduced plasma level of Cu,Zn-SOD after 2 h of soccer training [123]. Further investigations into the meaning of the Table 7. Variations in plasma enzyme values and hematocrit level at ~ 75% V Q , max on a cycle erg o m e t e r ( n = 10) [49]. Variable

Initial control

Immediately after 15 min exercise

After 15 min rest

24 h after exercise

Mn-SOD (|ig/l) Cu,Zn-SOD (|ig/l) AST-m (IU/1) CK (IU/1) Hematocrit (%)

110±6 38.1 ±5.1 5.73 ±0.30 91.2 ± 8 . 4 43.0 ±0.8

113±6 30.2 ± 6.3 7.97 ± 0.62^' 133 ± 14^ 46.6 ± 1 . P

104 ± 9 10.6 ±2.9^ 6.74 ± 0.70 109 ± 19 44.1 ±0.8

109 ± 7 12.6 ±4.5^ 5.62 ± 0.49 78.1 ± 14.7 43.4 ±0.7

Values are means ± S.E.M. SOD: superoxide dismutase; AST-m: mitochondrial aspartate aminotransferase; CK: creatine kinase. ^p< 0.05 vs. initial control values.

262


changes in the renal clearance of Cu,Zn-SOD after physical exercise seem to be required. Next, the ranger training was practiced for 93 h [124,125]. The soldiers moved about 80 km through a secluded place among the mountains, carrying 30—40 kg of equipment. Immediately after the ranger training there was a notable increase in Mn-SOD level in serum. The increased Mn-SOD seemed to be derived from skeletal muscles, since a positive correlation between Mn-SOD level and creatine kinase (CK) activity was noted (Fig. 1). CK is present mostly in the cytosolic fraction of cells. It is generally accepted, therefore, that the enzyme is released into the bloodstream even when there is a change in the cell membrane permeability [126]. Meanwhile, the increased serum Mn-SOD (which exists in the mitochondrial matrix) might reflect a disintegration of skeletal muscle in addition to increased O2 radical species. Despite the fact that Cu,Zn-SOD is predominantly localized in the cytoplasm like CK, it was unaltered, possibly because of increased renal clearance. Furthermore, cytokines may offer a partial explanation for these changes. For example, interleukin-1 (IL-1) induces the mRNA for MnSOD, not for Cu,Zn-SOD [127]. It has actually been shown that IL-1 activity in human plasma is significantly elevated several hours after exercise on an cycle ergometer (1 h at 60% of aerobic capacity) [128], confirmed by the same group [129]. During the recovery period, Mn-SOD level in serum appeared to rise, while CK activity had returned to the pre-experiment value within 8 days after the ranger training. In view of the intimate interplay between Mn-SOD on the one hand, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities on the other after an 8-day rest (Fig. 2), along with the reduced activity of cholinesterase (Ch-E; a good index of hepatic function), one may deduce that the elevation of Mn-SOD level was derived mostly from the liver. In 5,000h

4.67

L1 150 200 Mn-SOD (pg/l)

Fig. 1. Correlation between Mn-SOD level and CK activity in serum immediately after 93 h strenuous ranger training. The 95% confidence limits for the predicted mean value of Yare shown.

Chapter 11: Superoxide dismutases in exercise and disease 200h

2

263

Y = -16.1 + Q480X r = 0.703 F

lOOh

T T T i i i i -> -> -> -^

seven tissues of rats [69], or on Cu,Zn-SOD content in heart of rats [100]. Unexpectedly, on the other hand, the contents of Cu,Zn-SOD and Mn-SOD showed a downward trend in the kidney of young and old mice after a 6-week swimming training [122]. Very recently we have investigated the effect of a 6-week endurance swimming training on immunoreactive EC-SOD concentration (as judged by an ELISA) in serum from lean and obese (ob/ob) mice (Ookawara et al., unpublished data). As the result, serum EC-SOD concentration on either type of mouse was unaffected by the training. Meanwhile, interestingly, serum EC-SOD concentration was markedly higher in obese mice than in lean mice. Indeed, white adipose tissue had a high content of the enzyme and showed a relatively strong expression of the mRNA [36]. In addition, EC-SOD concentration in serum of lean mice increased significantly with age (Ookawara et al, unpublished data). These disparate results may be due to differences in intensity, duration, and mode of physical exercise, subcellular distribution of SOD isoenzymes, and tissue-specific expression of these isoenzymes. 3.5 Combined effects of dietary calcium restriction and acute exercise on SOD in animals A study was made of the combined effects of dietary calcium restriction and exhaustive exercise on Cu,Zn-SOD and Mn-SOD in rat soleus muscle [133].


267

Rats were assigned to the control rats or the calcium-restricted rats; they were restricted for 1 month or 3 months. Each group was subdivided into acutely exercised or nonexercised groups. Three-month dietary calcium restriction resulted in calcium deficiency and upregulated both SOD isoenzymes (activity and content). Unlike glutathione peroxidase (GPX) and catalase activities, exhaustive exercise did not decrease either Cu,Zn-SOD or Mn-SOD activity During the 3month calcium restriction, on the other hand, the mRNA expressions of both forms of SOD showed an initial upregulation, followed by a downregulation. Exhaustive exercise significantly increased their mRNA expressions only in the 3-month calcium-restricted rats. Also, exhaustive exercise markedly increased the activity of myeloperoxidase (an index of the infiltration of neutrophils in tissues) in soleus muscle from the 1-month and 3-month calcium-restricted rats as compared with the control rats; this significantly enhanced the ability of neutrophils to generate superoxide only in the 3 month calcium-restricted rats. The results obtained demonstrate that dietary calcium restriction upregulates both SOD isoenzymes in rat soleus muscle, indicating the potential for improvement of the resistance to the increase in intracellular reactive oxygen species. The results also suggest that exhaustive exercise may cause oxidative damage in soleus muscle of calcium-deficient rats through the activation of neutrophils. 3.6 Combined effects of hypoxia and chronic exercise on SOD in animals A number of studies have revealed that chronic hypoxia, by decreasing the total SOD activity [134,135] or Mn-SOD content [136,137], may lead to increased susceptibility to oxidant injury particularly in liver and soleus muscle. Thus, the oxidative stress-related consequences of physical training at high altitude were investigated [138]. As the result, the 4-week running training performed under hypobaric hypoxia, equivalent to an altitude of 4,000 m, increased the level of reactive carbonyl derivatives measured by anti-2,4-dinitrophenylhydrazone antibodies and spectrophotometry in both white and red types of skeletal muscle of rats compared with sea level trained rats and control groups. The altitude training also increased the activity of Mn-SOD in both types of muscle, whereas the Cu,Zn-SOD activity was not changed significantly (Table 11). Since GPX and catalase activities in either Table 11. Cu,Zn-SOD and Mn-SOD activities in white (WQ) and red (RQ) portions of quadriceps muscles of rats [138] Cu,Zn-SOD (U/mg protein) Group (n = 6) Control Sea level High altitude

Mn-SOD (U/mg protein)

WQ

RQ

WQ

RQ

9.06 ±0.7 9.16 ±0.4 9.46 ±0.6

11.5±0.5 14.9 ± 1.1 13.5 ± 1.3

5.24 ±0.5 8.31 ±0.7" 8.48 ± 0.6"

8.18 ±0.3 10.3 ±0.8 11.9 ± 1.1"

Values are means ± S.E.M."p< 0.05 vs. control group.

268

P^^t IV: Antioxidant defenses

white or red type of muscle of altitude trained rats did not vary substantially, it was suggested that the oxidative modification of certain amino acids is due to the increasing gap between activity of SOD and peroxide scavenging enzymes, which results in an increase in the number of hydrogen peroxide molecules. 3.7 Combined effects of SOD treatment and acute exercise on oxidative stress in animals An SOD derivative (SM-SOD), synthesized by covalently linking poly-(styleneco-maleic acid) butyl ester to a Cu,Zn-SOD, which circulates and is bound to albumin with a halflife of 6 h [139] was injected intraperitoneally into the rats prior to exhaustive running [140]. The exercise induced a marked increase in the activity of XO (which utelizes molecular oxygen as an electron acceptor and thus, generates 02^) in plasma, and an increase in thiobarbituric-reactive substances (TEARS), an indicator of lipid peroxidation, in the plasma, as well as in the soleus and tibialis muscles from nonadministered rats immediately after the exercise, indicating massive free radical formation. The immunoreactive content and activity of both SOD isoenzymes (Cu,Zn-SOD and Mn-SOD) of nonadministered rats increased significantly in the soleus and tibialis muscles immediately after running; this also suggested an increased output of O2. SM-SOD treatment definitely attenuated the degree of the increases in XO and TBARS in all the samples examined immediately after exercise. These findings suggest that a single bout of exhaustive exercise induces oxidative stress in skeletal muscle of rats, irrespective of its fiber type, and that this oxidative stress can be weakened by exogenous SM-SOD. Similar results were also given by the study on liver and kidney [141]. By contrast, Homans et al. [142] have shown that infusion of bovine serum SOD via the left atrial catheter in dogs affects neither the transient rebound function occurring early after exercise nor the prolonged period of stunning, indicating that the myocardial stunning that follows exercise induced ischemia is unlikely to be mediated by oxygen free radicals. The disparity noted between the studies by Radak et al. [140,141] and by Homans et al. [142] would probably be attributable to that in the half life of SODs administered (the former: 6 h and the latter: 5 min) [139]. Actually, a single injection of SM-SOD was effective under various oxidative stresses [143—146]. 4

SOD IN DISEASES

It has been strongly suggested that many of the cell alterations seen in normal aging process and various diseases including cancer, are due to oxidative damage from active oxygen species. SODs scavenge O2, and so, the study of these enzymes is of potential clinical interest. In this chapter we will attempt to summarize recent data on the chnical and pathological significance of Cu,Zn-SOD and Mn-SOD, especially in relation to neuronal degeneration, diabetes, cancer, and ischemia [28,29,147,148].


269

4.1 Knockout mice of SODs To analyze roles of SODs, several mice lines with targeted inactivation of SODs have been generated. These data provide new insight into understanding the significance of SODs in vivo. 4.1.1 Cu, Zn-SOD-deficient mice It has been reported that some cases of familial amyotrophic lateral sclerosis (FALS) are associated with mutations in the Cu,Zn-SOD gene (SODl) and that Cu,Zn-SOD protects neuronal cells from various damages including ischemia or apoptosis. In order to analyze the role of Cu,Zn-SOD, two groups generated mouse lines with targeted inactivation of Cu,Zn-SOD [149, 150]. Contrary to their expectations these mice developed normally and showed no phenotypic abnormality under normal conditions. However, Cu,Zn-SOD knockout mice exhibited marked vulnerability to motor neuron loss after axonal injury [149], and showed a high level of blood brain barrier disruption soon after 1 h of middle cerebral artery occlusion and 100% mortality at 24 h after ischemia, while wild-type mice showed 11% mortality [150]. These results suggest that Cu,ZnSOD is not necessary for normal development and function, but is required under stressful conditions following injury or ischemia. 4.1.2 Mn-SOD-deficient mice Mn-SOD-deficient mice were also generated by another two groups [151,152]. One mouse line with a targeted inactivation of Mn-SOD (deletion of exon 3) showed dilated cardiomyopthy, accumulation of lipids in the liver and skeletal muscle, and metabolic acidosis [152]. These mice were exceedingly hypotonic, hypothermic, and paler compared with wild mice. And they died within or on the first 10 days. A severe reduction in succinate dehydrogenase (the complex II) and aconitase activities were also found. Another mouse line with targeted inactivation of Mn-SOD (deletion of exons 1 and 2) exhibit reduced growth rate and survival (up to 3 weeks) [152]. Likewise, they exhibited severe anemia, dilated heart, degeneration of neurons in the basal ganglia and brain stem, and progressive motor disturbance (weakness, rapid fatigue, and circling behavior). These finding indicate that Mn-SOD is important for maintaining the integrity of mitochondrial enzymes and that its deficiency causes increased susceptibility to oxidative mitochondrial injury in many tissues, such as cardiac myocytes, skeletal muscles, central nervous systems, hepatocytes, and the like. 4.1.3 EC-SOD-deficient mice EC-SOD-deficient mice were also generated and developed normally and remained healthy up to the age of 14 months or more [153]. However, when

270

Pa^t IV: Antioxidant defenses

they were exposed to almost 100% oxygen, they considerably reduced a survival time compared to wild mice. 4.2 Cu,Zn-SOD 4.2.1

Clinical significance of Cu,Zn-SOD

Recently many investigators try to use recombinant Cu,Zn-SOD as a therapeutics, e.g., to reduce the infarct size in acute myocardial infarction. Many modified Cu,Zn-SODs have also been made, but in this section we will discuss the cHnical significance of endogenous Cu,Zn-SOD. The role of Cu,Zn-SOD under various pathogenic conditions has been widely studied. For example, it is well known that there may be a correlation between the life span and the Cu,Zn-SOD activity [154]. Recently, it was reported that mutations of Cu,Zn-SOD are related to the pathogenesis of FALS and that Cu,Zn-SOD is glycated and inactivated in diabetes. In both cases decreased activity of Cu,Zn-SOD may result in accumulation of damages by reactive oxygen species. Such slow and continuous damages by reactive oxygen species should be further studied in addition to the acute damages. As Cu,Zn-SOD is encoded by the human chromosome 21, it has been suggested that overexpression of Cu,Zn-SOD may play an important role in the neurobiological abnormalities of Down syndrome [155,156]. However, a partial trisomy 21 with overexpression of Cu,Zn-SOD was associated with none of the usual symptoms [157,158]. Therefore, this hypothesis seems unlikely Cu,Zn-SOD is widely and abundantly distributed in the cytosol of cells. Human liver samples contain approximately 0.65—1.5 mg/g of Cu,Zn-SOD in liver protein [159], whereas human erythrocytes contain 0.5—0.75 mg/g of Cu,Zn-SOD in hemoglobin. Therefore, particular attention must be paid to measure serum or tissue levels of Cu,Zn-SOD because hemolysis or contamination of erythrocytes leads to misinterpretation. 4.2.2 ALS ALS is a degenerative disorder of motor neurons in the cortex, brain stem and spinal cord. About 10% of cases are familial (an autosomal dominant trait). FALS cannot be clinically distinguished from sporadic ALS. As already stated, it has recently been reported that there is a tight linkage between FALS and various SODl missense mutations in different FALS famihes [160, 161]. Almost 50 mutations were found in patients with FALS (Fig. 3). However, definite mutations could not be identified in the exon 3, which forms active site region of Cu,ZnSOD. So, these mutations were assumed to alter interactions critical to the p-barrel fold and dimer contact rather than catalysis [161]. In addition, late onset progressive paralysis in the transgene mice was demonstrated [162]. The exact mechanism of motor neuron death, however, is still unclear and controversial

Chapter 11: Superoxide dismutases in exercise and disease .

1

10

Ac-A

T K A V

C V

• V/T

Exon I

L K G D G P V

Q G I

T • •

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F E

M S

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T

•

S/D

R

70

20

^M^

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2£

S N G P V K

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31 40 Exon II V W G S I K G L T E G L H G F H V 61

271

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fcî^5? T A G C T S A|

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Ex o n III

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80

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90

G D

^TlG

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T A

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T • T s

T A

V R 91

100

K D G V A D V S I

E D S V I

110

S L S G D H C I

120

1

T

T T

•

T

T

• •

A/C/R/D/SA/

G/KN/G

F

V

V

T T

121 lE

ExonlV

130 K A

D

D

L

G

KG

V H ^

L

•

H

^"t

G 150

140

G N E E S T K T G N A G S R L I A

TT^f

G R T

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f N

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V

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F/S T R

I

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I

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T

G/l

T

A

153 Q

D : Active Center lir : Deletion

Fig. 3. SODl mutation in FALS.

because both suppression and overexpression of SOD activity induced normal cell death and several groups reported a gain in function of mutant Cu,Zn-SOD. For instance, we have actually produced wild-type and six mutant Cu,Zn-SODs related to FALS in a baculovirus/insect cell expression system and showed that mutant Cu,Zn-SODs have a lower scavenging activity and more vulnerability to glycation reaction than wild-type [163]. Decreased scavenging ability, vulnerability to glycation, and free copper release from the inactivated enzymes are all likely reasons why the mutant enzymes might produce hydroxy radicals which may be a potent causative factor of FALS via the Fenton reaction. On the other hand, several groups reported that FALS was due to a toxic gain of function of mutant Cu,Zn-SOD rather than to lower SOD activity The mutant Cu,ZnSODs associated with FALS were shown to catalyze the oxidation of a model substrate by hydrogen peroxide at a higher rate than wild-type, these oxidative reactions catalyzed by mutant enzymes initiating the neuropathologic changes of FALS [164]. Moreover, other gains in function have also been reported: the ability to catalyze nitration of neurofilaments by peroxynitraite [165] and the ability of free radical formation from H2O2 [166]. Since each mutant Cu,Zn-SOD may have different properties, all these observations may be true in each mutant Cu,Zn-SOD. Further investigation will be expected, including the pathogenesis of sporadic ALS which is indistinguishable from FALS clinically even though mutation of Cu,Zn-SOD was also reported in sporadic ALS, even though mutation of Cu,Zn-SOD was also reported in sporadic ALS.

272


4.2,3 Diabetes mellitus 4.2.3.1 Glycation of proteins. Many proteins undergo nonenzymatic glycosylation reactions under hyperglycemic conditions and some of their activities are modified. These reactions are known as glycation to distinguish them from enzymatic glycosylation catalyzed by glycosyltransferase. Glycation is a post-translational modification that occurs in vivo through the direct chemical reaction between glucose and the primary amino groups of proteins. The initial product is a labile Schiflf base adduct, and then this adduct undergoes a slow Amadori rearrangement to a stable ketoamine derivative of the protein (Fig. 4). Glycation Protein+Glucose

It

C=NH-R HCOH

R: Protein

HOCH HCOH HCOH CHfOH Schiff Base

CH,-NH-R

CH,-NH-R

C=0

COH

HOCH

OHC

HCOH

HCOH

HCOH

HCOH

CH,-NH-R

Me"* Me'"-"* C-0' _ ^ ^ O H C HCOH

CH,-NH-R C=0 o, 0," V, ^ , 0 = C HCOH

HCOH

HCOH

CHiOH

CHrOH

CH,OH

CHjOH

Amadori Product

Endiol

Alcoxy Radical

Dicarbonyi

Protein

A

CH,

H C=0

CHfOH

R

C«0

C«0

C=0

NH

HCOH HCOH

c=6

CH,

C=0

CH,

HCOH

HCOH

CH,

COOH

HCOH

HCOH

HCOH

CH,OH

CH,OH

CH,OH

Deoxyosones (U3 and 4deoxygiucosones) L---Proteins AGE. Fig. 4. Reaction mechanism for glycation of proteins.

Carboxymethyl lysine (CML)

COOH

CH,OH erythronic acid


273

is considered to be the first step in a complex series of browning, or Maillard, reactions that occur in the presence of reducing sugar. Increased glycation and the subsequent Maillard reaction are thought to be involved in the structural and functional changes in body proteins that occur during normal aging and at accelerated rates in diabetes [167—169]. The reaction was found in the browning reaction of food in the 19th century. Glycation has also been seen in various proteins and in several enzymes such as ribonuclease [170], carbonic anhydrase [171], Cu,Zn-SOD [172], and Na"',K'"-ATPase [173]. 4.2.3.2 Glycation of Cu,Zn-SOD. Erythrocytes are subjected to a continuous flux of O2 and H2O2 due to hemoglobin auto-oxidation [154,174—176] and also undergo oxidative stress from environmental agents [177]. Cu,Zn-SOD in the erythrocytes may have some physiologically important role in combating these processes. Human erythrocytes contain glycated and nonglycated Cu,Zn-SOD, which can be separated by boronate affinity chromatoghraphy [178,179]. Actually, 50% or more of the erythrocyte Cu,Zn-SOD from diabetic patients was found to be glycated. Although such a high percentage of the Cu,Zn-SOD in the erythrocytes of diabetic patients was glycated, the total Cu,Zn-SOD activity did not differ much between the diabetic patients and the controls. Nonetheless, the specific activity of Cu,Zn-SOD in the erythrocytes of patients with diabetes was always lower than in nondiabetics [180]. These facts indicate that the Cu,ZnSOD is inactivated under hyperglycemic conditions. Indeed, when purified Cu,Zn-SOD was incubated with glucose under sterile conditions, the SOD activity showed a time- and dose-dependent decrease and the amount of ketoamine adduct increased simultaneously 4.2.3.3 Inactivation and Fragmentation of Cu,Zn-SOD by Glycation. The mechanism by which Cu,Zn-SOD undergoes glycation and inactivation has been studied [179—182]. Human Cu,Zn-SOD undergoes glycation reaction at specific lysine residues such as Lys-122 and Lys-128 which comprise a positively charged channel track. The glycation of the lysine residues resulted in a relatively negative charge, which may interfere with the electrostatic guidance of the substrate superoxide anion to the active site. The computer image of spinach Cu,Zn-SOD indicates that Lys-122 and Lys-128 are located on the surface of the enzyme molecule and appear to be easily attacked by glucose. In addition to the above mechanism, fragmentation of Cu,Zn-SOD was recently reported. Several groups reported that glycated proteins produce O2 in the presence of transition metal ions [183—186]. And it is well known that Cu,Zn-SOD is sensitive to H2O2 and undergoes random fragmentation after exposure to H2O2 [187—188]. Recently, both site-specific and random fragmentation of Cu,Zn-SOD was found following the glycation reaction [189]. The fragmentation proceeded in two steps. In the first step, Cu,Zn-SOD was cleaved at a peptide bond between Pro-62 and His-63, as judged by amino acid analysis and sequencing of fragment peptides, yielding a large (15 kDa) and a small (5


274

kDa) fragment (Fig. 5). In the second step, random fragmentation occurred. Electron spin resonance (ESR) spectra were also measured, the results suggesting that reactive oxygen species was implicated in both steps. 4.2.3.4 Increase of Glycated Cu,Zn-SOD in Diabetic Retinopathy and Cataracts. The glycation of proteins may play an important role in diabetic complications [167]. Cu,Zn-SOD is located in the lens epithelium [190,191], as are most of the drug-metabolizing enzymes and antioxidant enzymes [192]. Glycation of Cu,Zn-SOD in the lens may play an important role in cataractogenesis. In general, glycated Cu,Zn-SOD level seems to correlate with the level of HbAlc. In patients with diabetic cataracts the correlation is not apparent, whereas the level of glycated Cu,Zn-SOD is rather high compared to that in diabetic patients with no complications. An aged persons with senile cataracts, also have relatively higher levels of glycated Cu,Zn-SOD in their erythrocytes. It is unclear, however, whether the increased amount of glycated Cu,Zn-SOD is really related to the senile cataracts or due to other minor atherosclerotic changes in aged persons. If one separates younger and older populations of normal erythrocytes by centrifugation and compares the activities of those populations, the activity of normal younger erythrocytes is higher than that of aged erythrocytes. Stevens et al. [193] proposed that glycation might have a role in the browning and aging of lens crystallines associated with the development of senile and diabetic cataracts. Several investigators subsequently reported age-related increases in glycation of normal human [194—196], bovine [167], and rat lens [197], that is, they suggested that increased glycation of proteins with age could cause an age-related acceleration of glucose-dependent damage to protein. However, Patrick et al. [198] reported that glycation of human lens protein is essentially constant with age in normal lens. Streptozotocin, a nitrosourea compound produced by Streptomyces achromogenes, has been used to induce experimental diabetes. The drug also induces DNA strand breakage in islet cells. Streptozotocin injection into rats has been 9Ak 67k A3k

—

30k — 20k -^MBMHUaaMMP lA.Ak

—

•

-

- * Original Cu.Zn-SOD - * Large fragment - ^ Snnall fragrrênt

(Mr) 2 3 5 0 A 8 HOURS - DAYS" Incubation Time

Fig. 5. Fragmentation of Cu,ZnSOD after incubation with glucose.


275

observed to decrease the Cu,Zn-SOD activity of retina cells, erythrocytes, and islet cells [199,200]. However, the mechanism by which the Cu,Zn-SOD activity decreases in experimental diabetes is not yet known. Streptozotocin-induced diabetic rats also had high levels of glycated Cu,ZnSOD in the erythrocytes [201] and lens, as judged by affinity chromatography on a boronate column. As described above, much of the glycated Cu,Zn-SOD is inactive. This is one of the reasons that Cu,Zn-SOD activity is decreased in rats with streptozotocin-induced diabetes. Even in normal rat lens, approximately 40% of the Cu,Zn-SOD undergoes glycation. Under diabetic conditions, over 80% of the enzyme was found to have undergone glycation (Kawamura et al., unpublished data). Thus, under diabetic conditions, the glycated proteins produce 62^ on one hand, and the Cu,Zn-SOD undergoes glycation and inactivation on the other hand. These events may enhance the accumulation of ©2^ in the microenvironment of the tissues and, as a result, tissue damage and diabetic complications would be accelerated (Fig. 6). 4.2.4 Werner's syndrome Werner's syndrome is an autosomal recessive condition and is sometimes referred to as adult progeria. The disease is clinically characterized by accelerated aging and increased frequency of malignant tumors and diabetes [202]. At the cellular and molecular levels, cultured fibroblasts from patients with Werner's - ^ Amadori products

Proteins

metals

Reducing sugars

Glycated Cu,Zn-SOD Re lease \Catalase {Peroxidases

H2O, O2

Protein DNA reaction O H — r Lipid, etc. Attack !

Fig. 6. A possible mechanism for accumulation of O2 in a microenviroment due to inactivation of SOD.

276

Pcirt IV: Antioxidant defenses

syndrome have a markedly decreased replicative life span [203]. In addition, increased proportions of several enzymes in the fibroblasts have been reported to be heat-labile [203,204], as found in old fibroblasts. The etiology of the disease is still unknown, but an involvement of the free-radical scavenging system has been suggested [205]. An age-related reduction in Cu,Zn-SOD has been reported [206,207]. In patients with Werner's syndrome, erythrocyte Cu,Zn-SOD undergoes nonenzymatic glycosylation at multiple lysine residues, irrespective of the glycemic state. The enzyme purified from the patient was found to be unstable and had a very low specific activity due to nonenzymatic glycosylation. As already described, accelerated glycation reactions appear to bring about the production and accumulation of O2 and it may cause tissue damage and relate to the clinical feature of the progeria. 4.3 Mn-SOD 4,3,1 Clinical significance of Mn-SOD It is widely believed that Mn-SOD plays a role in various pathogenic conditions such as aging, carcinogenesis, inflammation, ischemia and others. Recently MnSOD-deficient mice showed that Mn-SOD gene (SOD2) is a lethal gene. MnSOD may protect mitochondria by protecting mitochondrial enzymes from reactive oxygen species. Moreover, very recently it has been reported that faulty localization of Mn-SOD may be related to progeria [208] and that Mn-SOD is inactivated by peroxynitrate (nitration), e.g., probably leading to chronic rejection of human renal allografts [209]. It is also widely known that Mn-SOD is induced by various cytokines, such as tumor necrosis factor (TNF)-a or IL-1, and that Mn-SOD is a protective enzyme against cytotoxicity of TNF [127,210]. This allows more interests to be focused on Mn-SOD. No direct evidence, however, has been presented linking Mn-SOD to these pathogenic conditions. Several laboratories found that, in cancer tissues or transformed cells, as well as in aged tissues, the activity of SOD decreased or disappeared as compared to that in uninvolved or younger tissues [211—217]. In a previous study [218] we found that the immunoreactive Mn-SOD levels in human lung cancer tissues are higher than those in uninvolved tissues from the same patients, whereas the level of the active enzyme does not increase. It still seems that the level of immunoreactive enzyme may provide a useful piece of information for monitoring cancer tissues. Recently we raised three monoclonal antibodies against human liver Mn-SOD. The epitope of one of these antibodies was found to be a COOH-terminal peptide, as judged by competitive inhibition assay using synthetic peptides [219]. Using this antibody we developed an ELISA method and found that the enzyme is also present in human serum [45]. Measurement of the serum immunoreactive Mn-SOD protein levels in various diseases revealed that the enzyme levels are increased in certain pathological conditions, such as acute myocardial infarction, primary biliary cirrhosis, primary hepatoma, gastric cancer, and acute myeloid


277

leukemia. Mn-SOD levels were also increased in the sera of patients with epithelial type ovarian cancer. In this section at first we will mention the induction mechanism of Mn-SOD in vitro and later explain linkages of each disease and Mn-SOD. 4.3.2 Expression of Mn-SOD in vitro 4.3.2.1 Expression of Mn-SOD in TNF-resistant and sensitive cell lines. TNF or IL-1 specifically induces mRNA for Mn-SOD, Mn-SOD is one of the protective proteins against TNF cytotoxicity [127,210,220]. This effect is blocked by actinomycin D, but not by cyclohexamide, indicating that the increase in MnSOD mRNA results from an increase in transcription of the Mn-SOD gene. Lipopolysaccharide (LPS) also induces Mn-SOD mRNA in pulmonary epithelial cells by a similar mechanism [221]. Recently it was reported that phorbol 12-myristate 13-acetate (TR\), a potent tumor promoter and protein kinase C activator, also induced Mn-SOD expression only in TNF-resistant cell lines and presented two hypothetical signal transducing pathways in this gene expression [222]. The importance of Mn-SOD for cellular resistance to TNF cytotoxicity has been reported [221]. However, no data were available regarding the levels of Mn-SOD protein after TNF treatment. The development of a monoclonal antibody and the ELISA technique made it possible to quantitatively determine MnSOD protein levels. The effect of TNF on the expression of Mn-SOD protein in TNF-resistant cells was examined. In the case of WI-38 (fetal lung cells), Mn-SOD protein levels increased dramatically, approximately 80-fold, after TNF treatment (Fig. 7). On the other hand, TNF treatment did not cause any changes in Cu,Zn-SOD expression in most of the TNF-resistant cells [223]. Induction of Mn-SOD was also seen in A549 cells (a human adenocarcinoma cell line). In ME-180, a human cervical epidermoid carcinoma cell line, and KYM-1, a human myosarcoma cell line, which are TNF sensitive, Mn-SOD levels are one order of magnitude lower than in TNF-resistant cells. However, ZR-75-1, a human breast cancer cell line, which is also TNF sensitive, contains a relatively high level of Mn-SOD, even though its level is not as high as that of TNF-resistant cells. The basal Mn-SOD protein levels in ME-180 and KYM-1 cells are very low, and even after treatment with TNF, no increase in Mn-SOD was observed. In ZR-75-1 cells, however, a tendency toward increased Mn-SOD levels was observed after TNF treatment. Therefore, in this cell line it seems likely that a different mechanism may control Mn-SOD expression. 4.3.2.2 Mechanism of Mn-SOD induction by TNF or TPA. Although several stimulators such as TNF, IL-1, and LPS have been reported to enhance MnSOD expression in some cell lines, the pathway that transduces a signal from corresponding receptors to Mn-SOD gene is not clearly understood. Recently, it was reported that phorbol ester (TPA), a protein kinase C activator, also induces

278


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WI-38

KURAMOCHI

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0 10 10 10

6,000

o) 4,000 h Q O 2,000

0

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0

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0

Dosage of rh-TNF (mg/ml)

4,000 r

A549

T24

WI-38

KURAMOCHI

3,000

2,000 Q

o CO

N

1,000

0

10 10'

0

10"'lO"'lO"'

0

10"'10"'10"'

0

10"'

Dosage of rh-TNF (mg/ml)

Fig. 7. Effect of TNFon MnSOD and Cu,ZnSOD levels in TNF-resistant cells.

Mn-SOD in various cell lines which are all resistant toTNF (Table 12) [222]. This gives us clue to investigate the intracellular signal transduction pathway. Since TPA enhanced Mn-SOD mRNA expression in TNF-resistant cell lines, in which other stimulators also induced the expression of the gene but did not affect TNF-sensitive cells, it is conceivable that protein kinase C is involved in this gene expression by TNF through phosphorylation of certain substrates. One possibility would be that activator protein-1 (AP-1) is responsible for this gene expression. Ho et al. [224] found the consensus sequence for AP-1 enhancer binding protein in the 5'-flanking region of the rat Mn-SOD gene. TPA acts to both activate AP-1 protein and to enhance protooncogene Jun/AP-1 expression. However, there would be more than one pathway because TNF could induce Mn-SOD in the cells that were desensitized to TPA by TPA-pretreatment. Thus, at least two pathways participate in Mn-SOD expression. One is triggered by pro-

Chapter 11: Superoxide dismutases

279

in exercise and disease

Table 12. Relative stimulation of Mn-SOD m R N A expression by T R \ , T N F , IL-1 and LPS in various cell lines^ [222]. Cell line

Control

TPA (lOng/ml)

TNFa (lOOng/ml)

IL-IB (1000 U/ml)

LPS (10 ^lg/ml)

HeLa^ A549^ Kuramochi*^ MCAS'' ME 180' HL60' K562"

1.0 1.0 1.0 1.0 1.0 1.0 1.0

18.7 9.0 6.7 11.2 1.2 0.4 0.9

7.8 18.5 2.7 1.4 0.8 0.6 1.0

8.1 25.3 19.8 6.0 1.3 0.7 1.3

3.0 1.0 1.4 16.5 0.4 0.6 1.0

±0.1 ±0.2 ±0.1 ±0.1 ±0.3 ±0.2 ±0.1

± 1.0 ± 1.2 ±0.1 ± 1.3 ±0.1 ±0.1 ±0.1

±2.5 ± 1.4 ± 0.5 ±0.8 ±0.4 ±0.1 ±0.3

±1.2 ±3.8 ±2.7 ± 1.4 ±0.2 ±0.1 ±0.1

±0.7 ±0.1 ±0.1 ±0.7 ± 0.2 ± 0.4 ±0.1

'Total RNA was prepared from various cells treated with 10 ng/ml TPA, 100 ng/ml TNF,1000 units/ml IL-1, or 10 |ig/ml LPS for 4 h. The amount of Mn-SOD mRNA was evaluated by scanning X-ray film exposed to Northern blot membrane filters. The mRNA levels relative to the control are presented as the means ± SD for three experiments. "TNF-resistant cell. "^TNF-sensitive cell.

tein kinase C activation, itself in the absence of new protein synthesis, and the other can be activated by TNF without protein kinase C activation [222] 4.3.2.3 Induction and release into serum of Mn-SOD from endothelial cells. Serum levels of Mn-SOD are elevated in patients with various diseases. However, the mechanism of serum increase of Mn-SOD was unclear. Recently we reported that Mn-SOD in human endothehal cells is also induced by TNF, TPA, IL-1 and LPS [225] these showed there is a possibility that Mn-SOD induced in endothelial cells is released into serum and thus, results in an increased serum levels of Mn-SOD in various diseases [226]. Mn-SOD in human endothehal cells, which were obtained from umbilical cords, is dramatically induced by TNF (Fig. 8) and this induction is partially blocked by H7, a protein kinase C inhibi700

1

10

TNF (ng/ml) Fig. 8. Induction of MnSOD and Cu,ZnSOD proteins in endothelial cells.


280

tor, and dexamethasone. Meanwhile, Cu,Zn-SOD levels are unchanged. Similar to cancer cell lines, there are at least two pathways in Mn-SOD expression in endothelial cells: one is triggered by protein kinase C and the other is activated by TNF without protein kinase C activation [225]. Mn-SOD levels in culture medium of endothelial cells are elevated after treatment of TNF (Fig. 9) [226]. These facts suggest that Mn-SOD induced in endothehal cells is released into serum of patients with various diseases especially in which cytokines have a major roles in pathogenesis including inflammation and ischemia [227].

/y

500

r

300

10

B

20 30 INCUBATION TIME (h)

600 Control IL-1( 5ng/ml) IL-1 (50ng/ml) 400 h

300

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Fig. 9. The MnSOD levels in the culture medium of cells stimulated with TNF and IL-1.


281

4.3.3 Mn-SOD in diseases 4.3.3.1 Mn-SOD levels in normal healthy controls. The levels of Mn-SOD in sera from 194 males and 207 females who were healthy adult individuals were examined by an ELISA [45]. The frequency distribution of serum Mn-SOD levels for the normal adult male was found to follow a normal distribution pattern. The distribution for the normal female adult was found to be slightly skewed, but the plotting of the cumulative frequency using normal probability paper gave a near-straight line. The mean level and SD for male and female were 99.8 ng/ml ± 24.8 and 88.8 ± 20.8 ng/ml, respectively. Assuming the upper limit of the normal male to be 150 ng/ml (equivalent to the mean value for normal male subjects plus 2 SD), the percentage of false positives was 2.1%. Similarly, assuming the upper limit of the normal female to be 130 ng/ml, the percentage of false positives was 1.0%. In children, the serum Mn-SOD levels are slightly lower than in adults and gradually increase in proportion to age. By 10 years of age the Mn-SOD levels are nearly at the adult level. In various diseases, including cancer, Mn-SOD levels are relatively high (Fig. 10). 4.3.3.2 Acute myocardial infarction. A great deal of interest has developed in the role of SOD modifying the toxic effects of 02^ arising in cardiac tissue during Mn-SOD (ng/ml) 0 Gastric Ca. Esophageal Ca. Lung Ca.

•^

100

200

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.

•

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Chronic Hepatitis Myocardial Infarction

ALL

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0/7

• ••

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iT

1/5 26/49

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6/15 6/8

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Malignant Lymphoma AML

500
heart > liver > spleen > brain > pancreas > lung. The concentration of lipoyllysine in bovine kidney and heart were 2.64 ± 1.23 and 1.51 ± 0.75 |ig/g dry weight, respectively [Lodge, 1997; see 37] Studies with human Jurkat T cells have shown that when added to the culture medium, lipoate readily enters the cell where it is reduced to its dithiol form, dihydrohpoate (DHLA). DHLA accumulated in the cell pellet, and when monitored over a 2 h interval the dithiol was released to the culture medium [45]. As a result of lipoate treatment to the Jurkat T cells and human neonatal fibroblasts, accumulation of DHLA in the culture medium was observed. The redox potential of the lipoate-DHLA couple is -320 mV. Thus, DHLA is a strong reductant capable of chemically reducing GSSG to GSH. Following lipoate supplementation, extracellular DHLA reduces cystine outside the cell to cysteine. The cellular uptake mechanism for cysteine by the ASC system is approximately 10 times faster than that for cystine by the x ~ - system [46]. Thus, DHLA markedly improves cysteine availability within the cell resulting in accelerated GSH synthesis [28,47]. Both lipoate and DHLA have remarkable reactive oxygen detoxifying properties [37]. Recent studies show that lipoic acid can potently stimulate glucose uptake in differentiated skeletal muscle-derived L6 myotubes. Glucose uptake in cytokine-treated insulin-resistant cells was enhanced by lipoic acid treatment suggesting that lipoic acid treatment may help in conditions such as infection-related disorders in glucose metaboHsm [48]. a-Lipoic acid is taken up by cells and reduced to its potent dithiol form, dihydrohpoate (DHLA), much of which is rapidly effluxed out from cells. To improve retention in cells, the lipoic acid molecule has been recently modified to confer a positive charge at physiological pH. This novel protonated form of lipoic acid is known as lipoic acid — plus or LA-Plus [49]. Structurally, compared to lipoic acid, LA-Plus more closely resembles the naturally occurring form of lipoic acid i.e., lipoyllysine. The uptake and reduction of LA-Plus by human Wurzburg T

Chapter 12: Antioxidants and physical exercise

305

cells was higher compared to that of lipoic acid. Several-fold higher amounts of DHLA-Plus, the corresponding reduced form of LA-Plus, were detected in LAPlus treated cells compared to the amount of DHLA found in cells treated with lipoic acid. On a concentration basis, LA-Plus has been observed to be much more cytoprotective than native lipoic acid [49]. 2.7 Selenium Forty years ago traces of dietary selenium was observed to prevent nutritional liver necrosis in vitamin E deficient rats [50]. At present, selenium is widely used in agriculture to prevent a variety of selenium- and vitamin E-sensitive conditions in livestock and poultry [51]. In animal tissues, selenium is either present as selenocysteine in selenoproteins such as glutathione peroxidase, selenoprotein P or thioredoxin reductase. Additionally, selenium may be present in animal tissues as selenomethionine that is incorporated in place of methionine in a variety of proteins. Selenomethionine containing proteins serve as a reservoir of selenium that provides selenium to the organism when dietary supply of selenium is interrupted. Selenocysteine is the form of selenium that accounts for its biological activity Perhaps the most prominent biological activity of selenium is that it is an essential cofactor for the critical hydroperoxide metabolizing enzyme glutathione peroxidase [52]. It has been suggested that selenium may also have direct antioxidant effects in biological systems [53]. Selenium content in food sources may markedly vary depending on the selenium content of the feed for animals, or selenium content in the agricultural soil. Organ meats, seafoods and muscle meat are considerable sources of selenium. Dairy products, and cereal and grains may also provide significant amounts to meet the RDA value of 70 and 55 |ig/ day calculated for adult men and women respectively [54]. 2.8 Recommended daily allowances The Food and Nutrition Board of the National Academy of Science and the National Research Council have developed guidelines for several dietary factors. These recommended daily allowances (RDA) were last revised in 1989 and at the time this work was written another process of revision was in progress. The allowances are intended to supply adequate amounts of these substances for an average daily intake over time and takes into account individual variations for most normal individuals who live in the US under usual environmental stresses. The RDAs can vary for infants, children, adults, elderly as well as for lactating women. According to values of 1989, the RDA for vitamin E is 10 mg for adult males and 8 mg for adult females. P-Carotene is not identified directly but its RDA is thought to be 1/6 the amount recommended for vitamin A (RDA 1 mg and 0.8 mg for men and women, respectively). Selenium has a suggested RDA of 0.07 mg for adult males and 0.055 mg for adult females. Vitamin C has a RDA of 60 mg for adults. There are no particular guidelines established for glu-

306

Pci^t JV- Antioxidant defenses

tathione. However, glutathione is composed of three amino acids, glycine, cysteine and glutamine. Adequate protein intake in the diet is needed but this does not guarantee glutathione synthesis as indicated previously [41]. 3

ANTIOXIDANT DEFICIENCY IN EXERCISE

The antioxidant deficiency model has been used to test the significance of various antioxidants in exercise-induced oxidative stress. Several studies have consistently indicated that vitamin E deficiency can lead to enhanced free radical formation resulting in compromised exercise performance and increased tissue lipid peroxidation [55—61]. These studies suggest that inadequate amounts of dietary vitamin E may decrease endurance performance by as much as 40% and lead to enhanced oxidative lipid damage of several tissues [57,62—64]. Also, vitamin E deficiency was associated with increased fragility of lysosomal membranes and greater hemolysis of red blood cells [57,62]. Vitamin E deficiency also decreased oxidative phosphorylation [59,63] in skeletal muscle, liver and adipose tissues. In female rats, however, vitamin E deficiency does not appear to influence the ability to run nor does it enhance tissue lipid peroxidation [65]. It has been suggested that female rats may be less susceptible to free radical damage compared to male rats because of higher levels of estrogen, a potential antioxidant, in circulation [61,62,66]. The effects of an ascorbate depleting diet on run time was examined in guinea pigs which do not synthesize vitamin C. Run time of ascorbate-depleted guinea pigs was significantly less than ascorbate-adequate animals [67]. Dietary selenium deficiency impairs tissue antioxidant defenses by markedly downregulating glutathione peroxidase activity in tissues such as the liver and muscle. This effect on the antioxidant enzyme did not influence endurance to a treadmill run, however. This suggests that muscle glutathione peroxidase activity is not a limiting factor in physical performance (Lang et al., 1987). Selenium deficiency has been also found to enhance lipid peroxidation in skeletal muscle mitochondria of rats that were exercised for 1 h [68]. Activity of antioxidant enzymes in both liver and skeletal muscle have been observed to adapt in response to selenium deficiency suggesting that the organs may have encountered and responded to an enhanced oxidative challenge. The relative role of endogenous GSH in the circumvention of exhaustive exercise-induced oxidative stress has been investigated using GSH deficient rats. GSH synthesis was inhibited by intraperitoneally administered L-buthionine-sulfoximine (BSO) to produce glutathione deficiency The BSO treatment resulted in: 1) ^ 50% decrease in the total glutathione pools of the liver, lung, blood and plasma, and 2) 80—90% decrease in the total glutathione pools of the skeletal muscle and heart. Compared to controls, glutathione deficient rats had remarkably higher levels of tissue lipid peroxides. A significant effect of glutathione deficiency on endurance to exhaustion was observed. Glutathione deficient rats could run for only about


307

half the interval when compared to the saline injected controls. This observation underscores the critical role for tissue glutathione in the circumvention of exercise-induced oxidative stress and as a determinant of exercise performance [69]. Increased susceptibility to oxidative stress was also observed in muscle-derived cells pretreated with BSO [70]. Leeuwenburgh et al. [71] studied the effect of chronic in vivo glutathione depletion by BSO on intracellular and interorgan GSH homeostasis in mice both at rest and after an acute bout of exhaustive swim exercise. BSO treatment for 12 days decreased concentrations of GSH in the liver, kidney, quadriceps muscle, and plasma to 28, 15, 7, and 35%, respectively, compared to GSH-adequate mice. GSH depletion was associated with adaptive changes in the activities of several enzymes related to GSH metabolism. Exhaustive exercise in the GSH-adequate state severely depleted GSH content in the liver (-55%) and kidney (-35%), whereas plasma and muscle GSH levels remained constant. However, exercise in the GSH-depleted state exacerbated GSH deficit in the liver (-57%), kidney (-33%), plasma (-65%), and muscle (-25%) in the absence of adequate reserves of liver GSH. Hepatic lipid peroxidation increased by 220 and 290%, respectively, after exhaustive exercise in the GSH-adequate and -depleted mice. It was concluded that GSH homeostasis is an essential component of the prooxidant-antioxidant balance during prolonged physical exercise. 4

ANTIOXIDANT SUPPLEMENTATION IN EXERCISE

4.1 Vitamin E Venditti et al. [72] observed that free radical-induced damage in muscle could be one of the factors terminating muscle effort. They suggested that greater antioxidant levels in the tissue should allow trained muscle to withstand oxidative processes more effectively, thus lengthening the time required so that the cell function is sufficiently damaged as to make further exercise impossible. Whether oxidative stress is the single most important factor determining muscle performance is certainly a debatable issue. The contention that strengthened antioxidant defense of the muscle may protect against exercise-induced oxidative stress-dependent muscle damage is much more readily acceptable [73]. Animal experiments studying the effect of vitamin E have shown mixed results on the prevention of lipid peroxidation [1] with the general trend that such supplementation may diminish oxidative tissue damage. Brady and coworkers [74] examined the effects of vitamin E supplementation (50 lU/kg diet) on lipid peroxidation in liver and skeletal muscle at rest and following exhaustive swim exercise. Vitamin E effectively decreased lipid peroxidation in liver independent of selenium supplementation, whereas skeletal muscle lipid peroxidation response was unaffected by the supplementation. Goldfarb et al. [75] observed that vitamin E supplementation can protect against run-induced lipid peroxidation in the skeletal muscle and blood. The effect in skeletal muscle was muscle fiber type-dependent. The

308


protective eflfect of vitamin E was more clearly evident when the animals were exposed to an additional stressor, dehydroepiandrosterone. Jackson et al. [76] examined the effect of both vitamin E deficiency and supplementation on the contractile activity of muscle. Male rats and female mice were given either a standard diet, a vitamin E-deficient diet with 0.5 mg/kg selenium or a diet supplemented with 240 mg a-tocopherol acetate/kg diet. The animals were given this diet for 42 to 45 days. Vitamin E deficiency, in both mice and rats, was associated with increased susceptibility to contractile damage. Vitamin E supplementation clearly protected against such damage. Despite the fact that vitamin E supplementation protected the muscles from damage as indicated by creatine kinase and lactate dehydrogenase leakage there was no apparent effect on muscle lipid peroxidation. Kumar et al. [77] noted that vitamin E supplementation for 60 days in female adult albino rats completely abolished the increase in free radical mediated lipid peroxidation in the myocardium as a result of exhaustive endurance exercise. They reported that exercise-induced lipid peroxidation in heart tissue increased in control rats but did not increase in the vitamin E supplemented rats. Consistently, it has been also observed that vitamin E supplementation for 5 weeks attenuated exercise-induced increase in myocardial lipid peroxidation [75,78,79]. Mcintosh et al. [80,81] observed that vitamin E supplemented diets protect against peroxisomal fatty acid oxidation-dependent leakage of hepatic enzymes into the plasma in response to physical exercise and dehydroepiandrosterone treatment. Vitamin E prevented dehydroepiandrosterone-induced increase of peroxisomal fatty acid oxidation and leakage of alanine aminotransferase and aspartate aminotransferase into the plasma. Exercised animals on a normal diet demonstrated similar peroxisomal fatty acid oxidation profile and plasma enzyme levels as the vitamin E supplemented group. Novelli et al. [82] examined the effects of intramuscular injections of three spin-trappers and vitamin E on endurance swimming to exhaustion in mice. Mice were injected on 3 successive days. It was observed that compared to either the control or placebo saline injected animals the spin-trap and vitamin E injected group had significantly increased swim endurance. In a study reported by Quintanilha and Packer [60] rats were given one of the following three diets and compared for liver mitochondrial respiration and lipid peroxidation: a diet deficient of vitamin E, a diet with 40 lU vitamin E/kg, or a diet with 400 lU vitamin E/kg. Hepatic mitochondrial respiratory control ratio were highest in the group supplemented with 400 lU/ kg. Additionally, liver lipid peroxidation in nuclei and microsomes was lowest in the vitamin E supplemented group especially when NADPH was present. Warren et al. [83] studied the effects of vitamin E supplementation, 10,000 lU/kg diet for 5 weeks, on muscle damage and free radical damage to membranes as indicated by alterations in plasma enzymes. Susceptibility of the skeletal muscles to oxidative stress was markedly decreased in response to vitamin E supplementation but this did not attenuate muscle injury triggered by eccentric contractions. It was concluded that vitamin E supplementation may be beneficial in protecting


309

against free radical damage, but that the injury caused by eccentric exercise may not be ROS mediated. The effect of dietary vitamin E on exercise-induced oxidative protein damage has been investigated in the skeletal muscle of rats. For a period of 4 weeks rats were fed with high vitamin E diet (10,000 lU/kg diet), a atocopherol and tocotrienol (7000 mg tocotrienol/kg diet) rich palm oil diet or control diet with basal levels of oc-tocopherol (30 lU/kg body weight). Uphill exhaustive treadmill exercise caused oxidative protein damage in skeletal muscles. A protective effect of vitamin E supplementation against exercise-induced protein oxidation in skeletal muscles was clearly evident [84]. a-Tocopherol level and fluidity has been studied in the neuronal membrane of rat brain after exhaustive exercise. The order parameter, 5-doxyl-stearic acid (5-DS), which is utilized for assessing the fluidity of the lipid bilayer closer to the hydrophilic face of the membrane, decreased in the pons-medulla oblongata; and the motion parameter, 16doxyl-stearic acid (16-DS) for the study of the lipid bilayer core, decreased in the cortex, hippocampus, hypothalamus and striatum, whereas it increased in the cerebellum after exercise. The w/s ratio of n- (l-oxyl-2,2,6,6-tetramethyl-4piperidinyl) -maleimido, used for the study of sulfhydryl protein conformation, also decreased in the hippocampus and midbrain after exercise. These exercisedependent changes were prevented by a-tocopheryl acetate supplementation (10,000 lU vitamin E /kg diet) [85]. Fish oils have been shown to have a beneficial effect on cardiovascular mortality based on numerous epidemiological studies [86], presumably via effects on triglyceride levels, membrane fluidity and platelet and leukocyte function [87]. Not all studies show beneficial effects, however [88]. Because the (n-3) fatty acids making up fish oil are highly polyunsaturated, concerns have been raised regarding increased oxidative stress from fish oil intake [89—93]. Furthermore, fish oils induce peroxisomal P-oxidation, in which fatty-acyl oxidation yields hydrogen peroxide (H2O2) as a normal byproduct, and upregulate the activity of the H2O2 decomposing enzyme catalase [89,90,94]. Under normal conditions up to 20% of cellular O2 consumption has in fact been estimated to occur in the peroxisome [95]. The beneficial effects of regular exercise on cardiovascular and overall mortality [96] may be decreased by uncontrolled exercise-induced oxidative stress. This may be particularly concerning in groups predisposed to oxidative stress, including that induced by fish oil [91—93]. Sen et al. examined the effect of fish oil and vitamin E supplementation compared to placebo soy oil and vitamin E supplementation on physiological antioxidant defenses and resting and exercise-induced oxidative stress in rat liver, heart and skeletal muscle [97]. The effects of 8 week vitamin E and fish oil supplementation on resting and exercise-induced oxidative stress was examined. Lipid peroxidation was 33% higher in fish oil fed rats compared to the placebo group in the liver, but oxidative protein damage remained similar in both liver and red gastrocnemius muscle. Vitamin E supplementation markedly decreased liver and muscle lipid peroxidation induced by fish oil diet. Vitamin E supplementation also markedly decreased oxidative protein damage in the liver and muscle. Exhaustive treadmill exercise

310

Pî^t IV: Antioxidant defenses

increased liver and muscle lipid peroxidation, and muscle oxidative protein damage. Vitamin E effectively decreased exercise-induced lipid peroxidation and protein oxidation [97]. A limited number of studies have examined the effect of vitamin E supplementation in humans. Exercise performance and physical fitness have multifactorial determinants and may not serve as reasonable end points to test the efficacy of antioxidant supplementation. Vitamin E supplementation (900 IUd~^ for 6 months) in trained swimmers did not alter their swim performance nor their lactate response in plasma [98]. Neither did vitamin E supplementation (800 IUd~^ for 4 weeks) alter the work load needed to run at 80% VO2 max in trained and untrained men [99]. Volunteers given 400 IUd~^ of vitamin E for 6 weeks showed no influence on cycle time, swim time, or step time [100]. Additionally no changes in VO2 max, a marker of physical fitness, was noted in humans following vitamin E supplementation [99,101,102]. However, Cannon et al. (1990) reported that 400 IUd~^ of vitamin E supplementation for 48 days decreased the amount of creatine kinase leakage from muscles during recovery from a downhill run. Sumida et al. [101] examined the effects of 4 weeks of vitamin E supplementation in 21 healthy college-aged males. The subjects ingested 300 mg of vitamin E daily and blood levels of several enzymes and lipid peroxides were determined before and for up to 3 h after cycling exercise to exhaustion. Exercise increased the level of lipid peroxidation byproducts in plasma immediately after the cycling. Such levels returned to normal at 1 and 3 h of recovery. Vitamin E supplementation significantly decreased the resting level of plasma lipid peroxides. Meydani et al. [103] reported that urinary excretion of lipid peroxidation byproducts tended to be lower in vitamin E supplemented individuals (400 lU doses, twice daily, for 48 days) compared to the corresponding placebo group. This effect was only significant 12 days after downhill running. The subjects ran 16% downhill at 75% of their maximum heart rate for three 15 min periods. Muscle biopsies were obtained from the vastus lateralis of young subjects. It was observed that exercise increased the level of lipid peroxidation byproducts increased in muscle of placebo group whereas in the muscle of the vitamin E supplemented group no such oxidative lipid damage was evident. Another study examined the effect of vitamin E supplementation (800 d"' for 4 weeks) and compared that to a placebo treatment in the same individuals at a specific exercise intensity [99]. Subjects were randomly assigned to either a placebo or vitamin E treatment group in a counter-balanced design. Subjects were exercised for 30 min at 80% VO2 max and blood samples were collected before and after the run. Vitamin E treatment attenuated the level of resting plasma lipid peroxidation byproducts and also protected against the exercise response. The effects of 5 months of a-tocopherol supplementation have been studied in 30 top class cyclists. Although the supplementation did not improve physical performance, it was evident that exercise-induced muscle damage was less in response to antioxidant supplementation [104]. In humans it has been observed that a combination of vitamin E and fish oil supplement delays oxidative modification of low density


311

lipoprotein [105]. In 1980, the US recommended daily allowance for vitamin E was reduced from 30 lU (recommended in 1968) to 15 lU. In the same year it was estimated that in the US, the amount of vitamin E supplied by a "normal" diet was about 11 lU (7.4 mg). Packer et al. have discussed that such dosages are insufficient for active athletes and that dosages of up to 400 lU/day may be reasonable recommendation for active athletes engaged in moderate to heavy exercise [106]. Vitamin E is proven to be safe at levels of intake up to approximately 3000 mg for prolonged periods of time [107]. However, individuals taking anticoagulants should refrain from taking very high doses (> 4000 lU) of vitamin E because vitamin E can act synergistically with this class of drug [108]. Long-term studies are necessary to determine safety issues related to vitamin E supplementation in humans. 4.2 Vitamin C Levels of vitamin C in the plasma has been shown to decrease in response to acute swim exercise [109]. Such effect as been noted to be proportional to the duration of exercise. This observation suggests that prolonged physical exercise is associated with enhanced consumption of vitamin C in the circulation [109]. Vitamin C supplements (3 g/kg diet) given to rats who were placed on a vitamin E deficient diet did not alter the run time to exhaustion in the vitamin E deficient animals. Vitamin C was also unable to counter the deleterious effects of vitamin E deficiency [57]. Oral vitamin C, however, did prevent exercise-induced blood GSH oxidation in rats [110]. In a prehminary report the effect of vitamin C supplementation in humans was documented. A mild protective effect of vitamin C supplementation, based on elevated total antioxidant capacity of the plasma, was observed [111]. In a double-blind, randomized crossover study, 19 healthy subjects (men and women) mean 35 years of age were given two separate bouts of eccentric work on their plantar flexors on separate legs 3 weeks apart [112]. Vitamin C (3 g/day) was supplemented for 3 days preceding exercise, and continued for 1 week. Vitamin C supplementation attenuated muscle soreness compared to placebo. In another study it was also noted that 400 mg of vitamin C supplement improves recovery from eccentric muscle damage [113]. Whether this effect of vitamin C was related to its antioxidant property is not clear. 4.3 Coenzyme Qio A few studies have examined the effects of coenzyme Qio to determine if additional amounts of this factor in the electron transport chain would be beneficial in preventing free radical damage [114—116]. Dietary coenzyme Qio supplementation protected against leakage of creatine kinase and lactate dehydrogenase from the muscles to serum following downhill run [114]. In two human studies, however, this beneficial effect of coenzyme Qio could not be observed [115,116].

312


The effects of ubiquinone supplementation (120 mg/day for 6 weeks) on aerobic capacity and lipid peroxidation during exercise has been investigated in 11 young (aged 22—38 years) and 8 older (aged 60—74 years), trained men. This crossover study was double-blind and placebo-controlled. Ubiquinone supplementation effectively increased serum ubiquinone concentration in both age groups but did not influence maximal aerobic capacity Consistent with previous reports, oral ubiquinone supplementation was ineffective as an ergogenic aid in both the young and older, trained men [117]. Kamikawa et al [118] noted, however, that 150 mg/day of ubiquinone supplementation for 28 days increases maximal exercise tolerance in angina pectoris patients. Karlsson and co-workers have also investigated the potential beneficial effects of ubiquinone on several physiological parameters and its relationship to other antioxidants [119—122]. They reported that 100 mg/day of ubiquinone supplementation for 6 weeks was beneficial in improving exercise capacity [122]. Swedish downhill skiers were noted to have decreased ubiquinone levels in their muscles and plasma following 3 days of skiing. A relationship with the amount of ubiquinone in muscle and exercise capacity in hypertensive and ischemic heart diseased individuals compared to age matched controls was suggested [120]. Ubiquinone supplementation may be beneficial but it may have certain limitations too. In one study it has been evident that supplementation of ubiquinone may enhance cellular damage during intense exercise [123]. Optimization of a proper ubiquinone supplementation regimen that would provide beneficial effects with minimum risk requires further research. 4.4 Glutathione Two brief rodent studies have shown that exogenous GSH may remarkably increase endurance to physical exercise [124,125]. Compared to placebo treated controls 0.5, 0.75 and 1 g/kg intraperitoneal doses of GSH increased endurance to swimming by a marked 102.4%, 120% and 140.7%, respectively [125]. At a dose 0.25 g/kg, GSH did not affect endurance when injected once but such a dose could significantly increase endurance when injected once a day for 7 consecutive days. In another study, oral GSH at dosages 0.25 to 1 g/kg caused a dose-dependent significant improvement in swim endurance [124]. Both abovementioned studies employed brief bursts of swimming as the exercise challenge and did not report any biochemical data related to either glutathione metabohsm or other indices of oxidative stress. Sen et al. [69] attempted to clarify the possible mechanism of such beneficial effect of GSH supplementation. Almost all evidence supporting the contention that a single bout of exercise may induce oxidative stress have been obtained from studies using exercise types that were long in duration, and mostly running or cycling in nature. Intraperitoneal injection of GSH solution (1 g/kg body weight) resulted in a rapid appearance of GSH in the plasma and was followed by a rapid clearance of the thiol. Following the injection excess plasma GSH was rapidly oxidized. GSH injection did not influence GSH status of other tissues studied. Following the repeated administration


313

of GSH, blood and kidney total glutathione levels were increased. Plasma total glutathione of GSH supplemented animals was rapidly cleared during exhaustive exercise. The GSH administration protocol, as used in this study, did not influence the endurance to exhaustive physical exercise of rats. It has been shown that following a treadmill run to exhaustion levels of immunoreactive Mn-SOD (manganese superoxide dismutase, a mitochondrial protein) remarkably increases in the plasma. Glutathione supplementation (500 mg/kg body weight) marginally suppressed such release of the mitochondrial protein to the plasma [126]. The inability of exogenous GSH to provide added antioxidant protection to tissues may be largely attributed to the poor availability of exogenous administered GSH to the tissues. In another part of this study, Atalay et al. [31] tested the effect of GSH supplementation on exercise-induced leukocyte margination and neutrophil oxidative burst activity Exercise-associated leukocyte margination was prevented by GSH supplementation. Peripheral blood neutrophil counts were significantly higher in GSH-supplemented groups compared to the placebo control groups. Also, exercise-induced increase in peripheral blood neutrophil oxidative burst activity as measured by luminol-enhanced chemiluminescence per volume of blood tended to be higher in the GSH-supplemented group, and lower in the GSH-deficient rats suggesting high plasma GSH may have augmented exercisedependent neutrophil priming. In these experiments, for the first time it was shown that GSH supplementation can induce neutrophil mobilization and decrease exercise-induced leukocyte margination, and that exogenous and endogenous GSH can regulate exercise-induced priming of neutrophil for oxidative burst response [31]. 4.5 N-acetyl-L -cysteine Blood glutathione homeostasis has been suggested to be a determinant of resting and exercise-induced oxidative stress in young men [127]. The effect of oral Nacetyl-L-cysteine (NAC) on exercise-associated rapid blood GSH oxidation in healthy adult males who performed two identical maximal bicycle ergometer exercises 3 weeks apart has been investigated. Before the second maximal exercise test, men took effervescent NAC tablets (200 mg x 4 /day) for 2 days, and an additional 800 mg in the morning of the test. The NAC supplementation protocol used in the study 1) increased the net peroxyl radical scavenging capacity of the plasma, and 2) spared exercise-induced blood glutathione oxidation [39]. The ability of oral N-acetyl-L-cysteine to prevent exercise-induced oxidation of blood GSH has been also observed in rats [110]. Reid and associates have tested the effect of NAC on muscle fatigue [128,129]. Fiber bundles were removed from diaphragms and stimulated directly using supramaximal current intensity. Studies of unfatigued muscle showed that 10 mM NAC reduced peak twitch stress, shortened time to peak twitch stress, and shifted the stress-frequency curve down and to the right. Fiber bundles incubated in 0.1 — 10 mM NAC exhibited a dose-dependent decrease in relative stresses

314


developed during 30 Hz contraction with no change in maximal tetanic (200 Hz) stress. NAC (10 mM) also inhibited acute fatigue. In a later experiment, this effect of NAC was tested in humans. Healthy volunteers were studied on two occasions each. Subjects were pretreated with NAC 150 mg/kg or 5% dextrose in water by intravenous infusion. It was evident that NAC pretreatment can improve performance of human limb muscle during fatiguing exercise, suggesting that oxidative stress plays a causal role in the fatigue process and identifying antioxidant therapy as a novel intervention that may be useful clinically [128,129]. 4.6 a-Lipoic acid The first study testing the efficacy of a-lipoate supplementation in exerciseinduced oxidative stress has been just reported. Khanna et al. [130] studied the effect of intragastric lipoate supplementation (150 mg/kg body weight for 8 weeks) on lipid peroxidation and glutathione-dependent antioxidant defenses in liver, heart, kidney and skeletal muscle of male Wistar rats. Lipoate supplementation significantly increased total glutathione levels in liver and blood. This information is consistent with results from previous in vitro experiments [41], and shows that indeed lipoate supplementation may increase glutathione levels of certain tissues in vivo. Lipoate supplementation, however, did not affect the total glutathione content of organs such as the kidney, heart and skeletal muscles. Lipoate supplementation-dependent increase in hepatic glutathione pool was associated with increased resistance to lipid peroxidation. This beneficial effect against oxidative lipid damage was also observed in the heart and red gastrocnemius skeletal muscle. Lower lipid peroxide levels in certain tissues of lipoate fed rats suggest strengthening of the antioxidant defense network in these tissues [130]. 4.7 Other nutrients Other nutrients that have been ascribed to be beneficial as antioxidants such as selenium and 6-carotene have not been examined individually but have been assessed in conjunction with either vitamin E deficiency or in combination with other antioxidants. The effects of selenium supplementation (0.5 ppm diet) or deprivation have been tested in liver, muscle and blood of swim exercised rats [74]. Some rats were additionally supplemented with vitamin E (50 d"^). Selenium supplementation increased the activity of the hydroperoxide metabolizing enzyme glutathione peroxidase in the liver. A tight regulation of tissue glutathione peroxidase activity by dietary selenium was observed because seleniumdeficient diet markedly downregulated the activity of the enzyme. Muscle glutathione peroxidase activity demonstrated similar responses to selenium intervention compared to the liver. Increased tissue lipid peroxidation was evident when both selenium and vitamin E were deficient. However, selenium deficiency had little effect when vitamin E was present. Selenium appeared to have minimal effects on swim-induced lipid peroxidation in the liver or muscle. Dietary sele-


315

nium supplementation in horses (0.15 ppm daily for 4 weeks) had minimal eflfects on exercise-induced lipid peroxidation as indicated by blood level of lipid peroxidation byproducts [131]. In a double bhnd human study no effect of selenium supplementation on human physical performance was observed [132]. Selenium poisoning is rare in the US, but the case of a man who was poisoned by selenium containing vitamin tablets has been described [133]. Strenuous physical exercise has been shown to decrease P-carotene levels in the serum. In a study of 574 women and 57 men from a rural city in Japan it was noted that the serum P-carotene lowering effect of exercise was independent of dietary factors, smoking and cholesterol intake as indicated by 3-day food records [134]. 4.8 Antioxidant interaction From the biochemistry of antioxidant action it is evident that antioxidants function in a network (see Chapter 10 by Sen and Powers in this volume, 219—242) and interaction between several major antioxidants have been clearly evident [1]. As a result, some studies have attempted to investigate the efficacy of a combination of several antioxidants as supplements [135—137]. Supplementation of individuals with a vitamin mixture containing 37.5 mg 6-carotene, 1250 mg vitamin C and 1000 lU of vitamin E for 5 weeks decreased the level of lipid peroxidation byproducts in the serum and breath, both at rest and following exercise at both 60 and 90% VO2 max [136]. In contrast, a previous study which used a similar mixture of antioxidants and exercised the subjects at 65% of maximal heart rate in a downhill run was unable to demonstrate any positive eflfects [135]. This inconsistency in observation was explained by differences in the nature and intensity of the exercise in the two studies. The eflfects of an antioxidant mixture (10 mg B-carotene, 1000 mg vitamin C and 800 lU of vitamin E) on human blood glutathione system and muscle damage has been determined [137]. A protective effect on the blood glutathione system and muscle damage was evident. A randomized and placebo-controlled study has been carried out on 24 trained long-distance runners who were supplemented with oc-tocopherol (400 lU /day) and ascorbic acid (200 mg /day) for 4.5 weeks before a marathon race. Serum content of ascorbic acid as well as oc-tocopherol were elevated in supplemented individuals. In this study, the antioxidant supplementation protocol was observed to significantly protect against exercise-induced muscle damage as manifested by the loss of creatine kinase from the muscle to the serum [138]. 5

PERSPECTIVES

Several lines of evidence consistently show that physical exercise may induce oxidative stress. The relationship between physical activity, physical fitness and total radical trapping antioxidant potential was examined in the Northern Ireland Health and Activity Survey. This was a large cross-sectional population study (n = 1600) using a two-stage probability sample of the population. A necessity for

316


antioxidant supplementation, especially in physically active and fit individuals, was indicated [139]. Depending on nutritional habits and genetic disposition susceptibility to oxidative stress may vary from person to person. Determination of tissue antioxidant status of individuals is thus recommended. Such information will be necessary to identify specific necessities and formulate effective strategies for antioxidant therapy. Nutritional antioxidant supplements are known to be bio-available to tissues and may strengthen defense systems against the ravages of reactive species. Results from antioxidant supplementation studies considerably vary depending on the study design and measures of outcome. Physical performance is regulated by multifactorial processes and may not serve as a good indicator to test the effect of antioxidant supplementation. The general trend of results shows no effect of antioxidant supplementation on physical performance. However, in a large number of studies it has been consistently evident that antioxidant supplementation protects against exercise-induced tissue damage. Diet of laboratory animals are often heavily enriched with antioxidant vitamins, particularly vitamin E. This may be one reason why antioxidant supplementation to animals fed a regular diet does not influence several measures of outcome. At present there is a growing trend among people to cut down the amount of fat in their diets. While this does markedly decrease caloric intake, in many cases this may also contribute to a marked decrease in the intake of fat-soluble essential nutrients including antioxidant vitamins. From available information we know that under regular circumstances antioxidants such as a-tocopherol, ascorbic acid and |3-carotene are well tolerated and free from toxicity even when consumed at doses several-fold higher than the recommended dietary allowances [140]. In view of this and the potential of antioxidant therapy, consumption of a diet rich in a mixture of different antioxidants may be expected to be a prudent course. 6

SUMMARY

1. The chemical nature of any antioxidant determines its solubility, and thus its localization in biological tissues. For example, lipid soluble antioxidants are localized in membranes and function to protect against oxidative damage of membranes. Water-soluble antioxidants, located for example in the cytosol, mitochondrial matrix or extracellular fluids, may not have access to ROS generated in membranes. 2. Studies with antioxidant deficient animals show that such deficiency may enhance exercise-induced tissue damage and may compromise work performance. 3. Results from antioxidant supplementation studies considerably vary depending on the study design and measures of outcome. Physical performance is regulated by multifactorial processes and may not serve as a good indicator to test the effect of antioxidant supplementation. The general trend of results shows no effect of antioxidant supplementation on physical performance.


317

However, in a large number of studies it has been consistently evident that antioxidant supplementation protects against exercise-induced tissue damage. 4. While further studies are needed to establish which antioxidant supplement regimen is appropriate for the physically active individual, consumption of a diet rich in a mixture of different natural antioxidants may be expected to be a prudent course. 7

ABBREVIATIONS

BSO: DHLA: GSH: GSSG: lU: NAC: ppm: RDA: SOD: 8

L -buthionine-sulfoximine dihydrolipoate reduced glutathione glutathione disulfide international unit N-acetyl-L -cysteine parts per million recommended daily allowances superoxide dismutase

REFERENCES

1. Sen CK. J Appl Physiol 1995;79,675-686. 2. Engelhardt JK Antiox Redox Signal 1999;1:5-27. 3. Sheppard A J, Pennington, JATand Weihrauch, JL. In: Packer L, Fuchs J (eds) Vitamin E in Health and Disease. New York: Marcel Dekker, 1993;9—31. 4. Burton GW, Joyce A, Ingold KU. Archives of Biochemistry and Biophysics 1983;221:281 -290. 5. Traber MG, Sies H. Annu Rev Nutr 1996;16:321-347. 6. Handelman GJ. In: Cadenas E, Packer L (eds) Handbook of Antioxidants. New York: Marcel Dekker, 1996;259-314. 7. Handelman GJ, van Kuijk FJ, Chatterjee A, Krinsky NI. Free Radic Biol Med 1991;10: 427-437. 8. Burton GW, Ingold KU. Science 1984;224:569-573. 9. Andersen HR, Andersen O. Pharmacol Toxicol 1993;73:192-201. 10. Kagan V, Nohl H, Quinn PJ. In: Cadenas C, Packer, L (eds) Handbook of Antioxidants. New York: Marcel Dekker, 1996. 11. Laaksonen R, Riihimaki A, Laitita J, Martensson K, Tikkanen MJ, Himberg JJ. Journal of Laboratory and Clinical Medicine 1995;125:517-521. 12. Aust SD, Morehouse LA and Thomas CE. Free Radic Biol Med 1985;1:3-25. 13. Buettner GR. Free Radic Res Commun 1986;1:349-353. 14. Meister A. Methods Enzymol 1995;251:3-7. 15. Meister A. Biochem Pharmacol 1992;44:1905-1915. 16. Meister A. J Nutr Sci Vitaminol. Tokyo 1992;Spec No: 1—6. 17. Sen CK, Hanninen O In: Sen CK, Packer L, Hanninen O (eds) Exercise and oxygen toxicity Amsterdam: Elsevier Science Publishers BV, 1994:89-126. 18. Thomas JP, Maiorino M, Ursini F, Girotti AW. J Biol Chem 1990;265:454-461. 19. Hayes JD, Pulford DJ. Crit Rev Biochem Mol Biol 1995;30:445-600. 20. DeLeve LD, Kaplowitz N Semin Liver Dis 1990;10:251-266. 21. Meister A. Pharmacol Ther 1991;51:155-194.

318 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60.

P

Part VIII •Chapter 19

Air pollution and oxidative stress Dianne M. Meacher and Daniel B. Menzel Department of Community and Environmental Medicine, 100 Faculty Research Facility, University of California at Irvine, Irvine, California 92697-1825, USA. E-mail: [email protected]

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5 6

7

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10 11 12 13 14

INTRODUCTION ATMOSPHERIC OZONE AND NITROGEN DIOXIDE DOSIMETRY MODELING OF OZONE AND NITROGEN DIOXIDE 3.1 Ozone 3.2 Nitrogen dioxide LABORATORY HUMAN EXPOSURE WITH EXERCISE STUDIES 4.1 Pulmonary function effects of acute ozone exposure 4.1.1 Concentration-response relationship 4.1.2 Reduction of exercise performance 4.1.3 Recovery of pulmonary function following acute ozone exposure 4.1.4 Mechanisms of pulmonary function effects of ozone 4.2 Pulmonary inflammation following acute ozone exposure 4.3 Airway responsiveness to ozone exposure 4.4 Pulmonary function effects of prolonged ozone exposure 4.5 Pulmonary function effects of mixtures of ozone and other air pollutants 4.6 Pulmonary effects of nitrogen dioxide exposure 4.6.1 Pulmonary function effects and airway responsiveness 4.6.2 Inflammatory response and epithelial cell permeability 4.7 Effects of urban air polluted with ozone and nitrogen dioxide OUTDOOR STUDIES OF HUMAN AMBIENT AIR EXPOSURES INFLAMMATORY RESPONSE TO OZONE 6.1 In vitro studies 6.2 Experimental in vivo studies EXTRAPULMONARY EFFECTS OF OZONE AND NITROGEN DIOXIDE 71 Ozone 72 Nitrogen dioxide MECHANISMS OF TOXICITY 8.1 Ozone 8.1.1 Lipid peroxidation 8.1.2 Protein oxidation 8.2 Nitrogen dioxide BIOCHEMICAL EFFECTS 9.1 Antioxidant enzyme activities 9.1.1 Ozone 9.1.2 Nitrogen dioxide 9.2 Antioxidants 9.2.1 Ozone 9.2.2 Nitrogen dioxide SUMMARY PERSPECTIVES ACKNOWLEDGEMENT ABBREVIATION REFERENCES

514 1

Pcirt VIII: Environmentalfactors INTRODUCTION

The health effects of ozone and nitrogen dioxide, the major oxidant air pollutants, remain controversial after many years of study. There is little doubt that this is a complex subject compared to others in toxicology This section is intended as a preface to the detailed data that will be presented in this chapter. The chapter is oriented to the topic of this volume so that some subjects regarding the toxicology of oxidant air pollutants have been deleted. We begin with the role of lung structure on toxicity and discuss features of the lung lining fluid. We propose that the toxicity of ozone and nitrogen dioxide can be explained from the direct chemical reaction of the oxidants with cellular constituents. Similarly, the protective effects of antioxidants can be ascribed to the lessening of oxidation in cells. We will also discuss the time course of ozone and nitrogen dioxide effects. Mathematical models pioneered by Miller et al. [1] are the first steps toward a predictive theory of the human toxicity of ozone and nitrogen dioxide. According to mathematical models [1,2], the major driving force affecting ozone and nitrogen dioxide toxicity is deposition of the gases in the lung. The relative deposition of ozone and nitrogen dioxide in the three major regions of the respiratory tract of humans is shown schematically in Figs. 1 and 2. Although ozone and nitrogen dioxide are deposited throughout the lungs, the absorption of the gases into the lungs is uneven among the regions. The nasopharyngeal region plays an important protective role. In humans using nasal breathing, the nasopharyngeal cavity removes a major fraction of ozone (about 50%) from the inhaled air. An even larger fraction of inhaled nitrogen dioxide is removed in the nasopharyngeal region because of the relatively greater water solubility of nitrogen dioxide compared to ozone. During moderate and heavy exercise, an obligatory switch to a combination of mouth and nasal breathing occurs in humans. The removal of ozone and nitrogen dioxide by the nasopharyngeal region decreases during exercise because of the lower efficiency of the oral cavity compared to the nasopharyngeal region in removing both gases. The regional uptake of ozone and nitrogen dioxide is controlled by the rates of reaction of the gases with the lung lining fluid. Even though the entire human lung is lined with fluid, the lung lining fluid is different in the alveolar region than it is in the conducting airways. Mucus, a glycoprotein mixture, covers the airways whereas lung surfactant, a protein-phospholipid complex composed mostly of phosphatidylcholine, covers the alveolar surface. Figure 3 is a cartoon illustrating some of the features of the respiratory bronchiole and alveolus. One complicating aspect of understanding the effects of air pollutants on the lung is the cellular diversity present in the lung. Due to its specialized functions of oxygen transport and carbon dioxide excretion, the lung has evolved into some 40 or more different cell types arranged anatomically to facilitate gas exchange in the alveoli. A few of these cell types are depicted in Fig. 3. The lung surfactant imparts ease of inflation and deflation to the alveoli so that all of the alveoli are inflated and collapsed at the same pace. Whereas most of

Chapter 19: Air pollution and oxidative stress

515

nasal cavity pharynx

- 4 0 % O3 Deposited

Fig. 1. A schematic representation of the deposition of ozone in the human respiratory tract. Ozone is poorly soluble in water so that removal in the nasopharyngeal region is less for ozone than for nitrogen dioxide. The deposition of ozone is mostly in the transitional zone of the lung, but all of the regions of the lung are damaged by ozone. In experimental animals, the most sensitive cells are the ciliated cells of the airways and type 1 cells of the alveoli. See Fig. 3 for detail on the transitional zone.

the fatty acid residues of the phosphohpids in the lung surfactant are saturated palmitic acid residues, up to 30% are unsaturated. The polyunsaturated fatty acids in the lung lining fluid are rapidly oxidized by ozone and nitrogen dioxide, decreasing the surface-tension-lowering ability of the lung surfactant. One hypothesis as to why ozone produces its greatest effects in the transitional or centriacinar zone is that ozone is more soluble in the lipid surfactant lining the alveoli than in the mucus lining the airways. The transitional zone includes the terminal bronchioles, respiratory bronchioles, and the immediately adjacent alveoli. As the lung surfactant contains mostly saturated fatty acids, it does not

Part VIII: Environmental factors

516 nasal cavity pharynx

Deposited

'^10%NO2 Deposited

bronchioles 10- 15%N02 Deposited

Fig. 2. A schematic representation of the deposition of nitrogen dioxide in the human respiratory tract. The nasopharyngeal region removes the majority of the inhaled nitrogen dioxide. The lower conducting airways and the pulmonary region also are sites of deposition. Nitrogen dioxide damage results mostly in the denuding of the ciliated regions of the transitional zone, but some damage to type 1 alveolar cells also occurs. See Fig. 3 for detailed structure of the transitional zone.

deplete the inhaled air of ozone or nitrogen dioxide before the gases can reach the cells lining the alveoli. The thin type 1 cells are low in mitochondria and protective antioxidants and thus are major targets for ozone and nitrogen dioxide toxicity. Type 2 cells, on the other hand, are secretory cells producing the lung surfactant. These cells are more metabolically capable than are the type 1 cells. Furthermore, in the transitional zone, bare patches or low coverage by mucus and lung surfactant result in greater ozone deposition in this region than in other regions of the lung. Ozone does injure the upper airways, but the most striking and critical long-term effect is in the transitional zone.


517

Mucus Layer

Ciliated Cells

Type 2 Cell Surfactant Secreting

Surfactant

Macrophage

Capillary Wall

Fig. 3. A cross-section of the transitional zone of the lung. The key feature leading to the sensitivity of this region to ozone is the discontinuation of the aqueous mucus layer and the beginning of the surfactant layer. The main component of an alveolus is a single type 1 cell that lies in close proximity to capillaries. Type 2 cells secrete phosphoHpid constituents of the lung surfactant. When type 1 cells die, type 2 cells differentiate into type 1 cells. The bronchiole is lined with ciliated cells and mucussecreting cells like the Clara cell. Clara cells contain most of the drug-metabolizing activity of the lung. Ozone and nitrogen dioxide exposure injures the Clara cells and decreases the drug metabolizing capacity of the lung. This simplified drawing also illustrates that multiple cell types (some 40 or more in total) make up the lung. All are affected by air pollution to differing extents due their physiology and position within the lung.

Moreover, although ozone may not survive transit through the type 1 cell, some products of ozonation can diffuse from the type 1 cell into the blood (Fig. 3). To increase oxygen transport, the lung has evolved to have a very small separation

518

P^^t VIII: Environmental factors

between the alveolar lumen and the capillaries. Products such as ozonides and aldehydes can diffuse across this junction into the blood. The small space between the alveolar lumen and the capillaries explains why both ozone and nitrogen dioxide can affect distal tissues even though the original reaction products, such as the initial ozonide or lipid peroxyl radical, are too short-lived to exit into the blood. As described in section 4, the contents of the fluid lining can be sampled using the procedure called bronchoalveolar lavage by introducing and withdrawing saline into one lobe of the lung. The lavage fluid contains cells, mucus, proteins, and small molecular weight compounds. The cell type most often found in the lavage fluid in a normal subject is the alveolar macrophage (Fig. 3). The alveolar macrophage is the first line of defense against microorganisms in the alveoli. The macrophages help kill and remove microorganisms from the lung by engulfing them and moving them to the mucus lining layer. The macrophages themselves can be killed by exposure to inhaled nitrogen dioxide and ozone. Exposure to these oxidants causes increased susceptibility of experimental animals to infection because the killing of macrophages by ozone and nitrogen dioxide allows microorganisms to multiply in the lung. Injury to the cells lining the airways by air pollution attracts alveolar macrophages to the middle and upper airways from their normal domain in the alveoli. The digestion of dead or moribund cells by macrophages followed by the release of cytokines, eicosanoids, and other factors appears to attract inflammatory cells, which are found in lavage fluid during inflammation. Attraction of inflammatory cells to the lung requires time for the circulation of cytokines and expansion of inflammatory cell populations. Interestingly, analysis of lung lavage fluid also yields ascorbic acid (vitamin C), glutathione, and vitamin E. The mechanisms by which these antioxidants are secreted into the lumen of the airways are unclear. During controlled human exposures to ozone and nitrogen dioxide, levels of some of these antioxidants decline possibly due to their destruction by reaction with inhaled ozone or nitrogen dioxide. Depletion or inhibition of transport of antioxidants into the airways could increase lung damage. There is little doubt that the lung lining fluid is the first protective barrier between an inhaled chemical or microorganism and the lung proper. The site of the toxic reaction (i.e., the target of the toxic agent) is as important as the extent of the toxic reaction. The kinetics and mechanisms of reaction of inhaled agents teach us much about the parts of the lung that are affected. Regional effects clearly are important because they define the extent of a physiological decrement. Antigens act mostly in the upper airways, whereas ozone and nitrogen dioxide act mostly in the transitional zone of the lung. Antigenic effects are mostly in the bronchi. Chronic ozone or nitrogen dioxide exposure of animals leads to the loss of structure and function in the centriacinar region. One of the challenges of extrapolating to humans the animal toxicity data from ozone and nitrogen dioxide exposures is the tremendous time scale over which


519

toxic effects occur in humans compared to experimental animals. Experimental animals can be exposed to a constant ozone or nitrogen dioxide concentration over most of their lifetimes. In ambient air, humans are exposed to episodes of air pollution consisting of mixtures of ozone, nitrogen dioxide, polyaromatic hydrocarbons, inorganic particles, and much more. The concentration of ozone, for example, varies over the course of the day (Fig. 4). Chemical reactions leading to the ozonation of unsaturated fatty acids or the oxidation of unsaturated fatty acids by nitrogen dioxide have lifetimes of 10^—10^ s whereas the health effects are seen chnically at 50—60 years or 10^—10^ s of exposure. Few other toxicants exhibit this time scale in toxicity. Consequently, antioxidant prevention of lung disease caused by oxidant air pollutants cannot be expected to be evident after a few months or years. Long-term studies in humans are needed. In Fig. 5, a hypothetical air pollution episode extending over 4 days is shown using the single-day ozone profile from Bakersfield, California (Fig. 4). The profile shown for Bakersfield is a very simple diurnal ozone pattern. Localities downwind from large metropolitan areas can have additional ozone peaks before dawn or at night because of so-called dark reactions contributing to ozone formation. Nitrogen dioxide concentrations follow more or less the same pattern as those of ozone except that nitrogen dioxide concentrations are greater at night than the ozone concentrations. The reason for this difference is that nitrogen dioxide is a reactant and hence a driving force in the formation of ozone by photochemical reactions. In the absence of light, nitrogen dioxide is not depleted by the reactions forming ozone. In the summer, 3- to 4-day cycles of air pollution can occur across the USA and Europe with longer cycles of relatively clean air. These episodes of air pollution occur because of inversion layers or stagnant air over metropolitan air sheds. 0.10 0.09 0.08

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Injury Arbitrary Units

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Fig. 5. Hypothetical lung injury from an air pollution episode. The lower chart shows a hypothetical 4day exposure episode using the time course of ozone levels in Bakersfield, California (See Fig. 4). The upper chart represents the injury to the lung from the exposure. The stippled area represents the time course of chemical reactions that occur rapidly upon ozone or nitrogen dioxide exposure. The chemical reactions occur again with each ozone exposure. Reactive intermediates are formed each day during inhalation of polluted air. The health effects are not observable until about 18 h later. The "injury" curve is displaced from the ozone curve by about 18 h. When injury exceeds the ability to repair, repeated exposures will result in an accumulation of damage as shown by the dashed line. About 20 years of repeated exposure to ozone and nitrogen dioxide at the current standard are needed before conventional histopathologic changes can be observed.

In our schematic in Fig. 5, a 4-day period is shown with no accumulation of ozone or nitrogen dioxide from one day to the next. Under some meteorological conditions, ozone and nitrogen dioxide may accumulate by carry-over from prior days or from nitrogen dioxide and ozone formed upwind that drift over a metropolitan area. Nonetheless, an ozone and nitrogen dioxide maximum occurs at about noon. Most in vitro studies indicate that the velocity of the oxidation of unsaturated fatty acids in cell membranes at low concentrations of ozone or nitrogen dioxide are likely to occur on the human lung surface is nearly first-order [3]. The formation of products from either ozone or nitrogen dioxide would be expected to rise as the exposure increases during the course of the day. The products of ozone and nitrogen dioxide reactions are also likely to decline in a first-order rate as do most other reactive intermediates formed in cells. The rates of formation and removal of reactive intermediates will result in a bell-shaped curve of concentration of reactive products vs. time as indicated in Fig. 5 by the stippled area. These reactive products will decline over time on their own. Probably, the antioxidants, particularly vitamin C in the lung lining fluid, will destroy them. More reactive products are formed in the lung on inhalation with each successive day of air pollution. When more products are formed than can be removed, toxicity occurs. The toxic effect produced is shown in arbitrary units of injury on the upper graph of Fig. 5. Repair of injury, including elimination of oxidized


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cell constituents, probably also follows a general first-order process. In humans, about 18 h elapses between a single exposure to ozone or nitrogen dioxide and the maximum toxic effect in the lung. The time course of injury will follow the time course of ozone or nitrogen dioxide concentration in the inhaled air, but with some delay to account for absorption, the kinetics of reactions between ozone or nitrogen dioxide and cell components, and repair. We set the delay at 18 h to account for the observed time course of biomarkers of toxicity Thus, the injury curve will rise and fall in the same manner as the ozone or nitrogen dioxide concentration rises and falls during the course of the day, however, the injury will be displaced in time by about 18 h. Breath-by-breath injury probably occurs but is obscured by the complexity of the process. Exercise, vs. rest, while breathing polluted air clearly increases lung deposition of pollutants in humans and may enhance the toxic effects of the diurnal ozone and nitrogen dioxide cycles. Thus, exercise at peak air pollution levels is inadvisable. The model we have constructed for this review assumes that repair occurs throughout an exposure period. Toxic injury occurs because the rate of repair is slower than the rate of toxic injury If the rate of the repair process is slower than the rate of formation of the toxic injury, some toxicity will be evident at a time when the air pollution rises again on the next day In the model, the toxic injury to the lung gradually accumulates as is evident by the rising baseline of the toxic injury with the time curve (shown by the dashed line in Fig. 5). As the episodes of air pollution occur again and again over the years, some forms of toxic injury are not repaired causing permanent damage to the accumulate. Consequently, preventing human exposures to peak air pollution levels is a useful strategy for protection of the public health. Because oxidizing air pollutants produce inflammation in the lung, repeated bouts of inflammation could result from air pollution episodes. The alarming rise in the prevalence and severity of asthma give added importance to the hypothesis that repeated bouts of air pollution cause inflammation leading to the initiation or exacerbation of bronchitis and asthma [4]. Viral lower respiratory tract infections are risk factors for both asthma and bronchitis. The lungs of young people living in areas with high air pollution, such as Los Angeles, and dying violent deaths show histopathologic abnormalities at about age 20 years. This data, together with the data on the transient effects on lung function in normal and asthmatic subjects of ozone levels at or near the 0.12 ppm averaged over 1 h in the USA EPA National Ambient Air Quality Standard, prompted EPA to determine that the standard did not provide an adequate margin of safety. The ozone standard was revised to a lower level and the averaging time was chosen to eliminate exposures to short-time, but recurring, peaks of ozone. By changing the averaging time for ozone, EPA expects to reduce human exposures to ozone significantly. In short-term studies of the protective effects of antioxidants on ozone or nitrogen dioxide toxicity it has been difficult to demonstrate unequivocally that antioxidants are protective in humans. As an indirect measure of the susceptibility

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of human red cells to oxidative changes following fatty acid ozonide exposures mimicking ozone inhalation, vitamin E was protective at 300 to 600 lU/day in a small group of volunteers [5]. Even in cell membranes bearing the maximum vitamin E concentration, the vitamin E does not reduce the ozone or nitrogen dioxide reaction with fatty acids although it does terminate propagation of the chain reaction of peroxidation. That is, some, albeit reduced, injury is occurring in the protected individual. On the other hand, near life-time studies in vitamin E deficient and vitamin E supplemented mice show that lung injury to the point of mortality is some 15- to 20-fold greater in the vitamin E deficient mice than in the vitamin E supplemented mice [6]. For the establishment of the essentiality of nutrients, data from experimental animal studies have been used as the best available data. In a similar manner, experimental animal studies show some of the best available data on the effects of long-term ozone and nitrogen dioxide exposure and of vitamin C and E protection. Whereas vitamins E and C protect animals from ozone or nitrogen dioxide exposure effects, the rates and chemistry of the reaction of ozone and nitrogen dioxide with cellular constituents indicate that high levels of the antioxidants are needed to provide protection. Unfortunately, vitamin E and C supplementation at the levels likely to be effective for protection against damage from polluted air are not attainable by consumption of foods [7]. Thus, supplementation of adults with 1,000 lU of vitamin E and 1,000 mg vitamin C per day may be required before such protection is likely Even in the presence of high levels of antioxidants, both ozone and nitrogen dioxide will react with cellular constituents to some small, but perhaps significant, effect [4]. Short-term effects of air pollution on pulmonary function and protection by vitamins C and E are difficult to demonstrate because of variations in pulmonary function among individuals are large compared to the air pollutant effects. Clearly, the preferable solution to the ozone and nitrogen dioxide problem is to reduce exposure to these air pollutants. Controlled human studies have demonstrated that exposure to oxidant air pollution, and particularly to ozone, can lead to pain on inspiration, reversible pulmonary function decrements, hyperresponsiveness to bronchoconstrictor agents, airway inflammation, and exercise performance reduction. Although knowledge of the mechanisms responsible for the effects of air pollutants is incomplete, exercise appears to increase the risk of air pollutant-related consequences. 2

ATMOSPHERIC OZONE AND NITROGEN DIOXIDE

The principal oxidants in photochemical smog are ozone and nitrogen dioxide. The gases are potent respiratory tract oxidants and are of major public health concern. Typically, ozone is in higher concentration in ambient air than in nitrogen dioxide. Ozone is also a more powerful oxidant than is nitrogen dioxide. Incomplete combustion of fossil fuels by both stationary sources (e.g., electric power plants) and mobile sources (e.g., cars and trucks) provides the precursors


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of ozone and nitrogen dioxide formation in the atmosphere. Motor vehicles are the primary mobile sources of the precursors, and electric utilities followed by industries are the main stationary sources. In many cities around the world, automobile traffic gives rise to most of the oxidant air pollution. Ozone and nitrogen dioxide are formed in the atmosphere through a series of reactions that involve nitric oxide, volatile organic compounds, and sunlight. Meteorological conditions such as strong ultraviolet radiation intensity, high temperature, and stagnated air can increase the production of photochemical smog. Ambient ozone levels in urban areas peak in the early afternoon (Fig. 4), whereas ambient nitrogen dioxide levels usually are highest in the late afternoon and evening hours [8,9]. An additional peak of nitrogen dioxide can occur in midmorning in cities with outdoor nitrogen dioxide levels greater than 0.2 ppm. Ozone levels are highest in the late spring or in summer in urban areas. In contrast, the maximum levels of nitrogen dioxide in outdoor urban air occur in the fall and winter months (Fig. 6). The time of day and site of exercising in an urban area are important in order to minimize exposure to oxidant air pollutants. Ambient levels of ozone (Fig. 7) and nitrogen dioxide have been declining in the USA because of reductions in precursor emissions. Nevertheless, many urban areas in the USA do not meet the 1979 USA National Ambient Air Quality Standard for ozone of 0.12 ppm (averaged over 1 h), which is to be exceeded only once per year [8]. Reductions in the use of fossil fuels will be necessary to decrease further emissions of photochemical smog precursors. The USA National Ambient Air Quality Standard that was announced in July, 1997, establishes an 8-h standard of 0.08 ppm ozone in order to protect the public against exposures longer than 1 h. Photochemical smog is not a phenomenon restricted to urban areas in the USA and occurs in highly populated cities around the globe. In Mexico City, ozone levels are high and typically range from 0.15 to 0.25 ppm [10]. The USA National Ambient Air Quality Standard for nitrogen dioxide is an annual arithmetic mean of 0.053 ppm. In much of the USA, annual average out0.2

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Fig. 6. Monthly 50th, 90th, and 98th percentiles of 1-h nitrogen dioxide concentrations at Long Beach, Cahfornia, 1986 to 1989. The episodic and cyclic nature of NO2 concentrations occurs in many other locations as well. Taken from [9].


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door levels of nitrogen dioxide range from 0.015 to 0.035 ppm. Nonetheless, average hourly concentrations of nitrogen dioxide range from 0.02 to 0.20 ppm in moderately polluted urban areas and from 0.2 to 0.5 ppm in those that are heavily polluted [9]. Outdoor levels of ozone are almost always higher than the indoor levels because outdoor air is by far the primary source of indoor ozone. On the other hand, nitrogen dioxide is produced indoors, as well as outdoors, so that indoor levels of nitrogen dioxide can exceed those outdoors when gas appliances are used. Nitrogen dioxide levels can reach 0.6 ppm during cooking on a gas range [11]. 3

DOSIMETRY MODELING OF OZONE AND NITROGEN DIOXIDE

Dosimetry is the process of measuring the amount of a substance that is absorbed at a target site. Absorption of air pollutants from the upper respiratory tract (nasopharyngeal region) and lower respiratory tract (tracheobronchial and pulmonary regions), as well as from the total respiratory tract, have been measured in experimental studies. Nevertheless, it has not been possible to determine absorption from smaller regions of the lung directly Therefore, mathematical dosimetry models are especially useful for predicting the dose to particular areas of the lung.


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3.1 Ozone Because ozone is nearly insoluble in water, the gas penetrates into the pulmonary (gas-exchange) region of the lung even during restful breathing. Miller et al. [1] and Overton et al. [2] modeled the effects of exercise on the regional uptake of ozone in the lung. Simulations with human morphometric airway data predict little effect of exercise on tracheobronchial uptake but a marked effect on pulmonary uptake [1]. Dosimetry models predict that with exercise the dose of ozone delivered to the tracheobronchial region is slightly increased while the ozone dose to the pulmonary region is markedly increased. The model of Miller et al. [1] predicts that heavy exercise will cause the total mass uptake of ozone to increase 10-fold. According to Miller [12], human clinical studies consistently have shown that exposures with exercise to levels below 0.2 ppm ozone affect minute ventilation only slightly because a decrease in tidal volume occurs along with an increase in breathing frequency Minute ventilation is the volume of gas exchanged in the lung per min. Ozone is transported in the upper respiratory tract primarily by convection, or bulk movement, whereas the gas is transported in the pulmonary region of the lung mainly by diffusion. During heavy exercise, however, convection can contribute to gas transport in the pulmonary region [12]. Surprisingly, Gerrity et al. [13] found a small (approximately 10%) decrease in uptake of ozone in the upper respiratory tract during nasal breathing by healthy young male volunteers compared with oral or oronasal breathing. Total respiratory uptake of ozone was reported by Wiester et al. [14] to be slightly greater during oral rather than nasal breathing, however. This latter group of investigators also determined that the total respiratory uptake of ozone was about 75% in adult males during breathing at rest. The percentage of uptake varied substantially among subjects whereas intrasubject variability was moderate. In another human study, Kabel et al. [15] determined that 80% of the inhaled ozone was absorbed in the upper airways during nasal breathing compared with 50% during oral breathing. The point at which 90% of the inhaled ozone was absorbed was more distal with oral breathing. This finding implies that during exercise a switch from nasal to oral breathing can increase the risk of injury to the lower respiratory tract because of a greater dose. Delivered dose of ozone, as determined mainly by minute ventilation, is suggested to account for part of the intersubject variability of pulmonary function response to ozone [16]. Subjects were exposed to 0.4 ppm ozone for 1 h during moderate intensity exercise and uptake efficiencies in the upper and lower respiratory tracts were measured. A significant relationship was found between either initial minute ventilation or average minute ventilation and forced expiratory volume in 1 s (FEVi). FEVi is the volume of gas that can be exhaled in 1 s following a full inspiration. In other words, ozone decreased the flow from the lung at maximum effort. Probably, this is due to a smaller effective airway diameter.

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3.2 Nitrogen dioxide The dosimetry models for nitrogen dioxide evolved from the models developed for ozone. The uptake of nitrogen dioxide and ozone in the respiratory tract are similar because both of the gases are highly reactive and poorly soluble in water. Like ozone, nitrogen dioxide is absorbed throughout the respiratory tract and maximally in the centriacinar region (the junction between the conduction airways and the pulmonary region). Because nitrogen dioxide is more water soluble than ozone, deposition of nitrogen dioxide in the airways is predicted to be somewhat greater than that of ozone. Total respiratory tract uptake of nitrogen dioxide has been measured experimentally in asthmatic and nonasthmatic humans. Healthy subjects exposed to 0.3—7.2 ppm nitrogen dioxide in a nitric oxide/nitrogen dioxide mixture absorbed 81—90% of the nitrogen dioxide while at rest [17]. Average uptake of nitrogen dioxide by adult asthmatic subjects exposed to 0.3 ppm nitrogen dioxide while at rest was 72% [18]. During exercise, uptake by both groups was similar, 81—90% for the healthy subjects and 88% for the asthmatic subjects. Uptake of nitrogen dioxide by the upper and lower respiratory tract was determined in subjects exposed to 2 ppm nitrogen dioxide [19]. Nasal uptake, which was 44% at rest, diminished to 15% during heavy exercise. The total respiratory uptake was calculated to be 80% at rest and 91% during heavy exercise. The increased ventilation during exercise leads to a decreased percentage of nitrogen dioxide absorbed in the upper respiratory tract. The results indicate both a greatly increased uptake of nitrogen dioxide by the lower respiratory tract during exercise, as well as an increase in total respiratory tract uptake. Despite the greater uptake, nitrogen dioxide is less toxic than ozone because it reacts more slowly with cell constituents than does ozone. 4

LABORATORY HUMAN EXPOSURE WITH EXERCISE STUDIES

In controlled human studies of air pollutants, human volunteers are exposed to a fixed concentration of a gas under carefully controlled conditions. Methods commonly used to assess effects of air pollutants in human exposure studies include pulmonary function tests, bronchoalveolar lavage, and airway inhalation challenge tests. Pulmonary function tests involve noninvasive methods such as spirometric measures of lung volumes, measures of lung resistance, and measures of breathing patterns. Dynamic spirometry tests such as FEVi and plethysmographic tests such as specific airway resistance measurements provide information about large airway functions primarily The coefficients of variation for these two specific tests are fairly low, about 3% for FEVi and about 10—20% for specific airway resistance measurements [11]. Because lung volume affects airway resistance, specific airway resistance measurements are calculated from resistance times volume. Bronchoalveolar lavage is performed using a fiberoptic bronchoscope that is


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inserted into an airway and secured in a subsegmental bronchus [20]. Then, sterile bufTered sahne is used to remove cells and fluids lining the respiratory tract distal to the bronchoscope. Valuable information about the types of immune system cells in the segment, as well as the amount of protein and various other molecules, can be obtained using this procedure. Airway inhalation challenge tests are used to examine the degree of responsiveness of the airways [21]. Responses include cough, bronchoconstriction, and increased mucus production. Commonly, the challenge is an inhaled agent such as methacholine, histamine, or carbachol that causes constriction of airways. Following each dose of a chemical provocation, airway resistance measurements or spirometry are performed. A target effect level, such as a 100% increase in airway resistance, is calculated from the concentration-effect curve. 4.1 Pulmonary function effects of acute ozone exposure 4.1.1 Concentration -response relationship Studies conducted in the 1970s [22,23] demonstrated that, at a given ozone concentration, minute ventilation elevated by exercise exacerbated the pulmonary response to ozone and reduced the threshold for the response, as well. In these early studies, very low levels of exercise were used. Later studies examined the effects of both multiple exposure concentrations and multiple intensities of either intermittent or continuous exercise [24,25]. The studies demonstrated that significant decrements in pulmonary responses result from exposure to 0.3 ppm ozone during moderate exercise. In addition, multiple regression analysis revealed that ozone concentration was primarily responsible for the pulmonary function variance [24]. A review of published data from pulmonary function studies that exposed normal subjects to ozone for 2 h during intermittent exercise confirmed that ozone concentration has a greater effect on pulmonary function than minute ventilation does[26]. In addition, the relationship between ozone concentration and alterations in pulmonary function was found to be nonlinear, and a threshold for pulmonary function effects caused by ozone could not be defined. More recently, a number of investigators have shown that significant reductions in pulmonary function occur upon exposure of healthy subjects to ozone concentrations ^ 0.2 ppm during heavy exercise [27—30]. In order to cause a significant decline in pulmonary function in these studies, a minimum level of 0.1 to 0.18 ppm ozone for 1—3 h during moderate to heavy intermittent or continuous exercise was necessary. In this chapter, the categories of ventilation levels will be described as follows: light exercise (^15 1/min), moderate exercise (20—40 1/min), and heavy exercise ( ^ 5 5 1/min). In addition, the word significant will be used to describe a statistical significance at p < 0.05. McKittrick and Adams [31] studied the effect of the exercise paradigm (inter-

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mittent or continuous exercise) on pulmonary function response when the ozone dose was held constant. Their results suggest that the exercise paradigm during exposures of 2 h or less has no effect on pulmonary function responses when the effective dose of ozone is equivalent at a given ozone concentration. The effective dose is a product of ozone concentration, ventilation, and exposure duration. The dose-response relationship found by McKittrick and Adams [31] is interesting from a mechanistic view. By dimensional analysis. Ozone effective dose (g) = Ozone concentration (g/1) x Ventilation (1/min) x Exposure duration (min) If the reaction rate of ozone with lung constituents was the rate-limiting factor determining the toxicity in these experiments, the dose would not be the amount of ozone alone but a more complex function of the ozone concentration at the target site. That is, the transfer of ozone to the underlying tissues follows Henry's law and the kinetics of chemical reaction with mass transfer [1]. The ozone dose would depend on the location in the lung where ozone absorption is maximal. Two factors may explain the differences found for exercise effects on ozone toxicity: 1) the endpoints used may be insensitive to ozone dose; and 2) the ozone reaction may be essentially instantaneous compared to the duration of subsequent biological measurements. It is our view that the latter is more likely. One should keep this possibility in mind when comparing chnical outcomes with the theory of how ozone produces its pulmonary toxicity. The matter is not trivial because the National Ambient Air Quality Standard for ozone has an averaging time-component. Generally, the component is chosen to provide the greatest protection of the population. The current averaging time of 8 h is aimed at reducing short-term exposures. Meteorology and air chemistry also influence the averaging time chosen. Nonetheless, further studies are needed to clarify the temporal relationships of ozone toxicity, especially during exercise. Limited data suggests that healthy persons over 50 years of age show less of a pulmonary function response to ozone exposure than young adults do, especially those less than 25 years of age [32]. Whether a difference exists in sensitivity to ozone between men and women is yet to be determined. A newly developed analyzer has been described that is expected to allow more accurate measurement of low doses of ozone (< 0.2 ppm) inhaled by human subjects during exercise [33]. The instrument can monitor ozone levels dynamically at the airway opening (nose or mouth). Such an instrument will be useful to confirm any interindividual differences in ozone sensitivity 4,1,2 Reduction of exercise performance As early as the 1960s, Wayne et al. [34] concluded from an epidemiologic study of high school cross-country runners that exercise performance is compromised by inhalation of oxidant air pollutants. Since that time, a number of controlled human studies have been conducted to examine the effects on exercise performance of acute (single 1- to 3-h) exposure to air pollutants and especially to


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ozone, the major oxidant in urban air pollution. Even though controlled human studies generally have a small sample size, they are valuable because they quantify concentration-response data. The endpoints of controlled human studies of the effects of short-term exposure to ozone on exercise performance include maximal oxygen uptake and endurance (i.e., the ability to finish strenuous exercise tests). Of the three studies in the late 1970s that evaluated the effects of acute ozone exposure on maximal oxygen uptake [35—37], only one reported a significant reduction in maximal oxygen uptake [35]. More recently, controlled human studies of the effects of acute exposure to ozone on highly trained athletes [30] and trained nonathletes [38] have found significant reductions in maximal oxygen uptake as well as in other maximal exercise endpoints. The decreases in pulmonary function and symptoms of respiratory discomfort that occurred during the studies may have been responsible for the reductions in maximal oxygen uptake [38]. Symptoms of ozone exposure include coughing, pain on deep inspiration, shortness of breath, throat irritation, and wheezing. Several controlled human studies of the effect of acute ozone exposures on endurance have shown that the ability of well-trained athletes to complete an exercise regimen is diminished [27—29,39]. In addition, subjective symptoms of respiratory discomfort are elevated in the subjects by the protocols. The mechanisms responsible for the effects of ozone exposure on maximal oxygen uptake and endurance are unclear. Although symptoms of respiratory discomfort have been suggested to be the cause, evidence for such a conclusion is lacking. A reflex inhibition of oc-motor neural activity during inspiration caused by ozone stimulation of neural receptors in the airways has been proposed [32]. Ozone reaction products could also reduce the airway caliber or thicken (via inflammation) the distance for diffusion of oxygen to the blood. 4.1.3 Recovery of pulmonary function following acute ozone exposure Studies of repeated daily short-term ozone exposures to ^ 0.2 ppm ozone have shown that ozone exposure during moderate-heavy exercise can cause enhanced pulmonary responses on the second day [32]. Within 3 to 5 days, an attenuation of spirometric responses is typically observed irrespective of the dose rate of ozone exposure. The attenuated response can last for up to two weeks. These observations support the accumulated-damage hypothesis given in the introduction. The relationship of pulmonary function responses and biomarkers of response in blood were measured in healthy subjects exposed to a pyramid-shaped profile of ambient ozone concentrations over three consecutive days (130 min/day) during light intermittent exercise [40]. The concentrations ranged from 0.25 ppm to 0.45 ppm ozone. Progressive decrements in forced vital capacity and FEVi began to diminish on the third day of exposure. These changes were accompanied by a

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P-Tyrosine may be a marker of direct ozone oxidation. 4.2 Pulmonary inflammation following acute ozone exposure An inflammatory response is of concern because of the presence of cells and factors in inflammatory exudate that can damage tissue. Neutrophils and macrophages can secrete substances capable of injuring lung tissue and compromising host defense mechanisms. The first evidence that ozone causes pulmonary inflam-

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mation was provided by Seltzer et al. [55] who performed bronchoalveolar lavage on subjects following exposure to 0.4 or 0.6 ppm ozone for 2 h during exercise. Later, a study by Koren et al. [46] confirmed the findings that exposure of humans to moderate levels of ozone during intermittent exercise results in enhanced levels of neutrophils and proinflammatory substances including prostaglandin E2 in bronchoalveolar lavage fluid. The presence of neutrophils in the lung is recognized as a marker of acute inflammation. Studies by Devlin et al. [56] indicate that inflammatory responses can occur in humans exposed to low levels of ozone (0.08 ppm) for 6.6 h while exercising moderately. The substantial range in response observed may indicate that some individuals are more susceptible to ozone damage than others are. Increases in inflammatory mediators in 18-h bronchoalveolar lavage fluid did not correlate with decrements in pulmonary function. Balmes et al. [57], too, found no correlation between the magnitude of decrements in pulmonary function and levels of inflammation in 18-h bronchoalveolar lavage fluid when healthy subjects were exposed to 0.2 ppm ozone for 4 h during exercise. The lack of a correlation between pulmonary function decrements and inflammatory response may indicate that different mechanisms are responsible for the two responses. On the other hand, Schelegle et al. [58] showed that the time course of ozone-induced neutrophilia (increase in neutrophils) and decrements in FEVi differs. Furthermore, the investigators have demonstrated that the time course of ozone-induced neutrophilia in bronchoalveolar lavage of subjects undergoing heavy exercise differs in the proximal and distal respiratory tract. Comparing measurements taken at 1, 6, and 24 h, FEVi decreased only at 1 h following ozone exposure. In contrast, the greatest increase in neutrophils occurred at 6 h in the in bronchoalveolar lavage of the proximal airways and at 24 h in bronchoalveolar lavage of the distal airways and alveolar region. Koren et al. [45] studied the time course of the inflammatory response following exposure of healthy subjects to 0.4 ppm ozone for 2 h during heavy exercise. Cellular and biochemical changes were detectable in bronchoalveolar lavage performed 1 h postexposure indicating that inflammatory response following ozone exposure develops quite rapidly Levels of neutrophils, interleukin-6, and prostaglandin E2 were higher at 1 h following ozone exposure than at 18 h. Other markers of inflammation including fibronectin, which has been related with fibrotic processes, and plasminogen activator, which in turn have been related with fibrinolytic processes, these were measured at higher levels at 18 h then at 1 h. These later changes could play a role in the long-term development of pulmonary fibrosis. Levels of tissue factor and protein were also elevated following ozone exposure, but the levels were similar at both time points. Tissue factor plays a role in fibrin formation, and an increase in protein in bronchoalveolar lavage indicates increased airway permeability. Similar results were reported by the same laboratories for another study in which subjects were exposed to 0.4 ppm ozone for 2 h while undergoing heavy exercise [59]. Therefore, maximal levels of the different inflammatory mediators in bronchoalveolar lavage fluid appear to vary tempo-


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rally following ozone exposure. Evidence of inflammation in airways 18 h after exposure of healthy athletes to 0.2 ppm ozone for 4 h during moderate exercise has also been found [60]. Significant increases in lactate dehydrogenase and the total number of cells were observed in lavage of mainstem bronchi, which was performed rather than bronchoalveolar lavage following the ozone exposure. Bronchoalveolar lavage is primarily a measure of conditions in the distal lung. Bronchial biopsies revealed that ozone exposure elevated the numbers of neutrophils present. Concern exists that there may be subpopulations of individuals who are particularly susceptible to adverse health effects from ozone. Ozone may induce a greater inflammatory response in asthmatic subjects compared with normal subjects. Following exposure to 0.12 ppm or 0.24 ppm ozone for 90 min during intermittent moderate exercise, leukocytes and epithelial cells were significantly increased in nasal lavage of asthmatic subjects but not nonasthmatic subjects [61]. Although there were no significant differences in pulmonary function responses between asthmatic and normal subjects exposed to 0.2 ppm ozone for 4 h during exercise, the asthmatic subjects showed significantly greater increases in a number of inflammatory endpoints in bronchoalveolar lavage than the normal subjects did[62]. Hence, asthmatics may be at increased risk of pulmonary injury from ozone exposure. By measuring clearance of radiolabeled technetium diethylene triamine pentacetic acid (^^"Tc-DTPA), Kehrl et al. [63] demonstrated increased epithelial cell permeability in subjects exposed to 0.4 ppm ozone while exercising heavily. Inflammatory cells and their products are thought to be involved in permeability changes in airways after ozone exposure [64]. Whether repeated periods in humans of ozone-induced acute inflammation progress into chronic pulmonary disease is unknown. Like pulmonary responses, attenuation of airway inflammation in humans seems to take place with repeated daily exposure to ozone [32]. Nevertheless, the extent of airway inflammation attenuation may be less complete and more gradual than the recovery of normal pulmonary responses. Some indicators of inflammation such as lactate dehydrogenase and elastase appear not to attenuate during repeated ozone exposure. Animal studies by Tepper et al. [65] showed that whereas decrements in pulmonary function attenuate with repeated exposure to ozone, inflammation and damage to epithelial cells advance and some biochemical markers remain elevated. 4.3 Airway responsiveness to ozone exposure In several previously cited studies, healthy subjects exposed to 0.12 ppm ozone for short periods during exercise demonstrated increased airway responsiveness to the bronchoconstrictors methachoHne or histamine [30,66]. Although acute increases in airway responsiveness are well documented following exposure to ozone, whether ozone causes prolonged increases in airway responsiveness, induces asthma, or increases susceptibility to asthma, is uncertain [32].

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Methacholine responsiveness in healthy subjects exposed to 0.2 ppm ozone for 4 h with moderate exercise was not associated with a decreases in FEVi [67]. Therefore, in the absence of respiratory symptoms, an individual may not be aware of even large pulmonary function decrements and may remain in an injurious environment unknowingly. Both healthy and mild-asthmatic subjects exposed to 0.4 ppm ozone for 2 h with intermittent exercise responded with an increase in the maximal degree of airway narrowing to methacholine 12 h after exposure [68]. The authors suggested that this temporal relationship between exposure and bronchoconstriction may explain the increase in hospital admissions for respiratory diseases 1 day after an ozone episode. The mechanism of ozone-induced increases in airway responsiveness has not been elucidated. Nevertheless, soluble mediators of inflammation such as cytokines and arachidonic acid metabolites may play a role [32]. That is, both the initial peroxidation products and later inflammatory agents combine to enhance the airway permeability. The increased permeability of the airways following ozone exposure would increase the diffusion of vasoactive agonists to the site of action. 4.4 Pulmonary function effects of prolonged ozone exposure A number of controlled human studies in which subjects were exposed to 0.08—0.12 ppm ozone for 6.6 h with moderate exercise found that FEVi was decreased compared to exposure to clean air [66,69,70]. Similar to the findings with acute ozone exposures, interindividual variation in response to prolonged exposures to ozone was considerable. Studies with prolonged ozone exposure protocols ( ^ 4 h) which result in modest changes in pulmonary function show evidence of a response plateau [66,71]. Responsiveness of subjects to exposure to varying levels of ozone suggests that the response plateau depends on ozone concentration, dose rate, and cumulative dose [71]. Based on results of this latter study, as well as those from numerous others, ozone concentration during prolonged exposures has a greater effect on pulmonary function than does exposure duration or volume of air inhaled [32]. In a study that examined pulmonary responses of asthmatic and nonasthmatic subjects to ozone, subjects were exposed to 0.16 ppm ozone for 7.6 h with intermittent light exercise [72]. Whereas decrements in FEVi were significantly greater for asthmatics than for nonasthmatics, no difference was observed for decrements in forced vital capacity between the two groups. The investigators concluded that bronchoconstriction was responsible for part of the ozone-induced pulmonary decrements. Attenuation of pulmonary responses with repeated prolonged exposure to 0.12 ppm ozone during moderate exercise were examined by Folinsbee et al. [69]. Pulmonary function decrements were observed on the first day of exposure. The decrements were lessened on the second day and were absent compared with the control values on the last 3 days of ozone exposure.


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4.5 Pulmonary function effects of mixtures of ozone and other air pollutants Although humans are exposed to mixtures of pollutants in ambient air, most controlled human studies have examined the effects of single air pollutants. In general, studies of mixtures of air pollutants have found no significant differences between exposure to ozone alone and exposure to the mixtures. Nevertheless, a few human studies of pulmonary function responses to mixtures of ozone and sulfur dioxide have demonstrated greater effects of the mixture than of either pollutant alone. In their study of allergic asthmatic adolescents, Koenig et al. [73] found that when the subjects were exposed sequentially to ozone and sulfur dioxide during intermittent exercise, significant changes in FEVi and total respiratory resistance occurred. No significant changes in pulmonary function occurred with sequential exposures to air and sulfur dioxide or to ozone and ozone. The response of asthmatic adolescents to sulfur dioxide may, therefore, be potentiated by exposure to ozone. Linn et al. [74] studied the effects of ozone and aerosols of sulfuric acid on the pulmonary function of normal, atopic, and asthmatic subjects undergoing exercise. Both the exposure to ozone alone and ozone mixed with sulfuric acid aerosol caused significant decrements in FEVi and bronchial reactivity to methacholine. The investigators concluded that the pulmonary effects in their study population were caused predominantly by ozone. Nonetheless, because a few of the subjects demonstrated significantly greater effects from exposure to the mixture compared with ozone alone, a subpopulation of individuals sensitive to ozone plus sulfuric acid may exist. Aris et al. [60] studied the exposure of human subjects during exercise to nitric acid gas followed by ozone (an exposure pattern typical in some coastal California regions). Nitric acid, the main acid air pollutant in the western USA, is capable of damaging the respiratory tract. Smaller reductions in pulmonary function were observed with air and ozone than with nitric acid and ozone. In other words, the nitric acid seemed to antagonize the effect of ozone. In a later study of simultaneous exposure of exercising human subjects to ozone and nitric acid gas, no significant differences in pulmonary function or cellular or biochemical components in bronchoalveolar lavage, proximal airway lavage, or bronchial biopsy specimens were observed in comparison with exposure to ozone plus air [75]. Low levels (0.05) in the young heart. Further, exercise increased heart MDA content in the old but not young rat. Thus, despite age adaptations of the antioxidant systems, aged hearts seem to produce more ROS and are more vulnerable to oxidative damage than young hearts at similar levels of metabohc stress. 6.0 SUMMARY 1. The heart is a highly aerobic organ characterized by a high density of mitochondria, a high rate of oxygen uptake, and a large energy turnover rate even at the resting state. These morphological and physiological properties favor a constant production of ROS as a byproduct of metabolism. During and shortly after maximal sustained myocardial work, ROS generation in the

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heart is probably augmented due to an increased electron leakage from the respirator chain, activated xanthine oxidatase and enhanced catecholamine secretion. However, the heart possesses only moderate levels of antioxidant enzymes compared to other aerobic tissues, therefore, its ability to remove ROS is rather limited. The nonenzymatic antioxidants, i.e., antioxidant vitamins and GSH, may thus, be more important in the heart. The delicate balance between production and removal of ROS makes the heart vulnerable to oxidative stress. 2. There is both direct and indirect evidence that high intensity physical exercise increases ROS production in the heart. Intrinsic myocardial antioxidant systems seem to be capable of metabolizing most of these deleterious species, therefore, acute tissue oxidative damage due to exercise probably does not occur to normal healthy subjects. However, antioxidant reserves of vitamin E and GSH in the heart have been shown to deteriorate due to vigorous acute and chronic exercise. Furthermore, there is preliminary evidence that ROS may target specific cellular and subcellular components susceptible to oxidative injury, such as mitochondria. These areas require further investigations. 3. A number of physiological, pathological and nutritional factors are known to increase myocardial susceptibility to exercise-induced oxidative stress by either increasing ROS production, or weakening antioxidant defense, or both. Research concerning the interactions of antioxidant deficiency/supplementation, drug metabolism and aging with exercise can greatly enhance our understanding of the etiologic mechanism of oxidative stress in this vital organ. 7

PERSPECTIVES

Due to the importance of the heart, vast resources have been put into cardiovascular research over the years. Yet, our understanding on free radical-antioxidant balance during exercise in the heart is still quite limited. The following areas appear to need particular attention. 1. Although there is some evidence that ROS production is increase in the heart during exercise, data in this respect are sparse and sometime equivocal. We need to gain definitive information as to whether heavy exercise generates sufficient amount of ROS in the heart to overwhelm the intrinsic antioxidant defense system, and the cellular sources of the ROS. 2. As a postmitotic organ, the heart has demonstrated limited adaptive response to acute and chronic exercise. However, recent research shows that gene regulation in the myocardium is altered due to a variety of physiological, pathological and nutritional conditions. Whether exercise could modulate gene expression of antioxidant enzymes and other proteins related to antioxidant function is a research field that is still wide-open . 3. Increasing evidence points to an intimate relationship between the various cardiovascular diseases and oxidative injury. Exercise is a well-known preventive measure of cardiovascular diseases, but also produces some risk of oxida-

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tive stress. This paradox requires further clarification using both epidemiological and experimental approaches. We need to consider the pros and cons of the various exercise modes, intensity and duration for different population to maximize the benefit while reducing the risks of exercise. This challenge has provided unlimited opportunity for future exercise physiologists and biochemists. 8

ACKNOWLEDGMENT

The author thanks the American Heart Association (AHA) National Center, AHA Illinois and Wisconsin Affiliates for supporting the research presented in the current chapter. 9

ABBREVIATIONS

ADM: ESQ: CS: DCFH: EPR: ETC: GCS: GGT: GPX: GR: GSH: GSSG: HX: I-R: MDA: PMN: RCI: ROS: SDA: SOD: XO:

adriamycin buthionine sulfoximine citrate synthase dichlorofluorescein electron paramagnetic resonance electron transport chain y-glutamylcysteine synthetase y-glutamyltranspeptidase glutathione peroxidase glutathione reductase glutathione glutathione disulfide hypoxanthine ischemia-reperfusion malondi aldehyde polymorphonuclear neutrophil respiratory control index reactive oxygen species semidehydroascorbate superoxide dismutase xanthine oxidase

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5. Veerkamp JH. In: Busch FM, Jennekens GI, Scholte HR (eds) Mitochondria and Muscular Diseases. The Netherlands: Beetsterzwaag, 1981;29—50. 6. Asayama K, Dobashi K, Hayashibe H, Kato K. J Nutr Sci Vitaminol 1989;35:407-418. 7. Miquel J, Fleming J. In: Johnson J,Walford R, Harman D, Miquel J (eds) Biology of Aging. New York: Alan R. Liss, Inc., 1986;51-76. 8. Cadenas E, Boveris A, Ragan CI, Stoppani AOM. Arch Biochem Biophys 1977;180:248-257. 9. Boveris A. In: Reich M, Coburn R, Lahiri S, Chance B (eds) Tissue hypoxia and ischemia. New York: Plenum, 1977;67-82. 10. Loschen G, Flohe L, Chance B. FEBS Lett 1971;18:261-264. 11. Chance B, Sies H, Boveris A. Physiol Rev 1979;59:527-605. 12. Master C, Holmes R. Physiol Rev 1977;57:816-882. 13. Godin DV,Wohaieb SA. Free Radic Biol Med 1988;5:165-176. 14. Van Bilsen M, Van der Vusse GJ, Coumans WA, De Groot MJM et al. Am J Physiol 1989; 257:H47-H54. 15. Ji LL, Fu RG, Mitchell EW,WaldropTG et al. Can J Physiol Pharmacol 1993;71:811-817. 16. Simpson PJ, Lucchesi BR. J Lab Clin Med 1987;110:13-30. 17. Downey JM. Ann Rev Physiol 1990;52:487-504. 18. Bindoh A, Cavallini A, Rigobello MP, Coassin M et al. Free Radic Biol Med 1988;4:163-167. 19. Hearse DJ, Manning AS, Downey JM, Yellon DM Acta Physiol Scand 1986;548:65-78. 20. HellstenY Acta Physiol Scand 1994;621:1-73. 21. Sahlin K, Ekberg K, Cizinsky S. Acta Physiol Scand 1001;142:273-81. 22. Brooks GA, FaheyTD. Exercise Physiology London: McMillan, 1985;290-291. 23. Freedman AM, Cassidy MM,Weglicki WB. Magn Reson 1991;4:185-189. 24. Pincemail J, Camus G, Roesgen A, Dreezen E, Bertrand Y, Lismonde M, Deby-Dupont G, Deby C. Eur J Appl Physiol Occ Physiol 1990;61:319-322. 25. Pyne DB. Sports Med 1994;17:245-58. 26. Petrone WF, English DK,Wong K, McCord JM. Proc Natl Acad Sci USA 1980;77:1159-1163. 27. Ji LL, Stratman FW, Lardy HA. J Am Coll Nutr 1992;11:79-86. 28. SchaibleT, Scheuer J. Progr Cardiovas Dis 1985;27:297-324. 29. Dhaliwal H, Kirshenbaum LA, Randhawa AK, Singal AK. Am J Physiol 1991;261:H632H638. 30. Leeuwenburgh C, Leichtweis S, Fiebig R, Hollander J, Gore M, Ji LL. Molec Cell Biochem 1996;156:17-24. 31. Cowan DB,Weisel RD,Winiams WG, Mickle DAG. J Molec Cell Cardiol 1992;24:423-433. 32. Lai CC, Peng M, Huang L, Huang WH, ChiuTH. J Molec Cell Cardiol 1996;28:1157-1163. 33. Yoshida T, Watanabe M, Engelman DT, Engelman RM, Schley JA, Maulik N, HoYS, Oberley TD, Das DK. J Molec Cell Cardiol 1996;28:1759-1767. 34. YoshidaX Maulik N, Engelman RM, HoYS, Magnenat JL, Rousou JA, Flack JE 3rd, Deaton D, Das DK. Circulation 1997;96(Suppl9):II-II21620. 35. Gohil K, Packer L, de Lumen B, Brooks GA et al. J Appl Physiol 1986;60:1986-1991. 36. Packer L. Am J Clin Nutr 1991;53:1050S-1055S. 37. Diplock AT Am J Clin Nutr 1991;53:189S-193S. 38. Sakanashi T, Sako S, Nozuhara A, Adachi K et al. Biochem Biophys Res Comm 1991 ;181: 145-150. 39. Diplock AT Free Radic Res 1997;26:565-583. 40. Rapola JM, Virtamo J, Ripatti S, Huttunen JK, Albanes D, Taylor PR, Heinonen OP. Lancet 1997;349:1715-1720. 41. Diaz MN, Frei B, Vita JA, Keaney JF Jr. N Engl J Med 1997;337:408-416. 42. Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine. Oxford, UK: Clarendon Press, 1985,73-75,104-106. 43. Ji LL, Fu RG, Mitchell EW J Appl Physiol 1992;73:1854-1859. 44. Ferrarri R, Ceconi C, Curello S, Cargnoni A et al. Am J Med 1991;91:95S-105S.

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92. Goldfarb AH. Med Sci Sports Exer 1993;25:232-236. 93. Scholz RW, Minicucci LA, Reddy CC. Biochem Molec Biol Int 1997;42:997-1006. 94. Diplock AT. Free Radic Res 1997;26:565-583. 95. Diaz MN, Frei B,Vita JA, Keaney JF Jr. N Engl J Med 1997;337:408-416. 96. Somani SM, Gupta SK, Frank S, Corder CN. Drug Develep Res 1990;20:251-275. 97. Arcamone F. Doxorubicin anticancer antibiotics. New York: Academic Press, 1981. 98. Olson RO, Mushlin PS. FASEB J 1990;4:3076-3086. 99. Davies KJA, Doroshow JH, Hochstein P FEBS Lett 1983;153:227-230. 100. Davies KJA, Doroshow JH. J Biol Chem 1986;261:3060-3067. 101. Langton D, Jover B, McGrath BP, Ludbrook J Cardiovasc Res 1990;24:959-968. 102. Nohl H. In: Johnson Jr. JE, Walford R, Harman D, Miquel J (eds) Free radicals, aging and degenerative diseases. New York: Alan R. Liss, Inc. 1986;77—98. 103. Hansford RG. Biochim Biophys Acta 1983;726:41-80. 104. Nohl H, Hegner D, Summer KH. Mech Aging Dev 1979;11:145-151. 105. Lakatta EG, Yin FC. Am J Physiol 1982;242:H927-H941.

('2000 Elsevier Science B.V. All rights reserved. Handbook of Oxidants and Antioxidants in Exercise. C.K. Sen, L. Packer and O. Hanninen, editors.

7 1 '2 ' ^-^

Part IX • Chapter 26

Influence of exercise-induced oxidative stress on the central nervous system Satu M. Somani and Kazim Husain Department of Pharmacology, Southern Illinois University School of Medicine, P.O. Box 19230, Springfield, IL62794-1222, USA. Tel.: +1-217-785-2196. Fax: +1-217-524-0145. E-mail: SSomani(a) wpsmtp. siumed. edu

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7 8 9 10

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INTRODUCTION 1.1 Oxygen and central nervous system 1.2 Reactive oxygen species in the central nervous system 1.3 Lipid peroxidation in the central nervous system 1.4 Antioxidant system and central nervous system OXYGEN-INDUCED CNS TOXICITY 2.1 Antioxidant-system/lipid peroxidations 2.2 Treatments EXERCISE-INDUCED OXIDATIVE STRESS 3.1 Acute exercise and central nervous system 3.2 Exercise training and central nervous system EXERCISE AND DRUG INTERACTION ON CNS CENTRAL NERVOUS SYSTEM DISEASES: REACTIVE OXYGEN SPECIES/ANTIOXIDANT SYSTEM 5.1 Alzheimer's disease 5.2 Parkinson's disease 5.3 Amyotrophic lateral sclerosis 5.4 Down syndrome 5.5 Multiple system atrophy 5.6 Huntington disease 5.7 Cerebrovascular injury STRATEGIES TO IMPROVE CNS FUNCTIONS 6.1 Antioxidant-supplementation 6.1.1 Vitamins 6.1.2 Glutathione 6.1.3 Antioxidant-enzymes 6.1.4 Other free radical scavengers SUMMARY PERSPECTIVES ABBREVIATIONS REFERENCES

INTRODUCTION

1.1 Oxygen and the central nervous system Oxygen is essential to most higher organisms for their survival, however, concentrations of oxygen greater than those present in normal air have been proven

714


harmful. The oxidative damage that results from high pressure oxygen is frequently experienced in professional diving, closed-circuit military underwater activity, hyperbaric oxygen (HBO) therapy for peripheral vascular ischemia, carbon monoxide (CO) poisoning, and severe localized infections [1]. Breathing highly pressurized oxygen can cause central nervous system (CNS) toxicity in humans with symptoms including nausea, paresthesia, tinnitus, twitching, tunnel vision, unconsciousness, and convulsions [2]. The convulsions of CNS oxygen toxicity, which resemble grand mal epileptic seizures, start in humans within a relatively short time of the oxygen exposure. Reactive oxygen species (ROS) that are produced in excess upon exposure to highly pressurized oxygen overwhelm the antioxidant system of the CNS and seem to mediate the hyperoxic injury [3]. The CNS is rich in lipids, and molecular oxygen dissolves 7- to 8-fold more in the nonpolar medium such as CNS than in the aqueous cell compartments. The brain's rate of oxygen metabolism is high, but the tissue oxygen stores are lacking. Neuronal cells are rich in mitochondria and have a high demand for adenosine triphosphate (ATP). Oxidative phosphorylation occurs extensively and involves ubiquinones, which are abundantly present in the CNS. Those ubiquinones autooxidize readily and are apparently involved in the production of ROS [4,5]. The microcirculatory blood vessels of the brain and spinal cord are frail and can be torn easily thus, resulting in the release of iron and copper into the tissues. These transition metals act as catalysts in the generation of ROS [6]. The gray and white matter of the CNS contain a high level of ascorbic acid [7]. These neuronal cells can concentrate ascorbate 100-fold more than the plasma because of the brain's particular active transport system for ascorbic acid. Although ascorbic acid is a powerful antioxidant, its high concentration in the CNS, in the presence of iron and copper (due to their release by CNS injury), could become pro-oxidant and produce ROS [8,9]. Thus, due to ROS produced by hyperoxic oxygen exposure the blood-brain-barrier breaks down and cerebral edema accumulates [10]. However, the prime site in the brain for ROS attack, which causes seizures is still obscure. Therefore, antioxidant therapy may provide protection against CNS oxygen injury. Since exercise increases oxygen consumption and uptake in vital tissues of humans and animals, it also enhances ROS formation in the various tissues of animals [11 —13]. We have shown that heart tissue from exercise-trained rats produces the ascorbyl-electroparamagnetic radical [14]. Since the brain's concentration of ascorbic acid is much higher in neuronal tissue than in the plasma, it is not known whether ascorbyl-electroparamagnetic radicals are produced in the brain specifically due to exercise training; but it would be interesting to study the effects of exercise intensity on ascorbyl radical formation in the CNS. Glucose interacts with proteins to produce oxygen radicals [15]. The brain utilizes glucose as its primary source of energy, and during exercise, there seems to be a greater demand for energy in the brain due to its increased oxygen consumption (uptake) and cerebral blood flow Various brain regions utilize glucose differently and superoxides are produced during the oxidative metabolism of glucose.

Chapter 26: Influence of exercise-induced oxidative stress on the CNS

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1.2 Reactive oxygen species in the central nervous system The CNS is particularly susceptible to the toxic effects of oxygen due to its generation of ROS. Among the various organ systems of the body, the CNS uses a disproportional 20% of the oxygen consumed by humans. Howêver, brain constitutes about 2% of the body wêight. Consequently, oxygen becomes harmful w^hen it is converted to ROS. ROS are very reactive, short-lived and have been know^n to cause damage to blood vessels in the brain [16], as wdl as to the brain's parenchyma [17]. The sequential univalent reduction of oxygen produces superoxide anion radical (O2-), hydrogen peroxide (H2O2), and free hydroxyl radicals (OH). O2- radicals can be produced in the CNS from a variety of sources, such as the mitochondrial electron transport chain and by dihydroorotic dehydrogenase [18]; it is also generated by xanthine oxidase, vs^hich is concentrated primarily in the endothelium of the cerebral blood vessels [19,20]. Superoxide is also produced in the CNS by prostaglandin H synthase, lipoxygenase and cytochrome P-450 oxygenase pathwâys [21,22]. In addition, enzymatic and non-enzymatic oxidation of catecholamines, L -amino acids, and hemoglobin have all been shown to produce dangerous levels of superoxide and H2O2 in the CNS [23—25]. Activated polymorphonuclear leukocytes generate superoxide from nicotimnamide adenine dinucleotide phosphate reduced (NADPH) oxidase in the CNS [26,27] H2O2 is generated in the brain's mitochondria entirely from the dismutation of superoxide in the CNS [28—30], while hydroxyl radicals can be generated in the CNS by superoxide-driven Fenton reactions or metal-catalyzed Haber-Weiss reactions [31,32]. Finally, nitric oxide (NO) is also produced in specific neurons of the CNS and in the brain microvascular endothelium by NO synthase [33]. NO can react with superoxide radicals to produce peroxy nitrite anions (ONO), which can cause brain injury. For example, ROS generated in the CNS may interact with aromatic amino acids (tryptophan, phenylalanine, and tyrosine) and sulfur containing amino acids (cystine and cystein) present at the active sites of the enzymes. Glutamine synthase and Ca'^'ÂTPase have been shown to be inactivated in the CNS of animals due to ROS-induced oxidation [34,35]. 1.3 Lipid peroxidation in the central nervous system Reactive oxygen species generated in the CNS rapidly react with lipids, proteins and nucleic acids [36]. The CNS is particularly vulnerable to the damaging effects of ROS on membranous lipids [37] due to its following characteristics: 1. High rate of oxygen consumption 2. High rate of oxidative metabolic activity 3. High concentrations of readily oxidizable substrates, such as polyunsaturated fatty acids and catecholamines 4. High content of iron 5. Low levels of antioxidant enzymes and glutathione ROS generated in the CNS can attack the unsaturated bonds of membrane lipids

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P^^t I^' Organ functions

and may undergo lipid peroxidation (LPO). Lipid peroxides may generate a peroxidation cascade that allows the peroxidative damage to spread to distant tissues and organelles. Transition metals, such as iron, seem to play an important role in the generation of ROS, as well as in the process of LPO in neuronal membranes. The impact of LPO on membrane lipids, membrane receptors and membrane-bound enzymes (ATPase and acetylchoUnesterase) can alter the function, structure and fluidity of membranes and may result in an altered calcium influx [38,39]. The aldehydes that produced lipid peroxide fragmentations (malondialdehyde, 4-hydroxy alkenals) are cytotoxic [40,41]. A plethora of evidence indicated that enhanced LPO contributes to the pathogenesis of oxygen toxicity and thus, several neurodegenerative diseases. Therefore, it is essential to inhibit LPO reaction by different means: iron-chelating agents, spin-trapping agents, free radical scavengers and chainbreaking antioxidants. 1.4 Antioxidant system and central nervous system Detoxification of ROS is one of the prerequisites of an aerobic life style. This detoxification is a part of the antioxidant defense system generated in the body Oxidative stress results when the production of ROS exceeds the ability of the antioxidant s5^stem to ehminate them [12]. The antioxidant system includes antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and an ancillary enzyme, glutathione reductase (GR). There are also non-enzymatic antioxidants in this bodily system which include: glutathione, vitamin E, ubiquinol (coenzyme Qio), ascorbic acid, thioredoxin, and a-lipoic acid. All of these are critical for protection against ROS. When the body's normal protective measures are active, oxygen consumed by the organs will form O2—, which can be readily dismuted by SOD to H2O2 and singlet oxygen (^02). H2O2 is then converted, by either CAT or GSH-Px, to H2O and ^02. Finally reduced glutathione (GSH) consumed in the GSH-Px reaction is recycled back to its reduced form by GR. These antioxidant enzymes and GSH are much lower in the CNS when compared to erythrocytes and peripheral tissues [42,43]. Glutathione, in its reduced state, plays an important role in protecting cells against free radical and oxyradical damage [44]. The turnover of GSH in the brain is considered to be slower than the turnover of GSH in the liver and kidney The half-life of GSH in rat brains is estimated to be 70 h [45] as compared to the 29-min halflife of GSH [46] in mouse kidneys. Glutathione deficiency, induced by buthionine sulfoximine (inhibitor of glutathione synthesis), leads to the enlargement and degeneration of mitochondria in the rat brain. Such a glutathione deficiency probably causes accumulation of H202-damaging mitochondria [47]. The brain stem and the spinal cord are more vulnerable to ROS injury because of their inherent low levels of GSH compared to the cerebrum and cerebellum [48]. Low levels of glutathione were found in six regions of the brain compared to the liver levels of young adult, middle-aged and aged rats [49]. The exposure


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of brain mitochondria to oxidative stress (caused by butyl hydroperoxide) results in glutathione depletion and the formation of glutathione-protein mixed disulfide [50]. In particular, the substantia nigra neurons showed an exceptional vulnerability to oxidative stress. When malondialdehyde (MDA) levels are measured as an index of endogenous lipoperoxidation, which represent oxidative stress, different brain regions exhibit varying concentrations of MDA formation under normal physiological conditions [51,52]. Thus, these findings suggest that the CNS has a great vulnerablity to oxyradical toxicity, and that it may possess unique coping mechanism(s) to deal with oxidative stress. Many pathologic conditions are caused by the nervous system's over production of ROS and the antioxidant system's lack of coping mechanisms: Parkinson's disease (PD), ischemic reperfusion injury, cataract formation, aging process, arthritis, amyotrophic lateral sclerosis, Huntington disease, cerebrovascular injury, asthma, carcinogenesis, Down syndrome (DS), Alzheimer's disease (AD), multiple system atrophy (MSA) and other ailments. 2

OXYGEN-INDUCED CNS TOXICITY

2.1 Antioxidant system/lipid peroxidation CNS oxygen toxicity depends upon the exposure time to oxygen and the partial pressure of oxygen [53]. ROS have been implicated in oxygen toxicity and were first reported by Gershman et al. [54]. They reported that the formation of ROS and lipid peroxides in the CNS increases after exposure to oxygen at increased levels of pressure [51,55]. In addition, increased CNS glutathione oxidation has also been reported during HBO exposure [56—59]. These effects, showing hyperbaric hypoxia on the CNS antioxidant system and LPO are depicted in Table 1. Most of the studies in rats and guinea pigs show increased SOD activity in the brain [60,61], which indicates superoxide generation due to the presence of excess oxygen at high-pressure levels. Conversely, the activities of CAT and GSH-Px decreased in the brains of rats and guinea pigs due to HBO exposure [60], thereby reflecting the accumulation of H2O2 and lipid hydroperoxide in the animals' CNS. The levels of GSH and protein sulfhydryl groups were depleted in the cerebal cortex (CC) and brain of the rats at various time intervals of HBO exposure [56,62]. We suspect that the inhibition of antioxidant enzymes and the depletion of GSH in the CNS may be due to ROS-mediated inactivation of the brain's enzyme proteins and the oxidation of GSH to glutathione oxidized (GSSG). Jenkinson et al. [63] have reported that hyperbaric hyperoxia increases GR activity in the rat brains and this may be related to increases in GSSG levels during oxidative stress, thereby sending intracellular signals to induce GR production. Such studies in rats have also demonstrated an appreciable augmentation of LPO in the entire brain, as well as in various brain regions of rats [51,61].

718

Table 1. Oxygen toxicity: antioxidant system and lipid peroxidation in central nervous system. Species

Condition

Duration

Brain / brain regions

YOChange (+ increase;

SOD

CAT

GSH-Px GSH/Protein-SH LPO

-

decrease)

Reference

Rat (SpragueDawley)

Hyperbaric hy- 4 h peroxiab 8.25 h

Brain Brain

+20% -2%

+11% -18%

+100/0 -21%

-

-

-

-

-

-

Guinea pig

Hyperbaric hyperoxia’

6.25 h 1 1.25 h

Brain Brain

+14% -2%

+2% -28%

-17% -34%

-

-

-

-

-

-

Normobaric hyperoxia”

12 h 36 h 48 h

Brain Brain Brain

-

-

-

-

-

+75%

+30% +35%

-

-

-

-

+99%

-

-

-

-

+138%

1h 3h 5h 8h 24 h

Cerebral Cortex Cerebral Cortex Cerebral Cortex Cerebral Cortex Cerebral Cortex

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-36% 45% -34% -24% -12%

Rats (Sprague- Hyperbaric Dawley) hyperoxiab

200 min

Brain

-

-

-

-

-1.5%

-

Jenkinson et al. [74]


200 min

Brain

-

-

-

-

+6%

-

Jenkinson et al. [63]


160 min 200 min

Brain Brain

-

-

-

-

-5% +4%

Rat (Wistar)

Rat (Wistar)

-

-

-

-

Harabin et al. [60]

Ahotupa et al. [61]

Gordon and Gajkowska [56]

-

-

-

Peacock et al. [62]


(Continued,)

Normobaric hyperoxia”

Harabin et al. [60]

Species

Condition

Duration

Brain / brain regions %I Change (+ increase;

SOD Rat (Wistar)

Hyperbaric hyperoxiab

20 min

20 min for 7 days

Brain Accumbens Amygdala Caudate Putamen Cerebellum Cerebral Cortex Corpus Callosum Globus Pallidus Hippocampus Hypothalamus Inferior Colliculus Olfactory Bulb Septum Spinal Cord Substantia Nigra Brain

CAT

-

decrease)

GSH-Px GSHiProtein-SH

Reference LPO +22% t1x1 t2% + 15Yu t3%1 t40% t 1 9% + 16% +20'%1

Noda et al. [51]

+11% +180/0 +2 1% +22% +42% + 13x1

+20%

'100% O2 at 1 or 2 atmospheric pressure; '100% O2 at 2-5 atmospheric pressure; SOD: superoxide dismutase; CAT: catalase; GSH-Px: glutathione per-

oxidase; GSH: reduced glutathione; LPO: lipid peroxidation.

Chapter 26: Influence of exercise-induced oxidative stress on the CMS

Table 1. Continued.

719

720

P^^t I^' Organ functions

2.2 Treatments Excess reactive oxygen species that are produced upon exposure to high pressure oxygen and deplete the antioxidant system seem to mediate hyperoxic CNS damage [3,55]. The severity of CNS toxicity and the lack of effective protective agents led to the use of several different compounds for protection in animals [54,64—66]. However, the actual mechanisms of protection that these agents provide remain unclear. Since ROS are produced in excess and the CNS antioxidant system is suppressed in CNS oxygen toxicity, ROS scavengers and exogenous antioxidants have been attempted as means of protection. Studies have shown that superoxide and H2O2 generation increases in oxygen toxicity [3,55]. Therefore, it is essential to attenuate the onset of CNS oxygen toxicity by increasing the concentrations of CNS enzymatic antioxidants to combat superoxide and H2O2. Free SOD and CAT injections have not been proven successful against CNS oxygen toxicity [67,68] due to the short circulating halflife of these enzymes and their inability to cross the blood-brain-barrier. However, Yusa et al. [69] have shown that liposome-entrapped SOD and CAT (injected intravenously), significantly protected against oxygen-induced CNS toxicity in rats. Some modified SOD can cross blood-brain barrier and provide protection to the brain against ROS. The endogenous tripeptide glutathione (GSH) is a well-known detoxifier of ROS and protected both rats and mice from CNS hyperbaric hyperoxia [65,66]. However, GSH has not protected rats from normobaric hyperoxic exposure [64]. A selenium deficiency has been known to decrease tissue GSH-Px activity [70] and increase the toxicity of oxygen in rats [71—73]. Since exogenous GSH administration did not protect selenium-deficient rats from hyperbaric hyperoxia [74], it is likely that GSH-Px activity is required for GSH-induced protection from CNS hyperbaric hyperoxia. As GSH is a peptide which can be easily degraded in the body (after administration), it is suggested that esters of glutathione may be more appropriately used to prevent CNS oxygen toxicity. Clearly, GSH appears to be an important endogenous antioxidant, since the depletion of tissue GSH has been demonstrated to augments oxygen toxicity [75,76]. The intracellular transport of GSH is another crucial factor in determining its availability in the CNS to counteract ROS produced during oxygen toxicity [62]. The dietary antioxidant, p-carotene, has also been reported to protect the rats against CNS oxygen toxicity [77]. Subsequently, during antioxidant therapy, ROS types their origin and their mode of action, as well as lipophilic antioxidants such as vitamin E and lipoic acid, must be carefully evaluated against CNS oxygen toxicity. 3

EXERCISE-INDUCED OXIDATIVE STRESS

Physical exercise is associated with enhanced respiration and increased oxygen uptake in the body's vital organs/tissues by as much as 15- to 20-fold. The brain consumes 20% of the oxygen taken in by the body Most of the oxygen consumed


721

by cells is reduced to H2O through mitochondrial electron transport chains; however, 2—5% of oxygen is converted into ROS via one electron reduction in the mitochondria. During the last decade, evidence has accumulated revealing the production of ROS and other free radicals during exercise and exerted oxidative stress on vital organs/tissues [11,13,78,79]. To protect themselves from ROS injury, cells contain several antioxidant defense mechanisms: endogenous antioxidants (glutathione, vitamins C, A, E, uric acid and iron binding protein) and antioxidant enzymes, such as superoxidase dismutase (SOD), CAT and glutathione peroxidase (GSH-Px). The AOE activities are well distributed in all organs of the body, which are dependent on oxygen consumption rate, metabolic rate, and metal ion and fatty acid amounts. AOE activities are prone to alteration with changes in bodily oxygen consumption (oxidative stress). Oxidative stress can be described as a disturbance in the antioxidant system rendering it unable to adequately scavenge free radicals/ROS and arrest LPO chain reactions. Antioxidant system responses to physical exercise are dependent on the type, mode, intensity, frequency, and duration of the exercise. The modulation of AOE activity depends primarily upon its substrate (ROS), cosubstrate production, nature of catalytic center, affinity of the enzyme to the substrate (K^, Michaelis Menton constants), and selectivity and specificity of the substrate. Exercise enhances the blood flow in most organs, but depletes the flow in the liver, which in turn influences the compartmentalization of GSH in the liver. Such changes in blood flow rates, during and after exercise may alter the transport of glutathione (extracellular and intracellular), and the synthesis or degradation of glutathione due to an increased uptake of oxygen in the blood. Consequently, the effectiveness of the antioxidant system varies after acute exercise and exercise training. Antioxidant activity in various tissues, specifically in different animal brain regions, is summarized in Table 2. 3.1 Acute exercise and central nervous systems Recently, Somani et al. [52] investigated the effects of acute exercise (100% V02max, Maximum oxygen consumption) on antioxidant enzymes and LPO studied in specific areas of the Fisher-344 rat brain. The changes in antioxidant enzymes and LPO in specific areas of rat brains after acute exercise are depicted in Table 2. Studies show that due to acute exercise, SOD activity decreased in the CC with no significant SOD changes in other brain regions. CAT activity increased, due to acute exercise, in the cerebal cortex and the corpus striatum (CS); whereas, GSH-Px activity increased profoundly in the cortex and decreased in the CS. GSH-Px activity increased profoundly in the cortex, a region with low basal activity Perhaps to cope with the oxidative stress of exercise, this enzyme is activated in the cortex to eliminate hydroperoxides. Glutathione reductase activity, which controls endogenous levels of reduced glutathione (GSH), was decreased in the cortex, due to acute exercise, suggests an inadequate level of reducing equivalents

722

Table 2. Exercise-induced changes in antioxidant system/lipid peroxidation in the central nervous system. Species

Rats (Fisher-344)

Rat (Fisher-344)

Rat (Sprague-Dawley)

Exercise mode and duration

Brain/ brain regions

% Change (+ increase;

SOD

CAT

GSH-Px

GR

Acute exercise1 treadmill (30 min)

Cerebral cortex Cerebellum Corpus Striatum Hypothalamus Medulla

-26 NC -1 5 -1 8 +8

+54 NC +40 NC NC

+147 +20 -66 NC +26

-17 -15 -10 NC -10

Cerebral cortex

NC

NC

+33

+62

Cerebellum Corpus striatum Hypothalamus Medulla

-1 5 +36 -2 5 -20

NC +25 -29 NC

-22 +22 +43

NC

Brainstem

+30

Corpus striatum Cerebral cortex Hippocampus

+30 NC +10

Exercise training/ treadmill (6.5 weeks)

Exercise training/ treadmill (7.5 weeks)

~~

-

decrease)

+214

Reference GSH GSSG -

~

-

+13 Red +10 Oxi NC NC -

+10

NC -20

-

NC

-

-

-1 3 NC NC

+9 Red +50 Oxi +10 Oxi +33 Oxi

-

NC

-

-

orLPO +66 NC +112 NC +22

Somani et al. [52]

-50

Somani and Husain ~ 3 1

-25 -3 1 -29 NC Somani et al. [81]

~


SOD: superoxide dismutase; CAT catalase; GSH-Px: glutathione peroxidase; GR: glutathione reductase; GSH: reduced glutathione; GSSG: oxidized glutathione; Red: reduced GSH; Oxi: oxidized GSSG; NC = no change.


723

NADPH and a failure to maintain GSH levels. Consequently, harm may incur as GSH is important in the regulation of brain function and is reportedly decreased during oxidative stress [50]. Acute exercise significantly increases LPO in the CS, which contains high basal MDA levels and controls motor activity It is notable that LPO is inversely proportional to GSH-Px activity in the CS which has a high dopamine content [80]. Since dopamine is labile to auto-oxidation and may be transformed to toxic intermediates with the generation of ROS, acute exercise may cause peroxidative injury to the striatal membrane, alter neurotransmitters and may interupt motor function. Interestingly, acute exercise selectively inhibits striatal acetylcholinesterase (AChE) activity and such an inhibition is correlated with an increase in LPO, thereby resulting in perturbation of motor activity [38]. 3.2 Exercise training and the central nervous system Somani and co-workers' [81] investigation of the effects of exercise training (7.5 weeks) on the antioxidant system in brain regions of Sprague-Dawley rats, show the regional variations in cerebral GSH and GSSG levels and GSH-Px and SOD activities in sedentary control rats. These variations were enhanced in specific brain regions after exercise training. The brain regions of sedentary control rats showed: 1) GSH levels were lower in the brain stem (BS) than the CC and CS — the latter two were comparable; 2) GSSG levels were also lower in brain stem than the CC and CS; 3) The GSH/GSSG ratio was observed in the decreasing order CS > BS > CC. In sedentary control rats, GSH-Px activity was found in the decreasing order CS> BS> CC> hippocampus (H); however, SOD activity levels registered in reverse order: C C > B S > C S > H . Thus, SOD activity was stimulated in brain regions due to exercise training (Table 2). This pattern is interesting in the brain regions of sedentary control rats when compared to level of changes in exercised rats. BS and CS seem to be more sensitive to oxidative stress, and SOD was induced in these regions during exercise. Evidently exercise does not produce significant alterations in GSH-Px activity in the brain regions, but SOD activity increases significantly following exercise training (Table 2). Examination of glutathione levels in each specific brain region of the rats showed that the cerebal cortex of sedentary controls has higher levels of GSH than other regions, and this suggests that this area may deal with oxidative stress better than other regions. The exercise training did not significantly change the GSH levels in this region, but the cerebal cortex showed a significant decrease in GSSG following exercise which indicates that the GSH to GSSG conversion did not occur due to exercise training. It seems, therefore, that exercise training results in a better redox ratio in this region and that the CC seems to be more capable of coping with oxidative stress because it has the highest concentration of GSH. This may be a biochemical correlation to the physical sensation of greater wellbeing and greater awareness following everyday exercise in humans. SOD and GSH-Px activity was not altered in this region due to exercise training

724


(Table 2). The level of GSH is significantly low in the BS when compared to CC and CS, thereby potentially predisposing it to greater oxidative stress. However, GSH levels slightly increased in this region due to exercise training. But, GSSG levels significantly decreased in BS indicating the turnover of GSH caused by exercise training. The GSH/GSSG ratio was highest in the BS of the exercise-trained rat when compared to the sedentary control regions. This ratio suggests that exercise training may contribute to the BS becoming more resistant to oxidative stress. This region also shows increased activity of SOD in conjunction with exercise training possibly due to enzyme induction. The oxidative stress seems to be predominantly due to the production of superoxide radicals, which are then scavenged by SOD. Since GSH-Px activity in BS is identical in exercised rats and sedentary control rats, there is no further change in GSH-Px activity, which suggests that no significant peroxidation takes place in the BS. These results show exercise training greatly benefits this region. Since the brain stem is a very vital area in autonomic functioning, cardiovascular regulation and respiration, exercise training may improve this region and its functions, which include controlling the heart rate and blood pressure [82]. Such improvements in physical fitness could result in the improved cardiovascular function that typically accompanies regular exercise training. The GSH levels and SOD activity in the CS region are comparable to levels in the CC region of sedentary control rats, although the size of the CS is much smaller than the CC [81]. However, the CS region has a significantly higher GSH-Px activity than the CC. Similarly, the CS region SOD activity increased, and GSSG levels increased slightly, after exercise training. The metabolic percentages of the corpus striatum (basal ganglia), in terms of O2 consumption by these tissues on a per g basis, exceed that of the CC. Thus, the highest levels of GSHPx activity and GSH concentration may help this part of the brain to cope with any oxyradicals formed during periods of high oxygen consumption such as exercise. The H has the lowest levels of GSH-Px and SOD among all the four brain regions studied [81]. Exercise training increases SOD activity, in this region, which scavenges the superoxides formed from the excess O2 consumed during exercise. The BS and CS show a significant increase in SOD enzyme activity (30%) due to exercise training where as the CC did not show any change in this enzyme activity Apparently, exercise training causes more oxidative stress in the BS and CS regions that have a better ability to induce antioxidant enzymes that can cope with the superoxides formed during exercise training. The BS and CS appear to be more sensitive to oxidative stress, due to levels of enzyme induction, whereas CC and H regions may have acquired more resistance to oxidative stress due to their higher levels of GSH. GSH was present 43% less in the BS than the CC. However, exercise training significantly increases GSH to GSSG ratios in BS and in CC, but this ratio does not change in the CS. Therefore, the benefit of exercise training is evident as GSSG levels decrease and GSH levels remain unaltered in these two regions in particular.


725

Recently, Somani and Husain [83] have reported the effects of exercise training (6.5 weeks) on the antioxidant system in various brain regions of Fisher 344 rats. In sedentary control rats, SOD activity decreases in the following order: cerebellum > medulla > cortex > striatum > hypothalamus. However, exercise training alters the decreasing order of SOD activity: cerebellum > striatum > medulla > cortex > hypothalamus. In other words, exercise training increase SOD activity only in the striatum, a deeper area of the brain which has low enzyme activity and controls motor function (Table 2). Perhaps the enzyme SOD is activated in the striatum to prevent superoxides in order to maintain and facilitate motor activity during exercise training. SOD activity is sensitive to tissue oxygenation, and its biosynthesis has been reported to be elevated in rats subjected to high oxygen tension [84]. Since the oxygen utilization has increased during exercise training, SOD activity is elevated in this brain region. Conversely, due to exercise training, SOD activity decreased in the medulla, cerebellum and hypothalamus, all superficial areas of the brain. Such a decrease in SOD activity may be due to either feedback inhibition of the enzyme or oxidative inactivation of enzyme proteins; feedback inhibition and oxidative inactivation of SOD have been documented by other investigators [85,86]. A significant augmentation of GSH-Px activity in the cortex and hypothalamus, which have low basal activity, is indicative of an efficient elimination of hydroperoxides after exercise training (Table 2). GSH-Px is a nonspecific enzyme for hydroperoxides, and this lack of substrate specificity extends its range of substrates from H2O2 to organic hydroperoxide [28]. A significant decrease in CAT activity, along with an increase in GSH-Px activity in the hypothalamus, suggests an enhanced removal of H2O2 due to exercise training. Additionally, GR activity, which controls the endogenous GSH levels, increased profoundly in the cortex and striatum, suggesting the maintenance of GSH due to exercise training. These results are consistent with our previous report on Sprague-Dawley rats [81]. Exercise training also causes a significant depletion of MDA in the cortex, striatum, cerebellum and hypothalamus which denotes an adaptive response to decreased LPO rates. No significant change in GSH and GSSG contents in brain regions (Table 2) is indicative of the regions adaptive response after exercise training, or a resistance to oxidative stress. Interestingly, exercise training selectively inhibits hypothalamic AChE activity, and the inhibition correlates with increased LPO, which indicates the perturbation of hypothalamic functions [87]. 4.

EXERCISE AND DRUG INTERACTION ON THE CNS

The biotransformation of chemicals can be both harmful and helpful to the body Under normal, healthy conditions, a chemical can be safely biotransformed and excreted from the body. However, several metabolic pathways may form electrophilic intermediate metabolites that generate superoxides and free radicals. Chemicals that contain nitro, amine, iminium, or quinone functional groups usually form these electrophilic intermediate metabolites which can covalently


726

Table 3. Drugs and chemicals which produce CNS toxicity mediated via ROS/free radicals. Drugs

Free radicals (ROS)

Reference

Cisplatin BCNU - carmustine Procarbazine Ethanol Physostigmine Quinones Morphine Parathione 6-Hydroxy dopamine

ROS methylradical free radical a-hydroxy-ethyl radical esroline to catechol to quinones reactive metabolites covalent binding reactive metabolite ROS/free radical

Clerici et al. [209] Reed eetal. [210] Sinha[211] Reinke et al. [92] Somanietal. [212] Kochhetal. [213] Nagamatsu et al. [214] Chambers et al. [215] Austetal. [216]

bind to macromolecules and produce tissue-specific toxicity. Table 3 summarizes the drug-induced neurotoxicity mediated via the production of ROS and free radicals. Two specific pathways that produce free radicals, or "O2, include NADPH-cytochrome P-450 reductase [88] and one electron reduction under low oxygen tension [89]. Once these reactive metabolites are generated, interaction with the ferrous ion could produce the hydroxy radical (OH), possibly leading to toxic effects. As exercise will increase the body's oxygen consumption and the levels of AOE, physical activity may reduce the toxicity of several chemicals. For example, ethanol and nicotine have been reported to induce cytochrome P450 II El in the brain of rats [90,91]. Ethanol has also been reported to generate a-hydroxyl ethyl radicals and exert oxidative stress [92]. We have recently demonstrated the interaction of exercise and ethanol on AOE activity in various brain regions of rats. In the brain, GSH-Px activity increases in the hypothalamus and striatum. CAT and GR activity also increased in the striatum, and a decrease in SOD activity in the striatum and medulla was shown. IMalondialdehyde levels increases in all brain regions after a single dose of ethanol when combined with acute exercise (Table 4). It is suggested, therefore, that the combination of acute exercise and ethanol exerts significant oxidative stress on specified brain regions of rats [52]. In addition, acute physical stress has been reported to increase the permeability of the blood-brain-barrier [93,94]. Thus, excess ethanol might have penetrated into the brain regions of exercised rats and caused oxidative injury Even peripherally acting drugs, such as pyridostigmine, have been reported to reach the brain and negatively affect the centrally controlled functions under stressful conditions [94]. The interaction of exercise training and chronic ethanol ingestion on the antioxidant system of rat brains has been recently studied [83]. The data indicate that SOD activity decreases significantly in the cortex, medulla, cerebellum and hypthalamus but increases in the striatum (Table 5). On the other hand, the GSH-Px activity significantly increases in the cortex, striatum and hypothalamus when intense exercise and alcohol are combined. GR activity profoundly increases in the cortex, striatum and cerebellum, whereas it decreases in the medulla under such conditions. (Table 5). IVLDA levels significantly decrease in the medulla and


727

Table 4. Interaction of acute exercise (AE) and single ethanol dose (Et) on antioxidant enzymes and lipid peroxidation in brain regions of rat.

SOD AE Et AE + Et CAT AE Et AE + Et GSH-Px AE Et AE + Et GR AE Et AE + Et MDA AE Et AE + Et

Cortex

Striatum

Medulla

Cerebellum

Hypothalamus

NC +33% +31%

NC NC -57%

NC -45% -41%

NC NC NC

NC NC NC

NC -54% NC

NC +92% NC

NC + 114% +42%

NC NC NC

NC -57% -65%

+ 147% NC NC

NC + 138% +69%

NC NC NC

NC NC NC

NC +189% + 176%

-17% NC NC

NC +52% +37%

NC NC NC

NC NC NC

NC NC NC

NC +73% NC

+ 112% NC NC

NC NC +58%

NC NC NC

NC NC NC

NC: no change; SOD: superoxide dismutase; CAT: catalase; GSH-Px: glutathione peroxidase; GR: glutathione reductase; MDA: malondialdehyde.

cerebellum in response to exercise training and chronic ethanol ingestion. The combination significantly depletes GSH levels in the cortex, striatum and medulla, whereas GSH/GSSG ratios decrease in the medulla only (Table 5). Therefore, we suggest that exercise training may protect certain brain regions from ethanolinduced oxidative injury. 5.

CENTRAL NERVOUS SYSTEM DISEASES - REACTIVE OXYGEN SPECIES/ANTIOXIDANT SYSTEM

Recent evidences have shown that ROS and free radicals are implicated in the etiology and pathogenesis of CNS diseases such as Alzheimer's disease, Parkinson's disease. Amyotrophic Lateral Sclerosis, Down syndrome. Multiple System Atrophy, Huntington diseases, neuritis, stroke, epilepsy and other cerebrovascular and neurodegenerative disorders. During CNS disease processes, ROS are generated and react with proteins, lipids and nucleic acids. Damage to functional groups in biological molecules causes molecular and cellular oxidative damage that leads to CNS disease. Brain cells are endowed with an elaborate antioxidant defense system to scavenge ROS in the process of CNS diseases. This section addresses the etiology of CNS diseases and changes in the antioxidant system during the development of specific CNS disorders.

728

P(^^t IX: Organ functions

Table 5. Interaction of exercise training (ET) and chronic ethanol ingestion (CEt) on antioxidant system and lipid peroxidation in brain regions of rat. Cortex SOD ET NC CEt -75% -31% ET + CEt CAT ET NC -34% CEt NC ET + CEt GSH-Px ET +33% NC CEt +41% ET + CEt GR +62% ET +43% CEt +84% ET + CEt MDA -50% ET CEt +30% ET +CEt NC GSH ET NC CEt -17% -24% ET + CEt GSSG ET NC CEt NC ET + CEt NC GSH/GSSG Ratio NC ET NC CEt NC ET + CEt

Striatum

Medulla

Cerebellum

Hypothalamus

+36% NC +58%

-20% -40% -41%

-15% -15% -53%

-25% -25% -32%

NC -44% NC

NC NC NC

NC NC NC

-23% -A2% NC

NC NC +42%

NC NC NC

-22% -22% NC

+43% NC +64%

+214% +221% +356%

-20% -40% -41%

NC +43% +49%

NC NC NC

-31% NC NC

NC -34% -40%

-25% NC -31%

-29% +40% NC

NC -42% -39%

NC -59% -52%

NC NC NC

ND ND ND

NC NC -33%

NC NC NC

NC ND NC

ND

NC NC NC

NC -62% -56%

NC -34% NC

ND ND ND

ND

NC: no change; ND: not detected; SOD: superoxide dismutase; CAT: catalase; GSH-Px: glutathione peroxidase; GR: glutathione reductase; MDA: malondialdehyde; GSH: reduced glutathione; GSSG: oxidized glutathione.

5.1 Alzheimer's disease (AD) AD is a major neurodegenerative disorder. It is characterized by the gradual, progressive formation of neurofibrillary tangles, senile plaques and loss of selective neurons [95,96]. The most severe of all AD symptoms seen in humans is a gradual loss of memory, reasoning and orientation. Although a number of hypotheses have been put forward as to the cause of AD, the pathogenetic mechanism is still obscure. One hypothesis speculates that ROS play a pivotal role in the patho-


729

genesis of AD [97]. It has been suggested that in AD, ROS interact with amyloid protein to form senile plaques, while amyloid protein itself may generate ROS within the membrane [98—100]. The activation of microglia by p-amyloid, and electron transport chain defects have also been proposed to generate ROS in AD [101,102]. The induction of heme-oxygenase-1 is associated with neurofibrilla tangles and senile plaques in AD [103]. Fibroblasts from AD patients have also shown increased sensitivity to ROS [104,105]. Thus, ROS can initiate LPO, an early stage of tissue damage implicated as one of the mechanisms by which oxidative damage occurs during the onset of AD [106]. Studies in humans have detected an increased LPO in diseased CNS tissues when they are compared with control samples (Table 6). Studies indicate that AD is associated with an increased basal and Fe'^^-induced formation of MDA in the frontal cortex but not in cerebellum [107]. An increased Fe"^"^/H202-induced formation of MDA in the cortex, but not in the cerebellum, has been reported in AD studies [108]. Other studies, as shown in Table 5, have also indicated an increased LPO product in the cortex of AD patients [109-114]. The changes in CNS antioxidant systems of AD cases are summarized in Table 6. This data indicates that SOD activity typically increases in the brain, and in specific areas of the brain, of individuals with AD [110,111,115—118]. However, a few studies demonstrated a decrease in brain SOD activity and cerebrospinal fluid (CSF) in AD cases [109,119]. Since both decreases and increases in SOD protein levels were noticed in the brain regions of AD cases [120,121], superoxide influx is likely to be increased in specific brain regions of AD patients. Likewise, a trend of increases and decreases in CAT activity has been shown in specific brain areas of AD cases [110,111,115], while CAT protein levels only increase in the H plus parahippocampal gyri [121]. Both increases and decreases in GSHPx and GR activity are seen in specific brain regions of AD cases [110]. Similarly, GSH and GSSG levels also fluctuate in particular brain regions of AD cases. Such studies of antioxidant system functioning during AD do not indicate consistency. Further correlative studies are required to resolve the issue of antioxidant enzyme activity, mRNA and protein levels in specific brain areas of AD patients and control subjects. 5.2 Parkinson's disease (PD) PD is a major progressive movement disorder that occurs most commonly in the elderly, and is characterized by selective degeneration of nigral dopaminergic neurons projecting to the caudate-putamen [122]. The etiology of PD is unknown, however, recent evidence suggests the involvement of ROS and oxidative stress in the development of this disorder [123,124]. The current hypothesis speculates the formation of ROS, which may lead to neuronal damage due to oxidative stress [125,126]. Results form postmortem studies have provided evidence supporting the involvement of oxidative stress in the pathogenesis of PD. Neurotoxin 1methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) is selectively toxic to nigros-

CNS disorder

Alzheimer’s disease

Species

Human

Braidbrain regions

730

Table 6. Central nervous system disorders and antioxidant system/lipid peroxidation.

YOChange (+ increase; - decrease)

References

SOD

CAT

GSH-Px GR

GSHorGSSG LPO

Cortex gyrus cinguli

+12C +10M

-

-

-

-

-

Hippocampus

-5c

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Hypothalamus Nucleus caudatus

+19M +4c +5M +23C +8M

Human

Amygdala Basal nucleus M. Caudate nucleus Entorhinal cortex Frontal cortex Hippocampus Occipital cortex Parietal cortex Putamen Temporal cortex

+8 +8 +19 +6 +11 +8 +I +8 -2 +15

-29 -1 -26 -2 -13 -6 -8 -24 -27 -19

-


Human

Cerebral cortex

-1 c p -22 Mp

-

-

Alzheimer’s disease Alzheimer’s disease

Human

Cerebrospinal fluid

-35

-

-

-

-

-

Human

Cerebellum Frontal cortex Hippocampus

-2 5 -3 5 -21

-

-

-

-

-

(Continued.)

Gsell et al. [116]

Kurobe et al. [120]

+300

Bracco et al. [ 1 191

Richardson [ 1091



Marklund et al. [ 1151

CNS disorder


Species

Human

Brainibrain regions


SOD

CAT

Amygdala Cerebellum Hippocampus Inferior parietal lobule Middle frontal gyrus Occipital pole Pyriform cortex Superior and middle temporal gyri

+55 +14 +51 +2 1 +28 +15 +120 +150

-42 +25 +I06 +I7 -19 +112 +82 +300

GSH-Px GR

References GSHorGSSG LPO

Love11 et al. [I 101

-

+112 +5 +I40 +6 +8 +93 +500 +40

+5 -6 +4 -2 1 -38 -30 +74 -18 -10 -24 -5 -14

+5 +6 +43 +22 +32 +16 +27 +75 +18 +32 +53 +40

Balaz and Leon [ 1 111

-

-


Human

Amygdala Cerebellum Frontal lobe Hippocampus Motor cortex Meynert N. Olfactory bulb Occipital lobe Parahippocampal gyrus Parolfactory gyrus Prepyriformgyrus Sensory cortex

+29 -1 5 +55 +22 +24 NC -10 +80 +3 1 +7 +9 +14

+loo +6 -5 +70 +16 +12 NC -1 5 +2 1 -6 +45 +50


Human

Brain

+30

-

Zemlan et al. [ 1 171

Human

Hippocampus plus +25P parahippocampal gyri

+50P

Pappolla et al. [121]

731

(Continued.)


Table 6. Continued.

Table 6. Continued. CNS disorder

Species

Braidbrain regions

% Change (+ increase; - decrease)



GSH-Px GR

GSHor GSSG LPO

Frontal cortex Hippocampus Occipital cortex Substantia innominta Substantia nigra

-

+12 +37 +2 1 +20 +6

Human

Temporal cortex

-

Human

Inferior parietal lobe Inferior temporal gyrus Occipital cortex Sensory/motor cortex Superior frontal gyrus Superior temporal gyrus

-

Human

CAT

-

-

-

-

-

-

-

-

-

-

-

-

-

732

SOD

References

Perry et al. [ 1441

-

-

-

+35

McIntosh et al. [I 121

+I2 +40 +5 +6 +9 +35

Palmer and Burns ~131

+42

Andorn et al. [114]

Human

Temporal cortex

Human

Hippocampus plus associative cortex

+50C

-

Delacourte et al. 11171

Parkinson’s disease

Human

Cerebrospinal fluid Amygdala Caudatus Cerebellum C. semiovale Frontal cortex Nucleus ruber Nucleus basalis Pallidum Putamen Substantia nigra Temporal cortex Thalamus

-2 -1C, -9M -7C, -15M +33C, +40M -2C, NC,M +22C, +11M +38C, +9M +97C, +2M +29C, -1 3M +3C, NC,M +55C, -6M +55C, +15M +27C, +11M

+9 +I3 -18 +33 -7 +9 NC +44 -1 +36 -1 +9 -20

Marttila et al. [ 1391)

(Continued.)

-



CNS disorder

Species

Brainibrain regions

CAT

GSH-Px GR

-32 -25 NC -40 -27 -2 1 -32 -30 -35 -20 -25 -14

-50 NC -9 -10 +6 -20 4 5 +7 -60 NC -30 -1 0

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-6

+4c -6M

-

-

-

-

+20 +29

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

SOD



Human

Human

Caudate Cerebellum Frontal cortex Globus pallidus Hypothalamus Occipital cortex Putamen Red nucleus Substantia nigra Temporal cortex Thalamus Vermis Cerebellum

Substantia nigra


Human

Caudate nucleus Cerebellum Cerebral cortex Globus pallidus Putamen Substantia nigra

-

-

-

-

-2.5 +4c -6M +4 -1c +33M

-1c +33M

GSHorGSSG

LPO

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Ambani et al. [140]

-

Saggu et al. [ 1371

-30 -8 -38 4 0

Jenner et al. [130]

+I0 +I9 +I70 -

733

(Continued.)

References


Chapter 26: Influence of exercise-induced oxidative stress on the CMS

Table 6. Continued.

734

Table 6. Continued. CNS disorder

Parkinson’s disease Parkinson’s disease


(Continued.)

Braidbrain regions

o/‘

Change (+ increase; decrease)

Reference

SOD

CAT

GSH-Px GR

GSHorGSSG LPO

-

-

-

-35

-

DiMonte et al. [ 1421

-17 -16 -14 -14 -19 -28

-

Perry and Yong [143]

-6T +I4 -20T +24 -14T -18 -14T +9 -33T -100

-

Human

Striatum

-

Human

Caudate nucleus Cerebellar cortex Frontal cortex Occipital cortex Putamen Substantia nigra

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Caudate nucleus

-

-

-

-

Cerebellar cortex

-

-

-

-

Frontal cortex

-

-

-

-

Occipital cortex

-

-

-

-

Substantia nigra

-

-

-

-

-

-

-

-

-

-

-7 -20 -26 -14 -19 -9

Human

Human

Caudate Frontal cortex Globus pallidus Putamen Substantia nigra Temporal cortex

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Perry et al. [144]

-

Kish et al. [I411



Species

Table 6. Continued.




Species

Human

Braidbrain regions

Caudate Cerebellum Cortex Globus pallidus (L) Globus pallidus (M) Putamen (lateral) Putamen (medial) Substantial nigra

Human

Substantia nigra

Human

Caudate nucleus Cerebral cortex Globus pallidus (L) Globus pallidus (M) Putamen Substantia nigra

Human

‘%) Change (+ increase;

-

SOD

GSH-Px G R

Putamen

(Continued.)

LPO +11

+24 -6 +21 +I 1 +4 +5 +35

-

-

-

-

-

-

Dexter et al. [ 1291

4 1 Red -26 Oxi

Sofic et al. [145]

4 Red

Sian et al. [ 1231

-

-

-

-

-1 7 Oxi -20 Red +11 Oxi -8 Red + I 2 Oxi -8 Red +2 Oxi -30 Red +26 Oxi 4 0 Red +29 Oxi

735

Substantia nigra

GSHorGSSG

-

Cerebral cortex

Globus pallidus (M)

References

-

Caudate nucleus

Globus pallidus (L)

CAT

decrease)


CNS disorder

Table 6. Continued. Species

Brainibrain regions

9’0Change (+ increase; decrease)

References

-

SOD

CAT

GSH-Px GR

736

CNS disorder

GSHorGSSG LPO

~~

~

Riederer et al. [I461


Human

Brain

Human

Cerebrospinal fluid

+2c +60M

Amyotrophic lateral sclerosis

Human

Cerebrospinal fluid

+21c + 1OOM


Human

Brain


Human


-

Yoshida et al. [I 381

-

Yoshida et al. [I381

43c

-

Rosen et al. [151]

Brain

+81C

-

Human

Cerebrospinal fluid

-63

-


Human

Brain

-38C, -lM (F) -1OC, -7M (S)

Down syndrome

Human

Cerebral cortex

+60C

Multiple system atrophy

Human

Caudate nucleus Cerebral cortex Globus pallidus (L) Globus pallidus (M) Putamen (L) Putamen (M) Substantia nigra

Bracco et al. [I 191

Bowling et al. [ 1501

-2 -

-

-

-

-

Feaster et a1 [ 1531

-

-

-

-

-

-

-

-

-

-

-

+40orNC -25 or +25 +96or -60 -10 or +25 -29 or -33 -30 or NC -15 or +I0

-

-

Brooksbank and Balazs [ 1581 Sian et al. [I231


(Continued.)

+36

CNS disorder

Species

Braidbrain regions

% Change (+ increase; - decrease)

SOD Huntington’s disease

Human

Cerebrospinal fluid

Huntington’s disease

Human

Caudate nucleus Cerebral cortex Substantia nigra

CAT

GSH-Px GR

+50C +60M -

-

-

Cerebrovascular injury

Human

Cerebrospinal fluid

+114


Rat

Cerebral cortex

+4c +63M


Rat

Hippocampus

Cerebrovascular iniurv

Rat

Isocortex

-

-

-

-

-

-

-

-

-

References GSHorGSSG LPO -

-

Yoshida et al. [138]

-10 or -50

-

Sian et al. [I231

-13 or 4 0 +5 or ND

-

Gruener et al. [ 1751

+23

+22

+56

-

+59

Singh and Pathak [1811

-

-

-

-

+9 13

Willmore et al. [ 1801

-

+56

Willmore and Rubin 11821

SOD: superoxide dismutase; CAT catalase; GSH-Px: glutathione peroxidase; GR: glutathione reductase; GSH: reduced glutathione; GSSG: oxidized glutathione; LPO: lipid peroxidation; C: CuZn-SOD; M: Mn-SOD; Mp: Mn-SOD protein; NC: no change; P: protein; T: total GSH + GSSG; Red: reduced glutathione; Oxi: oxidized glutathione; F: familial; S: sporadic; Cp: CuZn-SOD protein.


Table 6. Continued.

737

738

Part I^: Organ functions

triatal dopaminergic neurons, thereby providing a model and a clue to the mechanism in PD [127]. Oxidative stress in PD may also arise from the slow metabolism rate of dopamine [128]. Studies have indicated that LPO is involved in the death of neurons in PD patients [129—132]. The finding of an inverted iron content in the substantia nigra of a Parkinson's brain further supports this phenomenon [129,133,134]. The nature of ROS responsible for cell death in PD imphcates H2O2 and OH [135,136]. The alteration in antioxidant systems in select areas of the PD brain is summarized in Table 5. In most studies, manganese-superoxide dismutase (Mn-SOD) activity increased in the substantia nigra of PD cases [129,136] and also in the cerebrospinal fluid of PD patients [138] Both increases and decreases in CAT and GSH-Px activity has been reported in the substantia nigra of PD cases [123,139—141]. The consistent depletion of endogenous antioxidant GSH in the substantia nigra suggests that GSH plays a pivotal role in the pathogenesis of PD (Table 6). Such a depletion in human PD cases has been reported [123,129,142—145]. Other studies have also demonstrated a depletion of total glutathione and increased levels of oxidized glutathione (GSSG) [123,144,146]. Thus, the GSH depletion may initiate a cascade of events, such as enhanced LPO, leading to the destruction of nigrastriatal pathways. In addition, GSH depletion may also render the nigrostriatal pathway prone to a toxic insult. Such studies indicate that oxidative damage is present in the brain of PD. 5.3 Amyotrophic lateral sclerosis (ALS) ALS is a motor neuron-degenerative disease that is usually fatal within 5 years of the onset of symptoms. The paralysis of the victim is due to the degeneration of motor neurons in the brain and spinal cord, however, the underlying etiology of the neurodegeneration is not clear. About 10% of ALS cases are familiar whereas 20% of ALS cases are familial (FALS). FALS is clinically defined as an agedependent autosomal dominant trait [147,148]. Studies have demonstrated 11 distinct FALS-associated mutations in exon 2 and 4 of the gene encoding cytostoHc copper zinc superoxide dismutase (CuZn-SOD). Additional mutations have also been reported [149]. SOD activity in the postmortem frontal cortex of FALS patients with missense mutations is significantly decreased, but the decrease in enzyme activity did not occur in patients with sporadic ALS nor in a FALS case without a missense mutation of CuZn-SOD [150]. Protein carbonyl content, a measure of protein oxidation, increase in patients with sporadic ALS vs. a control group, which suggests that oxidative stress is a common feature of ALS. Alterations in the activity of the human brain's antioxidant enzyme SOD and cerebrospinal fluid in ALS cases are depicted in Table 6. Most studies indicate that CuZn-SOD activity in the brain and cerebrospinal fluid decrease in human ALS cases [119,150—152]. However, a few studies show an increase in brain CuZn-SOD activity [153], as well as an increase in CuZnSOD and Mn-SOD activity in the cerebrospinal fluid of ALS patients [138]. It


739

has been suggested that an altered, noval function of this enzyme, rather than a reduction of its activity, may be responsible for the pathogenesis of FALS [154]. Recent reports indicate that oxidative reactions that are catalyzed by mutant CuZn-SOD enzymes are likely to initiate the neuropathological changes in ALS patients [149,155]. Therefore, the antioxidant enzyme therapy may be useful for the treatment of ALS. 5.4 Down syndrome (DS) DS is a neurodegenerative disease, similar to dementia of Alzheimer's [156,157]. DS patients possess additional genetic material typically, an extra chromosome 21, which has been linked to various physical abnormalities. The genetic abnormality in DS individuals with trisomy 21, resulting in mental retardation, is not understood scientifically. Possibly, an overexpression of SOD may result in the excess production of H2O2 which is then followed by excessive hydroxyl radical production and a subsequent attack of cellular constituents. Table 6 depicts the changes in antioxidant systems and LPO in the brain of DS cases. An increased SOD activity, but no significant rise in GSH-Px activity, has been reported in the DS fetal brain [158]. Similar studies demonstrate an increase in SOD and GSH-Px activity in the fibroblast of DS patients [153,159], along with an increase in LPO [158,160]. These studies suggest that oxidative stress is involved in the pathogenesis of DS, and antioxidant therapy may be useful in DS cases. 5.5 Multiple system atrophy (MSA) Multiple system atrophy (MSA) is a neurodegenerative disorder similar to PD Pathologically there is nigrostriatal degeneration with marked gliosis and cell loss in the substantia nigra, caudate nucleus and putamen [123]. Table 6 summarizes the changes in antioxidant systems in the brain regions and cerebrospinal fluid of MSA cases. The GSH content of the lateral globus pallidus in MSA patients increases as compared to control subjects, whereas the GSSG content decreases [123]. The GSH level also decreases in substantia nigra and putamen. The nigral GSH/GSSG ratio decreased in MSA patients when compared to controls, indicating an oxidative stress condition in MSA patients. Yoshida et al. [138] have observed that CuZn-SOD and Mn-SOD protein levels increased in the CSF of MSA patients. Such results tend to suggest that the blood-brain barrier may be destroyed in MSA patients and CuZn-SOD in the blood may affect their CuZn-SOD levels. However, the increase in Mn-SOD levels may be caused by an induction in damaged tissue, since inflammatory cells are known to induce the biosynthesis of Mn-SOD by cytokines (161). Apparently, more detailed studies are required to examine the complete antioxidant system to clarify the etiology of MSA in humans.

740

Pî't ^^- Organ functions

5.6 Huntington's disease (HD) HD is a neurodegenerative and psychiatric disorder. It is characterized by atrophy with ghosis and marked neuronal loss in the caudate nucleus and putamen [123]. A number of hypotheses have been proposed, but the etiology of HD is still unclear. However, the oxidative stress hypotheses may be involved in the pathogenesis of this disease. Table 6 summarizes the alterations in the brain's antioxidant system and cerebrospinal fluid of HD patients. Sian et al. [123] demonstrated that GSH levels are slightly decreased in the CC and caudate nucleus, whereas GSSG levels were depleted in these regions of HD cases. Yoshida et al. [138] measured CuZn-SOD and Mn-SOD protein levels in the CSF of HD cases to find that both CuZn-SOD and Mn-SOD levels increase in the CSF of HD patients, thus, indicating the damage of the blood-brain barrier, inflammatory reactions in the damaged tissues, and the release of cytokines. The cytokines are known to involve the activation and biosynthesis of Mn-SOD [161]. Apparently, a complete analysis of antioxidant systems would yield more understanding of the mechanism involved in the pathogenesis of HD. 5.7 Cerebrovascular injury Head and spinal cord injury, as well as cerebral ischemia and stroke, are very common in society [162]. The severity of CNS injury appears to correlate with the incidence of post-traumatic epilepsy. CNS trauma initiates a sequence of responses such as altered blood flow and vasoregulation, disruption of the blood-brain-barrier, increases in intracranial pressure, ischemia, hemorrhage, inflammation and necrosis [163]. The pathophysiological process underlying disorders of CNS injury are not well understood. However, recent studies have emphasized the effect of free radicals on the pathogenesis of cerebrovascular injuries. Demopolous et al. [164] first proposed the free radical hypothesis associated with CNS injuries and it has been subsequently supported by other investigators [165—167]. Severe elevation of the arterial blood pressure causes cerebrovascular injury, which has been demonstrated to be a result of excess superoxide generation [168]. Ischemia, induced by occlusion of the arterial supply to the CNS, causes neurological dysfunction [169]. Suppressing generation has also been demonstrated in cerebral vessels, as well as in the CSF, after ischemia [170,171]. Many investigators have reported increased LPO following cerebral ischemia injury [164,172]. There are evidences implicating ROS generation in cerebral hemorrhages due to the fact that hemoglobin is capable of releasing superoxides [25] and increasing LPO during cerebral hemorrhages [173]. It has also been shown that traumatic injury to the brain or to the spinal cord is mediated by the production of superoxide [174,175]. There are indirect evidences of ROS generation as evidenced by enhanced lipid-peroxidation after traumatic CNS injury [176,177]. The precise mechanism of post-traumatic epilepsy remains unknown, although it has been suggested that blood in contact with cortical tissue is asso-


741

ciated with epileptogenesis. A contusion or cortical laceration causes the extravasation of red blood cells, with hemolysis and a deposition of hemoglobin within the neutrophil. Iron, liberated from hemoglobin and transferrin, can cause free radical generation and the initiation of LPO causing human post-traumatic epilepsy [178,179] and experimental epilepsy in animals [180,181]. Table 6 summarizes the changes in antioxidant systems following cerebrovascular injury in human and rats. Analysis of CSF from acute stroke patients reveals an increase in SOD contents indicating that superoxides are formed in these patients, and an increased synthesis of SOD protein may protect the oxidative damage of superoxide radicals [175]. Cerebrovascular injury in rats demonstrated an increase in antioxidant enzymes Mn-SOD, CAT, GSH-Px and GR in the cerebral tissues, as well as enhanced LPO [180—182]. These studies suggest that ROS are involved in the pathogenesis of cerebrovascular injury. 6

STRATEGIES TO IMPROVE CNS FUNCTIONS

6.1 Antioxidant supplementation Recent studies suggest that ROS and free radical-induced oxidative stress is involved in CNS oxygen toxicity, both during exercise and in the pathogenesis of CNS disorders. Therefore, antioxidant nutrients may play a significant role in the prevention and management of CNS injuries brought on by oxidative stress. During antioxidant therapy, it is essential to consider the types of ROS and free radicals and their modes of action and origin, as well as the use of hydrophilic vs. lipophilic antioxidants. There is convincing evidence that oxidative damage in the CNS and its functions can be improved by exogenous antioxidant supplements. 6,1.1 Vitamins Vitamin E is among the major lipid antioxidant vitamins and vitamin C is a major water-soluble antioxidant vitamin. Free radical-induced LPO has been shown to be suppressed in the CNS of animals during neuronal degeneration by vitamin E supplementation [165,183]. Vitamin E deficiencies enhance the effects of HBO on the CNS while vitamin E supplementation decreases it [59,60]. In experimental animals, iron-induced trauma was blocked by vitamin E and selenium [163,182]. Supplementing the body with vitamin E has proven to be most effective against Tardive dyskinesia [184]. Dietary vitamin E supplements in rats have also resulted in a reduction of LPO after swimming exercise [185]. In humans, dietary vitamin E supplementation for 2 weeks or a single dose reduced the extent of LPO caused by exercise-induced oxidative stress [78,186]. Unfortunately, however, vitamin E is an unsuitable therapy for acute CNS injury due to its low nervous system uptake levels [187]. Vitamin E supplements also decrease the incidence of intraventricular cerebrovascular injury [177] and cerebral ische-

742

P(^rt I^: Organ functions

mia [188]. Recently, vitamin E supplementation has been shown to be protective against neurodegeneration from PD [124]. More recently, it has been suggested that oral antioxidant supplementation may be preventive against LPO in the human brain [112]. However, as previously noted, vitamin E is less effective against acute brain injury and ischemia [189]. Vitamin C, or ascorbate, has dual effects. It acts as a pro-oxidant at low concentrations and as an antioxidant at higher concentrations [23]. Dietary administration of vitamin C has been shown to be effective in preventing exercise-induced oxidative stress [190]. The protective effect of vitamin C has also been demonstrated against oxidative injury in cerebral ischemia [188]. Preliminary data have revealed that ascorbic acid supplementation may improve the recovery of exercise-induced oxidative tissue injury. While there is no convincing evidence that oxidative damage in the CNS can be suppressed by vitamin C supplements, vitamin C can regenerate vitamin E, directly. Therefore, the combination of both may be more effective than ingesting either vitamin alone. P-carotenes, apart from being the best source of vitamin A, have been reported to be free radical quenchers, ^02 scavengers, and inhibitors of LPO. P-carotenerich diets have been shown to protect against CNS oxygen toxicity in rats [77]. It is suggested that P-carotene, or vitamin A, may also be beneficial to other CNS disorders. 6J,2 Glutathione (GSH) GSH is the most abundant thiol present in the cells. The roles of GSH include: 1) to serve as a cosubstrate for GSH peroxidase (GPX), wherein GSH is used as a hydrogen donor to reduce H2O2 and organic peroxide to water and alcohol, respectively; 2) to conjugate with exogenous and endogenous toxic compounds, catalyzed by GSH sulfur-transferase; 3) to reduce protein disulfide and GSH-protein mixed disulfide bonds, maintaining the sulfhydryl residues of certain proteins and enzymes in the reduced state; 4) to restore cysteine in a nontoxic form; and 5) GSH assumes a vital role in keeping a-tocopherol (vitamin E) and ascorbic acid (vitamin C) in reduced states. GSH administration in rats has proven effective in protecting against hyperbaric CNS oxygen toxicity [62,63]. Exogenous GSH administration may not be able to reach CNS in sufficient concentrations because of its degradation in the blood and tissues. Therefore, GSH esters have been more effective against cisplatininduced neurotoxicity [191]. GSH has been found to be effectively protective against CNS oxygen toxicity in rats and mice [62,63,65,74]. In addition, glutathione supplementation is also known to be protective against exercise-induced oxidative stress [192]. The depletion of GSH in PD brains suggests that GSH plays an important role in the disease's pathogenesis. Therefore, GSH, or its


743

esters supplementations, have therapeutic inphcations for the management and prevention of PD [144]. 6,1.3 Antioxidant enzymes (A OE) AOE are a significant defense mechanism for neuronal cells combating CNS oxygen toxicity [23]. Yusa et al. [69] have shown that treating rats with liposomes containing SOD and CAT, 2 h before HBO exposure, shifts the onset of convulsions. SOD has been reported to diminish reperfusion injury after spinal cord ischemia [193] and cerebral edema in rats [194]. Similarly, cerebrovascular injury caused by acute hypertension has been shown to be reversable with SOD and CAT treatment [168,195]. CNS traumatic injury has been reported preventable and reversable due to SOD and CAT administration [174]. Enzymatic antioxidants (SOD, CAT, and GSH-Px) usually act for only a short time, and have poor ability to cross the blood-brain-barrier and reach the specific site of injury. Therefore, more research is required to achieve maximum protective effects against CNS injury with antioxidant enzymes. Fortunately, Ebselen, a low molecular weight agent that has GSH-Px-like activity [183] and inhibits LPO. It has recently been shown to be otoprotective against cisplatin-induced ototoxicity [196]. 6.1 A Other free radical scavengers Other therapies have been studied for their potential role in ROS/free radicalinduced CNS diseases. For instance, cerebrovascular injury has reportedly been prevented by pretreatment with mannitol, a scavenger of hydroxyl radicals [195,197]. Evidently, Dimethyl sulfoxide (DMSO) prevents ischemic myelopathy and paresis in dogs subjected to ischemia of the spinal cord [198]. In addition, the use of chelating agents that prevent iron ions from participating in hydroxyl radical production and LPO has also been studied in association with CNS disorders. A new series of 21-amino steroids (Lazaroids) with built-in iron chelating activity has been developed for the acute treatment of CNS injury [199]. Such agents, with antioxidant properties, have been tested in head injury, spinal cord injury, stroke, neurotoxicity, hemorrhage, and reperfusion injury [189,200]. Free radical spin trappers such as 7V-tert-butyl%-phenyl nitrone (PBN) have also been tried as protectors against CNS disorders [34,201,202]. As a result of such studies, it is suggested that the derivatives of PBN will have therapeutic use in the future. Striatal lesions produced by malonate has reportedly been attenuated by coenzyme Qio [203]. The administration of coenzyme Qio for 1 month in HD patients reveals a significant protection against such lesions [204]. Nerve growth factor protects rat hippocampal and human cortical neurons against ironinduced injury [205]. Interestingly, endogenous cofactors %-lipoic acid and dihydrolipoic acid, which are well tolerated in humans are also neuroprotective against cerrebrovascular injury [206—208]. Recently, we have also demonstrated a dosedependent protection of %-lipoic acid against cisplatin-induced ototoxicity [217].

744


It is suggested that the use of antioxidants and free radical scavengers in the treatment of neurodegenerative diseases requires more detailed studies. 7

SUMMARY

1. Breathing oxygen is essential for human and animal life, but at high levels of pressure, oxygen can cause central nervous system (CNS) toxicity The generation of reactive oxygen species (ROS) and free radicals occurs due to an increase in oxygen pressure in the brain during exercise, as well as in the development processes of neurodegenerative diseases. 2. Several reports indicate that HBO exposure results in the formation of ROS and lipid peroxides in the CNS and depletes the antioxidant system. Studies show that exercise-induced oxidative stress commonly generates ROS and lipid peroxides and perturbs the antioxidant enzymes and glutathione in specific brain regions of animals. Recent evidences have also demonstrated perturbation of the CNS antioxidant system in many CNS disorders. 3. The combination of exercise training and alcohol ingestion on CNS antioxidant systems has also recently been studied. It is suggested that exercise training may protect certain brain regions against ethanol-induced oxidative injury Even peripherally acting drugs pyridostigmine and others have been reported to cross the blood-brain-barrier and affect CNS functions during physical stress. 4. Antioxidant nutrients appear to play a significant role in the prevention and management of CNS injuries that are caused by oxidative stress. There is convincing evidence that oxidative damage in the CNS and its functions can be improved by exogenous antioxidant supplements, such as antioxidant vitamins, glutathione and antioxidant enzymes. Other therapies, such as the use of radical scavengers, iron chelators, free radical spin trappers and other endogenous antioxidant a-lipoic acids have also protected the brain against CNS injuries. 5. It is suggested that during any type of antioxidant therapy, as well as during the use of hydrophilic or lipophilic antioxidants, the types of ROS/free radicals and their origin and mode of action in the CNS must be considered. 8

PERSPECTIVES

The implication of ROS/free radicals in CNS oxygen toxicity, exercise and drug metabolism, as well as in the pathogenesis of several CNS disorders and injuries, has been studied. However, the roles of endogenous antioxidants, as well as exogenous supplementation in CNS disease processes and injuries have not been well explored. Since GSH plays a pivotal role in the maintenance of the intracellular redox status and AOE functions, during acute exercise and exercise training, it is essential to study results of the exercise performance and intracellular-interorgan homeostasis, especially in the CNS. Recent work on adenosine A3 recep-


745

tors, through which adenosine enhances the activity of antioxidant enzymes, has opened a door for studying the interactions of agonists and antagonists of adenosine on antioxidant enzymes and LPO in the CNS during exercise, HBO exposure and neurodegenerative processes. The use of more selective agonists and antagonists, of adenosine Ai A2 and A3 receptors, will provide a better understanding of the mechanisms responsible for the activation or inhibition of antioxidant enzymes in the CNS during exercise, HBO exposure and during neurodegeneration. Exercise, oxygen exposure and CNS injuries may enhance the production of nitric oxide (NO) in cerebrovascular tissues. It is not known whether NO synthase inhibitors or activators would have antagonistic or synergistic effects on adenosine receptor-induced responses in CNS antioxidant systems. Both adenosine and NO may play a role during exercise, cerebrovascular oxygen exposure and during the development of CNS disorders. Therefore, the actions and interactions of both agents, through modulatory receptors on antioxidant systems would be an important future study. Moreover, it is likely that Ca'^'^-dependent enzymes, such as protein kinase C and NO synthase, may contribute to exercise, HBO and CNS injury-induced oxidative stress on cerebrovascular systems. The exploration of calcium channel-blockers, nucleoside transport blockers, protein kinase C and NO synthase inhibitors will provide a better understanding of adenosine receptor-mediated antioxidant responses in cerebrovascular tissues. In addition, further studies of the role of pro to-oncogene Bcl-2, transcription factors activator protein-1 (AP-1) and nuclear factor-KB ( N F - K B ) , and heat shock proteins may reveal the intracellular regulation that occurs in the CNS antioxidant defense system during exercise, HBO exposure, as well as during CNS disease processes. 9

ABBREVIATIONS

AChE: Acetylcholinesterase AP-1: Activator protein-1 AD: Alzheimer's disease ALS: Amyotrophic leteral sclerosis AOE: Antioxidant enzyme ATP: Adenosine triphosphate BS: Brain stem C: Copper zinc superoxide dismutase CAT: Catalase Cp: Copper zinc superoxide dismutase protein CNS: Central nervous system CSF: Cerebrospinal fluid CC: Cerebral cortex CuZn-SOD: Copper zinc-superoxide dismutase CS: Corpus striatum CO: Carbon monoxide


746 DMSO: DS: F: FALS: GSH: GSSG: GSH-Px: GR: H: HD: H2O: H2O2: OH: HBO: Km: LPO: M: MDA: Mn-SOD: MPTP: Mp: MSA: NADPH: NF-kB: NO: ^02: O2 —: Oxi: ONOO-: PBN: P: PD: Red: ROS: S: SOD: T: V02 max:

Dimethyl sulfoxide Down syndrome Familial Familial amyotrophic lateral sclerosis Glutathione reduced Glutathione oxidized Glutathione peroxidase Glutathione reductase Hippocampus Huntington disease Water Hydrogen peroxide Hydroxy radical Hyperbaric oxygen Michaelis menton constant Lipid peroxidation Manganese -superoxide dismutase Malondialdehyde Manganese-superoxide dismutase 1 -methyl-4-phenyl-1,2,3,6-tetrahydropyridine Manganese-Superoxide dismutase protein Multiple system atrophy Nicotimnamide adenine dinucleotide phosphate reduced Nuclear factor -kappa B Nitric oxide Singlet oxygen superoxide anion Oxidized Peroxy nitrite anion N-tert-butyl-1-phenyl nitrone Protein Parkinson's disease Reduced Reactive oxygen species Sporadic Superoxide dismutase Total Maximum oxygen consumption

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PartX Aging


©2000 Elsevier Science B.V. All rights reserved. Handbook of Oxidants and Antioxidants in Exercise. C.K. Sen, L. Packer and O. Hiinninen, editors.

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Part X • Chapter 27

Oxidants and aging Kenneth B. Beckman and Bruce N. Ames Department of Molecular and Cell Biology, 401 Barker Hall, University of California at Berkeley, CA 94720-3202, USA. Tel.: +1-510-642-5163. Fax: +1-510-643-7935. E-mail: kbeckman @ uclink4. berkeley. edu

1 INTRODUCTION 2 AN OVERVIEW OF THE FREE RADICAL THEORY OF AGING 2.1 The origins of the free radical theory 2.2 Sources of oxidants 2.3 Targets of oxidants 2.4 Antioxidant defenses 2.5 Repair of oxidative damage 2.6 Synthesis: the interaction of oxidant generation, oxidative damage, and repair 3 REFINEMENTS AND COROLLARIES OF THE FREE RADICAL THEORY 3.1 Oxygen radicals and evolutionary theories of aging 3.2 Oxygen radicals and the somatic mutation theory of aging 3.3 Oxygen radicals and mitochondrial theories of aging 4 OXIDATIVE PHENOMENOLOGY: AGE-ASSOCIATED TRENDS 4.1 Accumulation of oxidative end-products 4.2 Steady-state levels of oxidative modification 4.3 Oxidative depletion of biochemical pools 4.4 Age-associated trends in antioxidant defenses and repair 4.5 Age-associated trends in oxidant generation 5 INTERSPECIES COMPARISONS 5.1 Oxidative damage and maximum life span potential 5.2 Antioxidant defenses and maximum life span potential 5.3 Generation of reactive oxygen species and maximum life span potential 6 ENVIRONMENTAL MANIPULATION AND LIFE SPAN 6.1 Dietary restriction 6.2 Activity restriction 6.3 Oxygen tension 7 SUPPLEMENTATION AND LIFE SPAN 7.1 Dietary antioxidants 72 Pharmacological supplements 8 SUMMARY 9 PERSPECTIVES 9.1 Nitric oxide 9.2 Regulatory roles for oxidants and antioxidants: signal transduction 9.3 Oxidants and apoptosis 9.4 Exercise and life span 10 ACKNOWLEDGEMENTS 11 ABBREVIATIONS 12 REFERENCES

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Part X: Aging

INTRODUCTION

The study of aging has been characterized by a huge phenomenological hterature, and at the same time, the lack of estabhshed primary causes. The diverse hfe histories of animal species, which manifest aging in very different ways, have been an obstacle to testing unified theories. In order for experimental gerontology to provide more than a catalog of age-related changes, it has been necessary to define the alterations that are common to most old cells, tissues, and animals, while simultaneously respecting that there is not a single phenomenon of aging or a single cause. This has taken some time, and from the outside it may have appeared that the field has been mired in phenomenology Throughout this time, though, there has never been a shortage of unified theories attempting to reduce aging to something more tractable. In fact, gerontologists have been prolific in this regard [1,2]. Whereas some researchers have believed that a small number of random, deleterious mechanisms could explain degenerative senescence, others have opted for theories of "programmed" aging, in which senescence is the final destination in a developmental pathway In the course of these debates, a number of scientists have rallied around a set of ideas called the free radical theory of aging — loosely, the belief that damage by reactive oxygen species is critical in determining life span. This theory inspired many experiments in which evidence of oxidative damage in aged animals was sought. In the last 10 years or so, the nature of aging research has changed dramatically; one might say that the field has entered early adulthood. The tools of molecular biology are now sophisticated and accessible enough that researchers within gerontology have adopted them. At the same time, molecular biologists situated on the edge of aging research have made inroads, and have discovered that fruit flies and nematodes are amenable to the study of aging. Also, medical researchers investigating human diseases of aging, such as Alzheimer's disease and inherited progerias, have overcome long-standing roadblocks. It has been gratifying, therefore, that many of the preliminary studies in what might be called "molecular gerontology" lend credibility to the free radical theory Results from disparate experimental systems have recently shown that oxygen radicals play a role in degenerative senescence, and the pace of discoveries is quickening. The likely result of this combination of approaches will be the unraveling of the physiological tangle of aging, and it seems safe to say that one of the important knots will turn out to be oxidative stress. However, there is a danger that in the excitement of theoretical confirmation, certain nuances are lost. For instance, the revelation that oxygen radicals may be involved in neurodegeneration does not mean that oxidative stress determines life span. The free radical theory has sought not only to explain the mechanisms of senescence, but it has also attempted to explain differences between species' life spans in terms of oxidants. So although many recent studies indicate that oxygen radicals play some kind of role in aging, only a small number of these sup-

Chapter 27: Oxidants and aging

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port the more ambitious version of the free radical theory. On the other hand, there is no reason to ding to such a stringent version of the free radical theory, and it is becoming apparent that whether or not they determine life span, oxygen radicals are certainly important players in aging's pathophysiology. In other words, the scope of the free radical theory of aging should include aging-associated oxidative stress in general, rather than limiting itself to those oxidative events which may determine life span. In fact, many current articles indicate that such a blurring of distinctions has already occurred and that as it is commonly used, "free radical theory" encompasses a broad set of ideas. Therefore, our first purpose here is to delineate these different conceptions of the free radical theory. Due to the recent popularity of free radical research, a large number of reviews have addressed various aspects of the interplay between oxidants and aging [3—40]. Rather than merely updating this literature, our aim in this chapter is to provide a systematic categorization of the types of experiments that have been performed. The phenomenon and study of aging are incredibly diverse, and although it is a diversity of evidence which makes the free radical theory appealing, the menagerie of animals and techniques used can be confusing. By breaking the literature down into smaller pieces, we hope to make it easier to digest. In this chapter, then, we will briefly outHne the evolution of the free radical theory, and then describe the different areas of evidence. We will focus on recent experiments, and point to the areas which we feel are most likely to provide future insights. The way in which we have categorized the literature is outlined in Table 1. Although any such system is somewhat arbitrary, we hope that ours will make it easier to assimilate the existing literature and envision future experiments. ^ 2

AN OVERVIEW OF THE FREE RADICAL THEORY OF AGING

2.1 The origins of the free radical theory In 1956, Denham Harman suggested that free radicals produced during aerobic respiration cause cumulative oxidative damage, resulting in aging and death. He noted parallels between the effects of aging and of ionizing radiation, including mutagenesis, cancer, and gross cellular damage [41,42]. At the time, it had recently been discovered that radiolysis of water generates hydroxyl radical (•OH) [43], and early experiments using paramagnetic resonance spectroscopy had identified the presence of •OH in living matter [44]. Harman, therefore, hypothesized that endogenous oxygen radical generation occurs in vivo, as a by^ In reviewing as broad a topic as the free radical theory, we have been forced to Hmit both the content and the number of references cited. Ahhough we have done our best to include recent work, omissions were inevitable. We apologize to all authors whose work we have not managed to include, and direct readers to other recent reviews for material we have left out.

Experimental approach

Chapter section

Strengths of the approach

Weaknesses of the approach

Oxidative phenomenology

4.0

Simplicity Large existing data set. Elucidation of the basic biochemistry of oxidative stress. Technical foundation for other approaches. Negative result may be instructive.

Results are merely correlative (oxidative damage may be a consequence of ageing). Negative results, considered uninteresting, may fail to appear in the literature.

Interspecies comparisons

5.0

Specific testable predictions. Deviations from predictions may refine theory. Use of different species avoids conclusions that are species- or strain-specific. Negative results may be instructive.

Logistical problems in animal handling. Quantitative comparisons complicated by qualitative interspecies differences in oxidative defenses and/or repair.

Dietary restriction

6.1

Very well established model of life span extension. Straightforward methodology Probably relevant to human aging and cancer.

Results are somewhat correlative (alterations in oxidative stress may be consistent with but incidental to a more fundamental cellular switch).

Activity restriction

6.2

A direct test of the rate of living theory, with specific testable predictions. Straightforward methodology.

To date, limited to relatively simple organisms, such as invertebrates.

Manipulation of oxygen concentration

6.3

A direct test of the rate of living theory, with specific testable predictions. Straightforward methodology.

Results hard to interpret. Are positive results (life span attenuation by hyperoxia, extension by hypoxia) relevant to normoxic aging? Not applicable to most mammals.

Supplementation with dietary antioxidants

7.1

(Potentially) a test of the free radical theory

Results hard to interpret, due to unknown fates of supplements, potential adverse effects, complexity of overall antioxidant defenses.

Administration of pharmacological antioxidants

7.2

(Potentially) a test of the free radical theory.

Results hard to interpret, due to unknown fates of supplements, potential adverse effects, complexity of overall antioxidant defenses.

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Table 1. A categorization of the strengths and weaknesses of approaches to the testing of the free radical theory of aging.

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product of enzymatic redox chemistry. He ventured that the enzymes involved would be those "involved in the direct utilization of molecular oxygen, particularly those containing iron". Finally, he hypothesized that traces of iron and other metals would catalyze oxidative reactions in vivo, and that peroxidative chain reactions were possible, by analogy to the principles of in vitro polymer chemistry. All of these predictions have been confirmed during the past 40 years. The theory gained credibility with the identification in 1969 of the enzyme superoxide dismutase (SOD) [45], which provided the first compelling evidence of in vivo generation of superoxide anion (02* ), and from the subsequent elucidation of elaborate antioxidant defenses [46]. The use of SOD as a tool to locate subcellular sites of 02« generation led to a realization which buttressed the free radical theory, namely, that mitochondria are a principal source of endogenous oxidants [47]. Gerontologists had long observed that species with higher metaboHc rates have shorter maximum life span potential (MLSP): they age faster [48]. In fact, it had been proposed at the turn of the century that energy consumption per se was responsible for senescence, a concept referred to as the "rate of living" hypothesis [48—50]. The realization that energy consumption by mitochondria may result in 02* production linked the free radical theory and the rate-of-living theory irrevocably: a faster rate of respiration, associated with a greater generation of oxygen radicals, hastens aging. By now, the two concepts have essentially merged. 2.2 Sources of oxidants Ground state diatomic oxygen, (^Sg~02 or more commonly, O2), despite being a radical species and the most important oxidant in aerobic organisms, is only sparingly reactive itself due to the fact that its two unpaired electrons are located in different molecular orbitals and possess "parallel spins". As a consequence, if O2 is simultaneously to accept two electrons, these must both possess antiparallel spins relative to the unpaired electrons in O2, a criterion which is not satisfied by a typical pair of electrons in atomic or molecular orbitals (which have opposite spins according to the Pauli exclusion principle). As a result, O2 preferentially accepts electrons one at a time from other radicals (such as transition metals in certain valences). Thus, in vivo, typical two- or four-electron reduction of O2 relies upon coordinated, serial, enzyme-catalyzed one-electron reductions, and the enzymes which carry these out typically possess active-site radical species such as iron. One- and two-electron reduction of O2 generates superoxide {P2^) and hydrogen peroxide (H2O2), respectively, both of which are generated by numerous routes in vivo, as discussed below In the presence of free transition metals (in particular iron and copper), 02*" and H2O2 together generate the extremely reactive hydroxyl radical (#011). Ultimately, •OH is assumed to be the species responsible for initiating the oxidative destruction of biomolecules. In addition to 02* , H2O2, and ©OH, two energetically excited species of O2 termed "singlet oxygens" can result from the absorption of energy, (for instance, from

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ultraviolet light). Designated by the formulas Âg02 and ^Sg"^02, both of these species differ from the triplet ground state (^2g"02) in having their two unpaired electrons in opposite spins, thereby eliminating the spin restriction of groundstate O2 and enabling greater reactivity. The chemistry of oxygen and its derivatives has been extensively discussed elsewhere [51,52]. Since all of these species (02«", H2O2, •OH, Âg02, and î:g'^02), by different routes, are involved in oxygen's toxicity, we will collectively refer to them as "oxidants". It is now beyond doubt that oxidants are generated in vivo and can cause significant harm [27,46,47,53—55]. There are numerous sites of oxidant and oxygen radical generation, four of which have attracted much attention: mitochondrial electron transport, peroxisomal fatty acid metabolism, cytochrome P450 reactions, and phagocytic cells (the "respiratory burst"). Before discussing the potential contributions of different sources of oxidants, it is worthwhile briefly to outline them. In the textbook scheme of mitochondrial respiration, electron transport involves a coordinated four-electron reduction of O2 to H2O, the electrons being donated by NADH or succinate to complexes I and II, respectively, of the mitochondrial electron transport chain (ETC). Ubiquinone (coenzyme Q, or UQ), which accepts electrons from complexes I (NADH dehydrogenase) and II (succinate dehydrogenase), undergoes two sequential one-electron reductions to ubisemiquinone and ubiquinol (the Q cycle), ultimately transferring reducing equivalents to the remainder of the electron transport chain: complex III (UQcytochrome C reductase), cytochrome c, complex IV (cytochrome c oxidase), and finally, O2 [51]. However, it appears mitochondrial electron transport is imperfect, and one-electron reduction of O2 to form 02* occurs. The spontaneous and enzymatic dismutation of 02*~ yields hydrogen peroxide (H2O2), and so a significant by-product of the actual sequence of oxidation-reduction reactions may be the generation of 02# and H2O2. How much 02* and H2O2 do mitochondria generate? In classic experiments during the 1970s, measurements of H2O2 generation by isolated mitochondria indicated that it is maximal when ADP is limiting and the electron carriers are consequently reduced ("state 4" respiration) [56]. Estimates of state 4 H2O2 generation by pigeon and rat mitochondrial preparations amounted to 1—2% of total electron flow [56,57]. One problem with this estimate of mitochondrial H2O2 generation is its rehance on the use of buffer saturated with air (20% O2). In vivo, the partial pressure of O2 is about 5%, and so these calculations may overestimate the flux of oxidants in vivo. Even disregarding the use of air-saturated buffer, the initial estimate of percentage ETC flux leading to H2O2 can be challenged on the grounds that in these experiments, the concentrations of substrates fed to mitochondria were higher than occurs physiologically [58,59]. When H2O2 is measured with more physiological concentrations, the flux is about 10fold lower [58], and experiments using subcellular fractions of SOD-deficient E. coli suggest in vivo leakage of 0.1% from the respiratory chain [59]. What proportion of mitochondrial H2O2 ultimately derives from ETC 02#~


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generation? Unfortunately, the measurement of 02* generation by intact mitochondria is prevented by the presence of mitochondrial SOD (mSOD). Therefore, the isolation of submitochondrial particles from which mSOD has been removed (by sonication of the intact organelles followed by extensive washing), was used for the detection of ETC 02* . In these experiments, stoichiometric estimates of the ratio of 02» generation (by submitochondrial particles) to H2O2 generation (by the intact organelles) fell between 1.5 and 2.1 [60—64]; since two 02* molecules dismutate, (either spontaneously or with the help of mSOD), to form one molecule of H-O2, such results suggest that virtually all mitochondrial H2O2 may originate as 02« [65]. Moreover, as most cellular H2O2 originates from mitochondria, 02* from the ETC may be a cell's most significant source of oxidants [47]. In a recent discussion of the classic in vitro work [66], some of the original experimenters take issue with the idea that free 02» exists in mitochondria as a result of normal flux through the ETC. They point out that in addition to having removed mSOD from mitochondria, the sonication they employed also resulted in the loss of cytochrome c, which rapidly scavenges 02« in vitro and is present in mitochondria at local concentrations from 0.5 to 5 mM. In mitochondria, they argue, mSOD and cytochrome c rapidly scavenge 02* (in the matrix and intermembrane spaces, respectively). More to the point, the authors stress that unless the ETC was poisoned with inhibitors such as antimycin A, 02* generation was not detected in their experiments [60,61]. Arguing that mSOD should act to increase the rate of 02* generation in vivo (by accelerating product removal by dismutation to H2O2) they suggest that the actual role of mSOD in vivo may be to increase hydrogen peroxide generation (with 02# as a rapidly consumed intermediate) [66]. Ultimately, there remains a good deal of uncertainty surrounding the mechanisms, quantity, and meaning of mitochondrial C)2* generation in vivo [67], a mystery which is deepened by recent reports of enzymatic nitric oxide (NO«) generation in mitochondria [68—70]. Since Oj^ and NO« react to form the oxidant peroxynitrite (ONOO), mitochondrial 02»" generation may soon need to be considered in the light of its ability to destroy NO* and form ONOO , as is discussed below. A second source of oxygen radicals is peroxisomal (3-oxidation of fatty acids, which generates H2O2 as a by-product. Peroxisomes possess high concentrations of catalase, and so it is unclear whether or not leakage of H2O2 from peroxisomes contributes significantly to cytosolic oxidative stress under normal circumstances. However, a class of nonmutagenic carcinogens, the peroxisome proliferators, which increase the number of hepatocellular peroxisomes and result in liver cancer in rodents, also cause oxidative stress and damage [71—74]. Interestingly, during the regeneration of the liver following partial hepatectomy, there exist peroxisomes which do not stain for catalase activity [75], hinting that during rapid cell proliferation, oxidant leakage from peroxisomes may be enhanced. Microsomal cytochrome P450 enzymes metabolize xenobiotic compounds, usually of plant origin, by catalyzing their univalent oxidation or reduction.

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Although these reactions typically involve NADPH and an organic substrate, some of the numerous cytochrome P450 isozymes directly reduce O2 to 02#" [76,77], and may cause oxidative stress. An alternative route for cytochrome P450-mediated oxidation involves redox cycling, in which substrates accept single electrons from cytochrome P450 and transfer them to oxygen. This generates 02# and simultaneously regenerates the substrate, allowing subsequent rounds of 02« generation [51]. Although it is unclear to what extent cytochrome P450 side-reactions proceed under normal conditions, it is possible that such chronic 02» generation by cytochrome P450 is the price animals pay for their ability to detoxify acute doses of toxins [9]. Lastly, phagocytic cells attack pathogens with a mixture of oxidants and free radicals, including 02#", H2O2, nitric oxide, and hypochlorite [78—80]. Although the massive generation of oxidants by immune cells differs from the above three sources of free radicals to the extent that it is the result of pathology, it is, nevertheless, a normal and unavoidable consequence of innate immunity Chronic inflammation is, however, unique among the endogenous sources of oxidants because it is mostly preventable [81—83]. In addition to these four sources of oxidants, there exist numerous other enzymes capable of generating oxidants under normal or pathological conditions, often in a tissue-specific manner [51]. To give a single relevant example: the deamination of dopamine by monoamine oxidase generates H2O2, in some neurons, and has been implicated in the etiology of Parkinson's disease [84]. Lastly, the widespread catalytic generation of the radical nitric oxide (NO#), achieved by various isozymes of nitric oxide synthase and central to processes as diverse as vascular regulation, immune responses, and long-term potentiation, increases the potential routes for destructive oxidative reactions [85]. The interaction between 02#~ and NO# results in ONOO", which is a powerful oxidant. As originally articulated by Harman, the free radical theory of aging does not distinguish between different sources of oxidants. However, the rate of living hypothesis clearly singles out mitochondrial 02«~ and H2O2 generation, as it is the mitochondrial respiration rate which negatively correlates with MLSP Also, many other established sources of oxidants are tissue specific (associated with hepatic, neuronal, and other specialized functions), and are less likely to explain aging across a broad range of species. For this reason, mitochondrial 02«~ and H2O2 have captured the lion's share of attention. However, it may turn out that for some age-associated disorders, nonmitochondrial oxidants are critical. In the expanded sense of the free radical theory, any oxidants, mitochondrial or not, may play a role. Therefore, despite the great number of intracellular sources of oxidant that have been identified in a qualitative way, in terms of ranking their relative importance the field is in its infancy 2.3 Targets of oxidants What are the targets of endogenous oxidants? The three main classes of biologi-


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cal molecules; lipids, nucleic acids, and proteins, are susceptible to free radical attack, and there is plenty of evidence to suggest that all suffer oxidative damage in vivo. Although it is well beyond the scope of this review to treat the biochemistry of oxidative damage in any great depth, the area has been expertly reviewed [51]. A synopsis of the better known pathways of oxidative damage, however, is warranted and the most familiar end-products are described here. The earliest research on the destruction of biological molecules by oxidants involved lipids [86]. Food chemists have long understood that the rancidity of fats results from peroxidative chain reactions in lipids ("autoxidation"): a lipid hydroperoxyl radical abstracts a hydrogen atom from the double bond of a neighboring unsaturated lipid, forming a hydroperoxide and an alkyl radical, the latter which combines with O2 to regenerate a lipid hydroperoxyl radical capable of initiating another round of oxidation. Ultimately, intramolecular reactions and decomposition yield cyclic endoperoxides and unsaturated aldehydes, the latter of which are reactive and may act as mutagens [87], inactivate enzymes [88,89], or operate as endogenous fixatives, reacting with proteins and nucleic acids to form heterogeneous cross-links [90]. Moreover, a primary effect of lipid peroxidation is decreased membrane fluidity, which alters membrane properties and can significantly disrupt membrane-bound proteins [91]. Oxidative damage to nucleic acids includes adducts of base and sugar groups, single- and double-strand breaks in the backbone, and cross-links to other molecules. The spectrum of adducts in mammalian chromatin oxidized in vitro and in vivo includes more than 20 known products, including damage to all four bases and thymine-tyrosine cross-links [92—94]. The electrochemical properties of the adduct 8-oxoguanine (8-oxogua) and the deoxynucleoside 8-oxo-2,7-dihydro-2'-deoxyguanosine (8-oxodG), which have permitted the coupling of extremely sensitive electrochemical detection to HPLC chromatography, have resulted in hundreds of studies concerning its formation, accumulation, and excretion [95]. The identification of specific enzymatic repair of oxidative lesions has recently provided both proof of the significance of oxidative DNA damage, as well as tools to manipulate the load of damage in vivo by genetic knockout [95-100]. The oxidation of proteins is less well characterized, but several classes of damage have been documented, including oxidation of sulfhydryl groups, reduction of disulfides, oxidative adduction of amino acid residues close to metal-binding sites via metal-catalyzed oxidation, reactions with aldehydes, protein-protein cross-hnking, and peptide fragmentation [101,102]. A particularly intriguing recent development has been the realization that a number of enzymes possessing active-site iron-sulfur clusters are acutely sensitive to inactivation by 02* [103,104]. For example, E. coli aconitase is inactivated by 02# with a rate constant of 10^ M ' s ^ [105,106]. Mammalian mitochondrial aconitase is inactivated in vitro and in vivo by treatments which increase mitochondrial 02# generation, such as growth under hyperbaric conditions [107,108]. Since aconitase participates in the citric acid cycle, its inhibition would be expected to have pleio-

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tropic effects. Moreover, the mechanism of aconitase inhibition by 02» has been demonstrated to involve the release of free iron from the enzyme [103]. Free iron atoms catalytically exacerbate oxygen stress (see below), and it has been proposed that superoxide's genotoxicity is a function of its ability to liberate protein-bound iron [109,110]. Unlike lipids and nucleic acids, proteins represent a very diverse target for oxidative damage. Although protein oxidation has been demonstrated at the level of the peptide backbone and amino acids, there has been relatively little scrutiny of differences between proteins in their sensitivities. A detailed quantitative comparison of bovine serum albumin and glutamine synthase has shown susceptible residues of the former (methionine and the aromatic amino acid residues) to be oxidized about twice as fast as those on the latter, implicating all four levels of protein structure in relative susceptibility [111]. A study of the oxidation sensitivities of various cloned K"^ channels from T lymphocytes, cardiac cells, and neurons, revealed that whereas five of the cloned channels were highly sensitive to oxidation, an equal number were resistant [112]. Differential sensitivities raise the possibility that the loss of homeostasis which is a hallmark of aging could result from the selective oxidation of proteins. In the context of aging, a particularly relevant aspect of oxygen's toxicity is its promotion by some metals and by elevated O2 partial pressure. Iron and copper catalyze the homolytic cleavage of ROOH (the Fenton reaction), leading to the generation of •OH [51]. It is •OH which is the most reactive oxidant, reacting at diffusion-limited rates. The catalytic properties of iron and copper explains why cells possess metal chelating proteins such as ferritin and transferrin, which reduce the concentration of redox-active metals [113,114]. In humans, the body's content of iron increases with age (in men throughout their lives, and in women after menopause), and it has been suggested that this accumulation may increase the risk of oxidative damage with age [115,116]. Lastly, oxidative stress in vivo is aggravated by increasing O2 partial pressure, due to a more pronounced flux of mitochondrial 02^ [47]. Consequently, the manipulation of O2 partial pressure is a relatively simple tool that has been used to test the free radical theory. 2.4 Antioxidant defenses Cells are equipped with an impressive repertoire of antioxidant enzymes, as well as small antioxidant molecules mostly derived from dietary fruits and vegetables [46,117]. These include: (i) enzymatic scavengers such as SOD, which hastens the dismutation of 02^ to H2O2, and catalase and glutathione peroxidase (GPX), which convert H2O2 to water; (ii) hydrophilic radical scavengers such as ascorbate, urate, and glutathione (GSH); (iii) lipophilic radical scavengers such as tocopherols, flavonoids, carotenoids, and ubiquinol;


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(iv) enzymes involved in the reduction of oxidized forms of small molecular antioxidants, (GSH reductase, dehydroascorbate reductase), or responsible for the maintenance of protein thiols (thioredoxin reductase); and (v) the cellular machinery which maintains a reducing environment, (e.g., glucose-6-phosphate dehydrogenase, which regenerates NADPH). The complement of defenses deployed differs not only between organisms or tissues, but even between cellular compartments. For instance, GPX plays an important role in mammals but is absent from flies and nematodes [118,119], and there exist in humans three forms of SOD (cytosolic Cu,Zn-SOD, mitochondrial MnSOD, and extracellular SOD), encoded and regulated independently [55]. As far as supporting the free radical theory of aging is concerned, the universality of antioxidant defenses is good news. Although the nature of these defenses varies between species, the presence of some type of antioxidant defense is universal. In fact, some antioxidants, such as SOD, are very highly conserved. Clearly, an indifference to oxygen free radicals is inconsistent with life, underlining the centrality of oxidative damage. Moreover, the fact that antioxidant defenses are not uniform has been incorporated into the free radical theory; differences in antioxidant defenses between species have been put forth to explain differences in life span. Although there is something uncomfortably ad hoc in these two different interpretations of the data, they are not inconsistent. Whereas aerobic life requires organisms to cope with oxidation to some extent, different evolutionary pressures appear to have selected for more or less investment in these defenses, as is discussed in section 3 below. Lastly, a persistent problem in testing the free radical theory is that antioxidants are both parallel, (different antioxidants can play similar roles, e.g., catalase and GPX), and serial, (enzymes operate in tandem to decompose radicals to harmless products, e.g., SOD and catalase). Consequently, measurements of individual antioxidant activities do not have great relevance. In fact, as is discussed below, measurements of age-related changes in individual antioxidants have led to conflicting results [14]. For this reason, aggregate assays have been devised, such as the susceptibility of crude cellular homogenates to in vitro oxidation by ionizing radiation [120,121]. Although these assays do not provide any information about the specific mechanisms of defense, they conveniently measure overall effectiveness. 2.5 Repair of oxidative damage Unlike defenses against oxidants, which have been extensively characterized, the machinery for repairing oxidative damage is relatively unexplored. Nevertheless, it is clear that cells repair oxidized lipids, (e.g., phospholipase A2 cleaves lipid peroxides from phospholipids) [122], oxidized nucleic acids (e.g., glycosylases specifically recognize and excise oxidized bases from double-stranded DNA) [97,123—125], and oxidized proteins [126—129]. The comparative biochemistry of cellular repair is very fertile ground for the free radical theory.

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2.6 Synthesis: the interaction of oxidant generation, oxidative damage and repair The existence of multiple intracellular sources of oxidants and complex defenses has led to refinements of the free radical theory. For example, it is clear that the metabolism of oxygen radicals is dynamic, with damage resulting from an increase in oxidant generation or a decrease in antioxidant defenses. Consequently, a difference in life span between species or individuals could be due to different rates of living, or to different "rates of scavenging" [3,10]. The picture has been further complicated by the discovery of specific enzymatic repair of oxidative damage, leading to "repair" or "fidelity" versions of the theory, in which life span is determined by the failure to correct oxidative damage [122,130]. The relationship between these three components of oxidative stress; oxidant generation, antioxidant protection, and repair of oxidative damage, and the way in which they have been investigated in testing the free radical theory, is illustrated schematically in Fig. 1. Increases in oxidant generation, and decreases in antioxidant protection and repair systems, are amongst the theory's testable predictions, and have been examined both as a function of age in individuals of the same species, as well as between species of differing MLSP. Lastly, an extremely important, (if experimentally recalcitrant), aspect of the interactions between oxidants, antioxidants, and repair are feedback loops, positive and negative, between them. Antioxidant defenses and cellular repair systems have been shown to be induced in response to oxidative challenges [131 — 133], and are of course potential targets of oxidative destruction [134]. Also, the generation of oxidants may be enhanced by the malfunctioning of oxidatively damaged molecules [135,136]. Therefore, by examining Fig. 1 it is not difficult to envision ways in which primary oxidative destruction of any target, (e.g., the components of the mitochondrial ETC, scavenging enzymes such as SOD, or DNA repair enzymes), might promote further oxidative damage in what is frequently called a "catastrophic vicious cycle." Although such cycles are intuitively appealing, their documentation awaits future work and will be extremely difficult from a technical standpoint. An alternative to lab-based approaches, namely the computational modeling of these complex interactions in what has been termed a "Network Theory of Ageing," is being pursued by theoretical gerontologists [137]. Ultimately, a question of obvious importance is whether or not such cycles, if they exist, could be broken, and modeling may help pinpoint weak links for therapeutic intervention. 3

REFINEMENTS AND COROLLARIES OF THE FREE RADICAL THEORY

As the free radical theory has gained ground, it has incorporated other ideas. For example, as mentioned above, the rate of living hypothesis dovetailed with the free radical theory once mitochondrial free radical generation was confirmed.

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*l

X^

Damage

^y

Fig. 1. The ultimate outcome of oxidative stress is a function of: (a) oxidant generation, (b) antioxidant defenses, and (c) repair of oxidative damage. Bolds arrows denote damage by oxidants, and dashed arrows routes for its prevention or repair. Due to the ways in which these processes may interact, (e.g., mitochondrial oxidants may damage the enzymes responsible for repairing oxidized mitochondrial components, thereby worsening mitochondrial oxidant generation), multiple positive and negative feedback loops are possible. Aging (A) is situated at the intersection of these processes. In testing the free radical theory, changes in a—c have been measured both as a function of age, and as a function of species maximum life span potential. The similarity of the figure to the international emblem of radiation is not a coincidence; the free radical theory has its roots in radiation biology

Three other ideas that have been influential are the evolutionary concept of antagonistic pleiotropy, the somatic mutation theory of aging, and the mitochondrial theory of aging.

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3.1 Oxygen radicals and evolutionary theories of aging The intracellular generation of oxidants capable of limiting life span may appear paradoxical. It seems reasonable to expect that natural selection might have devised aerobic cells which do not leak toxic by-products. Evolutionary biologists have contributed to the free radical theory by suggesting why physiologically harmful generation of oxygen radicals occurs. They have argued that natural selection favors genes that act early in life and increase reproduction, rather than genes which act to preserve nongerm cells (the "disposable soma"), a principle called "antagonistic pleiotropy" [138—142]. The concept of antagonistic pleiotropy stresses that in the wild, reproductive success is principally a function of external factors. With the exception of modern-day humans, individuals do not usually die of old age, but are eaten, parasitized, or out-competed by others. Therefore, preserving the cells of the "disposable soma", otherwise known as the body, may be disadvantageous if it detracts from more pressing problems. The toxicity of respiratory burst oxidants, for instance, cannot easily be eliminated by evolution, since this would result in death from childhood infection. Similarly, an investment in improved antioxidant defenses maximizes fitness only if the resources are not better invested in strength, beauty, speed, and so on. The potential relevance of this concept to exercise-associated oxidative damage is clear-cut. The human capacity for strenuous exercise at peak performance, a product of evolution, was probably "selected for" because it served short-term goals, such as hunting, escaping predators or conflict, sexual display, etc. In other words, the hypothesis that exercise might have a toxicological "cost" is well in line with current evolutionary theory. 3.2 Oxygen radicals and the somatic mutation theory of aging The somatic mutation theory holds that the accumulation of DNA mutations is responsible for degenerative senescence [97,143—146]. In the case of cancer, which results from both point mutations in oncogenes and the loss of tumor suppressor gene function, (often by deletion), the role of mutations is unquestionable [117]. It remains to be seen whether or not the argument is equally valid for nonproliferative senescence. For instance, whereas significant age-related increases in somatic mutations in a reporter transgene (lacZ) have been measured in a mitotic tissue of transgenic mice, (the liver), no increase was detected in the largely post-mitotic brain of the same animals [147], suggesting that neurodegeneration, at least, is unlikely to be the result of accumulated somatic mutations in nuclear DNA. Moreover, the accumulation of mutations in the liver tissues was not dramatic, suggesting that mutagenesis may be of little functional consequence to mitotic tissues as well [148]. In light of these data, what evidence is there that somatic mutations are related to aging? A compelling argument for the somatic mutation theory of aging was provided years ago in the discovery that DNA repair ability correlates with species-specific life span [149], a phenomenon which


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has recently been reconfirmed [150]. However, it has been noted that DNA repair, which is necessary for the prevention of tumorigenesis, is necessary but not sufficient for longevity [150]. Ultimately, arguments about the physiological significance of somatic mutations hinge upon how disruptive a given mutational burden is to a cell or animal; with current methods, this is an unanswerable question. In any case, it has been demonstrated in numerous studies with prokaryotes, yeast, and mammalian cells that oxidants are mutagens, against which cells actively protect their genetic material [151,152]. Although it is not yet clear what fraction of mutations can be attributed to oxidative damage, the characterization and cloning of defense genes against oxidative mutagenesis [95], and the development of in vivo mutagenesis assays [153], has finally opened up avenues for definitive experiments. 3.3 Oxygen radicals and mitochondrial theories of aging The mitochondrion has also long attracted attention as one of the cell's weak links, an organelle whose dysfunction has profound negative pleiotropic effects [154]. Mitochondria supply ATP and also sequester potentially toxic Ca^"^ ions and yet, due to their generation of 02» and H2O2, they are on the front lines of respiratory oxidative stress. The idea that the mitochondrion is, therefore, uniquely vulnerable was embraced early on by proponents of the free radical theory [155]. In the early 1980s, Miquel and his colleagues proposed that oxidative damage to mitochondrial DNA in postmitotic cells would lead to mutations and blocks to replication, and consequently to mitochondrial dysfunction and physiological decline [143,156,157]. This "mitochondrial DNA mutation hypothesis of aging", which incorporates free radicals, somatic mutations, and the central role of mitochondria in homeostasis, is presently under intense scrutiny [16,158—171]. Of course, the fact that aerobic exercise exerts profound effects on muscle mitochondria makes these mitochondrial theories of aging of particular relevance to the topic of this volume. 4

OXIDATIVE PHENOMENOLOGY: AGE-ASSOCIATED TRENDS

The phenomenological approach to the free radical theory has involved looking for traces of oxidative damage in vivo. Phenomenology is not well suited to critically testing the free radical theory, since the data, (which are voluminous and generally supportive), mainly represent correlations. Documented increases in oxidative damage, no matter how impressive, may be a consequence of a primary, nonoxidative event. Nevertheless, phenomenology is the foundation upon which more powerful experiments depend, since the analytical methods developed for it have been used to compare species, genetic mutants, and populations with differing life spans. In fact, almost all of the biomarkers of oxidative stress described in this section have been found to accumulate at a faster rate in short-lived spe-

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cies, and in many cases, this rate correlates with O2 consumption. Famiharity with the most frequently measured endpoints is a prerequisite to assessing the free radical theory. If oxidative damage is a significant cause of cellular degeneration, then one expects to see more of it in older individuals. Oxidative damage has been described in terms of "accumulation, modification and depletion"; accumulation of end-products of oxidative damage (such as lipofuscin), modification of existing structures (such as oxidative adducts in DNA) and depletion (such as the loss of enzymatic activity or reduced thiols). 4.1 Accumulation of oxidative end-products The gradual and steady accumulation of intracellular yellow-brown fluorescent pigments, referred to as lipofuscin, occurs in numerous phyla. Lipofuscin arises prominently in postmitotic cells, where it is argued that it remains undiluted by rounds of cell division [172], and is located in small granules in secondary lysosomes. Lipofuscin is structurally complex and variable, consisting mostly of cross-linked lipid and protein residues [172—174], and is ubiquitous, documented in species as diverse as nematodes, fruit flies, rats, bees, crab-eating monkeys, crayfish. Most important, it is abundant in aged tissues, where it may occupy more than half of the volume of the cell [38]. Early on, it was discovered that incubation of amino acids with the lipid peroxidation product malonaldehyde under acidic conditions leads to the formation of lipofuscin-like fluorophores [90]. The plausibility of such a reaction, given the contents and low pH of lysosomes, suggested that lipid peroxidation in vivo leads to the formation of lipofuscin [91]. Other in vitro studies of lipid peroxidation have since uncovered a great number of routes to fluorescent, cross-linked products via promiscuous oxidative chemistry, which suggests that lipofuscin is a biomarker of lipid peroxidation [38,172,175]. Despite extensive in vitro experiments, it is not known with certainty how lipofuscinogenesis occurs in vivo, nor how lipofuscin comes to accumulate with age. Lipid peroxidation could occur throughout the cell and be followed by lysosomal phagocytosis and cross-linking of peroxidative by-products, in which case an age-related increase in lipofuscin content could be seen as the result of oxidative damage. Alternatively, an age-associated decline in lysosomal activity, (due to something besides oxidation), might increase the residence time of phagocytosed material enough to enhance lipofuscinogenesis in situ from constant amounts of peroxides [176]. In support of the latter possibility, infusion into rat brains of lysosomal proteinase inhibitors leads to the rapid accumulation of lipofuscinlike granules [177]. This scenario suggests that lipofuscinogenesis may be a consequence, not a cause, of aging. Experiments with cultured cardiac myocytes have established roles for both oxidative damage and lysosomal turnover [136]. Lipofuscin accumulates in these cells in culture, and growth under increasing O2 partial pressure from 5% to

Chapter 2 7: Oxidants and aging

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40% markedly enhances its accumulation [178]. Inclusion of iron in the growth medium further increases lipofuscinogenesis, and the iron chelator desferal depresses it, suggesting that Fenton reaction-generated •OH is an initiator [179]. Lastly, antioxidants inhibit lipofuscin formation in cultured cardiomyocytes, whereas lysosomal protease inhibitors increase it [180—182]. Even if oxidative damage is primarily responsible for depositing lipofuscin in the lysosomes of senescing animals, is it more than a biomarker of aging? It has been theorized that lipofuscin accumulation is likely to impair autophagy, as more lysosomal volume is occupied by the indigestible material [136]. As lysosomes are responsible for the recycling of materials and organelles, their failure may include the following: (i) a delay in mitochondrial turnover (with a concomitant decrease in mitochondrial efficiency or an increase in mitochondrial oxidant generation); (ii) an accumulation of oxidatively modified proteins and lipids in the cytosol awaiting degradation (potentially aggravating cytosolic lipid peroxidation); (iii) an accumulation of lipofuscin-bound iron in a redox-active form (which might promote further intralysosomal lipid peroxidation); and (iv) the disruption of lysosomal membranes (and the spillage of hydrolytic enzymes into the cytosol). Although these speculations [136] remain to be substantiated, it has been shown that when treated with sublethal doses of H2O2, cultured cells display lysosomal disruption, and leakage of the lysosomal compartment into the cytosol [183]. Also, it has been demonstrated that the sensitivity of cultured primary hepatocytes to oxidation, which was associated with a loss of GSH and an influx of calcium ions, was prevented by the iron chelator desferal [184]. What is intriguing about these results is the fact that whereas desferal stabilized the lysosomes, it did not prevent the loss of GSH nor the increase in intracellular calcium, and so it may be that it is lysosomal leakage per se, rather than peroxidative damage, which is the actual lethal step in this model of oxidative killing. 4.2 Steady-state levels of oxidative modification Unlike cytosolic proteins whose half-lives are measured in minutes or hours, some extracellular proteins are rarely recycled, and oxidative modification of these old macromolecules occurs. A class of fluorescent cross-linked molecules that is distinct from lipofuscin forms on long-lived proteins such as collagen and lens crystallin [185]. These modifications are initiated by the reaction of reducing sugars with free amino groups (glycation), a chemical sequence which is unrelated to oxidation and results in a molecule known as an Amadori product. Further nonoxidative rearrangements result in stable, cross-linked "advanced glycation end products" (AGEs) [186], whose absolute abundance appears to be an excellent biomarker of age [187]. Recently, it was discovered that oxidation is one fate of the Amadori product. Pentosidine, the name given to a cross-link involving arginine, lysine and pentose moieties, is one such a "glycooxidation pro-

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duct," the formation of which requires the presence of O2 [188]. It appears as if Amadori products themselves are a source of H2O2 in vitro, which then accelerates glucose-mediate fluorogenic collagen cross-linking in a catalase-sensitive fashion, although it is unclear to what extent this occurs in vivo [187,189]. As is the case with other AGEs, the tissue burden of pentosidine is elevated in diabetics, as a consequence of hyperglycemia. Pentosidine has been found to accumulate as a function of age in shrews, rats, dogs, cows, pigs, monkeys, and humans, yielding equivalently shaped curves in all cases [190]. It is not clear, however, how glycooxidative modifications might contribute to degeneration. It has been proposed that cross-linking in cartilage is related to its decreased elasticity and relative resistance to proteolysis in old animals [191]. However, the absolute amount of collagen pentosidine cross-hnks attained at death is much higher in long-lived than in short-lived species: 6—7 pmol/mg in 3.5-year-old shrews, 15—18 pmol/mg in 25-year-old monkeys, and 50—100 pmol/mg in 90-year-old humans [190]. In other words, it appears that the rate of pentosidine accumulation may merely be a measure of more rapid oxidative damage in short-lived species, rather than an actual cause of dysfunction. An intriguing twist to this story has been the cloning of a specific cellular receptor for AGE, called RAGE, (Receptor for AGE), that belongs to the immunoglobulin superfamily and is expressed by mononuclear cells and the vascular endothelium [192,193]. One of the effects of AGE binding by RAGE is the generation, (in mononuclear cells), of intracellular oxidants, the activation of the oxidant-sensitive transcription factor N F - K | 3 , and the induction of downstream events linked to atherogenesis [194,195]. It has recently been shown that RAGE, which is highly expressed by microglial cells in the brain [196], is a receptor for amyloid-P-peptide (AP). RAGE binding of Ap results in oxidant generation, implicated in the etiology of Alzheimer's disease [20,197]. Several amino acid residues in proteins are susceptible to oxidative modification, forming side chain carbonyl derivatives [102]. The development of sensitive methods for the analysis of protein carbonyls by Stadtman and his co-workers [198] enabled them to study oxidative modification in human brain tissue and cultured fibroblasts, as well as in rat liver. They found a 2- to 3-fold rise in protein carbonyl content between a young and old age and an increase from 10% to about 30% of the total protein pool [8]. The increase was exponential and correlated well with decreased activity of the oxidation-sensitive enzyme glucose-6phosphate dehydrogenase (G6PD). By comparison, the rise in protein oxidation in the mongohan gerbil was less dramatic, increasingly significantly in brain, heart, and testis, but not in kidney. As in human tissues, trends for the activity of G6PD correspond to the increased damage, falling in brain and heart but not in kidney [121]. Similar results have been reported in an insect model. An age-associated 2.5-fold increase in the protein carbonyl content of old vs. young houseflies has also been documented [199]. As in the case of humans, the increase occurs exponentially during the life span. The similarity of the degree and pattern of increase in insects and mammals is striking, considering the enor-


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mous diflference in their MLSP, (40 days vs. 100 years). Moreover, protein carbonyl levels increase similarly in mitochondrial extracts from the thoracic flight muscles of these animals [135]. Mitochondrial aconitase is particularly prone to oxidative modification during aging in vivo, and was identified by the immunoblotting of housefly mitochondrial protein extracts with a monoclonal antibody designed to detect protein carbonyls [200]. Carbonylation of this key citric acid cycle enzyme increased in parallel with a decline in its activity. Somewhat stronger evidence that protein oxidation may play a causative role in senescence comes from comparisons of "crawlers" vs. "fliers" of the same aging cohort. Although the two groups share the same chronological age, crawlers are phenotypically senescent individuals which have lost the ability to fly and have a shorter remaining average life span than fliers, (e.g., 9.0 days vs. 13.3 days for 10-day-old crawlers and fliers, respectively). The protein carbonyl content of crawlers was 29% higher than that of fliers [199], reflecting their greater phenotypic age, as was the degree of carbonyl modification of mitochondrial (but not cytosolic) aconitase [200]. Humans suffering from Werner's syndrome, a disease characterized by premature senescence, are individuals whose phenotypic aging is also accelerated, and they too appear to have more extensive protein oxidation. Fibroblasts from Werner's patients of all ages have a level of protein carbonyls equivalent to that in 80-year-old controls [201]. In a creative study attempting to correlate protein oxidation to a physiologically relevant endpoint, it was shown that in old mice, inter-animal variation in protein carbonyl content of two different areas of the brain, (cerebral cortex vs. cerebellum), was associated with parallel inter-animal variation in memory and motor function deficits [202]. Are protein carbonyls physiologically relevant, or are they merely markers? What are the actual consequences of protein modification? Unfortunately, there is little quantitative data with which to answer this question, although qualitative data exists. The fate of oxidized proteins may depend upon the form of damage. For example, metal-catalyzed oxidation of G6PD by iron/citrate results in a thermolabile enzyme which is a better substrate for proteolysis than is the native enzyme [203]. Rapid turnover of metal-oxidized G6PD may, therefore, proceed efficiently On the other hand, G6PD modification by 4-hydroxy-2-nonenal, a lipid peroxidation product, also inactivates the enzyme, but does not render the enzyme thermolabile or increase its degradation by proteases [88]. This diflference exists despite the fact that in both cases, the same lysine residue is affected. To make matters more complex, the cross-linking of G6PD multimers by 4-hydroxy-2-nonenal, (which predictably results in a product with lipofuscin-like fluorescence), produces a molecular species which actually inhibits the multicatalytic protease [204]. The cost to a cell of protein oxidation is presently an unknown quantity. The appearance of protein-bound 3,4-dihydroxyphenylalanine (DOPA) on •OH-damaged proteins has been characterized. When converted to a quinone, protein bound DOPA can undergo redox cycling, generating 02» . It has, therefore, been proposed that protein oxidation may contribute to the progression of

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aging not merely by the loss of protein function, but also by an acceleration of the flux of oxidants [63,205-209]. The oxidative modification of DNA has also been studied in animals of different ages, with conflicting results. Although some studies have reported a modest increase in specific oxidative adducts, single-strand breaks and abasic sites, others have been negative [97,210—213]. The failure to detect an age-related increase in oxidative adducts by the analytical chromatographic techniques typically employed may have been due to the difficulty of working close to the limit of sensitivity [95]. In fact, it has become apparent that the measurement of the adduct 8-oxodG is frequently plagued by artifacts [214—218], and that these may have compromised some published experiments. Of particular concern are measurements of 8-oxodG in mitochondrial DNA (mtDNA) [219], which have generally been higher than in nuclear DNA, but which may be particularly prone to artifacts associated with the analysis of small samples [216,219]. Moreover, it is noteworthy that even among the highly variable published estimates of 8oxodG in mtDNA are values that are equivalent to the lowest measured values of 8-oxodG in nuclear DA [220]. Due to the small number of studies of mtDNA, and the high variability between the measured values, it is not yet possible to conclude that mtDNA is, in fact, more heavily oxidized than nDNA. Encouragingly, alternative PCR-based methods for measuring oxidative damage have recently been used to compare oxidation of mtDNA and nDNA by exogenous oxidants, with the result that the former appears more sensitive than the latter [221,222], although these studies could not quantify baseline values of damage. With methodological improvements, future experiments may be more conclusive. For instance, the use of single-cell gel electrophoresis, (the comet assay), to measure single-strand breaks and abasic sites in whole rat hepatocytes in situ, revealed a statistically significant 1.5-fold increase in old rats compared with young rats [223], (although this experiment did not distinguish between oxidative and nonoxidative damage). In any case, even if the burden of oxidative adducts does increase with age, there is virtually no information about the likely effect of oxidative DNA damage in vivo, apart from the knowledge that it leads to mutations and cancer. The fact that there is active DNA repair in post-mitotic tissues (in which the danger of mutation due to replication is nonexistent), and that such repair is often targeted to transcribed regions of the genome, suggests that DNA damage itself interferes with gene expression, and is not tolerated [224,225]. This important question deserves more attention. 4.3 Oxidative depletion of biochemical pools The oxidative depletion of molecules with increasing age has not been well documented in senescent animals, since the destruction of molecules does not often leave traces. Luckily, some pathways of oxidative damage do leave biochemical fingerprints. The loss of integrity of lipid bilayers due to peroxidation is one of


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the most salient eflfects of oxidative damage [91], and results in the generation of aldehydes and alkanes. Unfortunately these are not easily measured, the widespread use of the simple and nonspecific thiobarbituric acid test notwithstanding [86]. Nevertheless, countless studies have reported an increase in thiobarbituric acid-reactive substances (TEARS) with age. Combined with other more reliable assays, these have demonstrated that there is a greater degree of lipid peroxidation in older animals [226]. The measurements of exhaled ethane and pentane is a technique that has the advantage of being applicable to humans [227]. Unlike lipofuscin and TEARS, which measure the size of a pool of destroyed molecules and require a tissue biopsy, the assay of exhaled hydrocarbons measures the rate of damage, and is noninvasive. Ereath pentane has been found to increase significantly with age in humans, suggesting that increased lipid turnover occurs with age due to peroxidation [228—230]. Refinement of the technique and elimination of the associated artifacts [231] should facilitate further testing of the free radical theory in humans. The loss of activity of several oxygen-sensitive enzymes, (G6PD, glutamate synthetase), has been reported in mammalian models of aging [8]. In houseflies, a decline in G6PD, glutamate synthetase and alcohol dehydrogenase activities has also been documented, and coincides with a dramatic loss of protein sulfhydryls [232]. Another commonly reported age-related loss is an increase in the ratio of oxidized to reduced glutathione, which may reflect a disruption of the cell's redox state [37,233]. 4.4 Age-associated trends in antioxidant defenses and repair What is the cause of age-related oxidative damage? It could result from less active antioxidant defenses and repair, but studies which have measured age-related changes in antioxidant defenses have generated conflicting results. Recent measurements of antioxidants in mongohan gerbils [121] and mice [233] are representative of the types of patterns which have been uncovered in many other studies [28,234—241]. In various tissues of gerbils, there was not a consistent pattern of change; increases in SOD and decreases in GSH were observed, whereas GPX was equivalent at different ages and catalase increased or decreased, depending upon the tissues and the age at analysis. In mouse brain, on the other hand, significant decreases in SOD, catalase, and GSH reductase were observed, although GPX levels were unchanged. Another complication is that defenses are induced in response to stress. Therefore, a higher level may indicate better protection, or alternatively, greater need for antioxidant defenses due to an increase in oxidant generation. Studies of antioxidants in rat heart and skeletal muscles illustrate this point. In heart, decreases in cytosolic SOD and GPX and increases in mitochondrial SOD and GPX were noted in older animals, and several indices of oxidative damage were also elevated [242]. From these results, it was concluded that although overall myocardial antioxidant defenses were weakened in the older animals, they were induced

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in mitochondria as a compensatory response. In skeletal muscles, in contrast, increases were observed in both cytosolic and mitochondrial forms of all of the enzymes studied [243], despite the fact that indices of lipid peroxidation were again elevated. In this case, it was concluded that both cytosolic and mitochondrial antioxidants were induced. The credibility of these hypotheses is not in question, but it is hard to see how they could be disproved. When these and similar studies of age-related antioxidant levels are combined, what remains is a confusing assemblage of ambiguous trends. Of course, interactions between antioxidants are complex, which aggravates the problem. In order to avoid the problems posed by assays of individual antioxidants, aggregate measures of antioxidant defenses have been devised. A crude but integrative measure of antioxidant defenses, for instance, is the susceptibility of a homogenate to induced oxidation. X-irradiation of a whole body homogenate of houseflies results in a linear, dose-dependent increase in protein carbonyls. When homogenates of old and young flies are compared, the rate of induction of protein carbonyls by X-irradiation is 45% higher in 14-day-old than 5-dayold flies. This suggests that the antioxidant defenses in older flies are less able to cope with oxidative stress. Moreover, the activity of G6PD, an enzyme known to be sensitive to oxidation, decreases upon X-irradiation of living flies, and does so to a greater extent in old than young animals [120]. When this assay was applied to the gerbil samples described above, in which no overall change in antioxidants was seen, a clear difference between young and old tissues emerged. Whereas 6 krad of X-irradiation induced a 20—38% increase in protein carbonyls in 5-month-old animals, it induced a 152—211% increase in 26-month old animals [121]. Similarly, although synaptosomes from young and old mice contain equivalent amounts of ATP and GSH, those of old mice were far more sensitive to GSH depletion by the diethyl maleate than those from young mice [244]. Lastly, reperfusion injury is a well-established model of oxidative stress associated with the re-establishment of blood flow following ischemia, and it causes greater oxidative damage to heart tissues of old rats than young ones [245]. The use of a polyclonal antiserum specific for adducts between lipid peroxidation end-products and proteins detected such covalent modifications of mitochondrial proteins from old but not young animals, which was associated with a more dramatic loss of respiratory capacity in the former. Whereas the baseline mitochondrial respiratory parameters (before ischemia-reperfusion) did not differ between young and old animals, the administration of a physiologically relevant stress revealed a probable age-related decline in antioxidant defenses. Another alternative to measuring absolute levels of antioxidants in old vs. young animals is to investigate the ability of animals of different ages to induce antioxidants, an approach that has been applied to the analysis of SOD in the nematode C. elegans [246]. Whereas in young animals, challenge with hyperoxia or the redox cycling compound plumbagin resulted in an increase in SOD activity. In middle-aged or old animals it actually resulted in a net loss of activity. What about repair of oxidative damage? Does its activity decrease with age?


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The bulk of evidence suggests that there is probably not an overall age-associated change in the intrinsic ability of cells to degrade damaged proteins [247,248]. Although a dramatic decrease in the activity of the oxidized protein-specific alkaline protease has been reported in old rat hepatocytes [101], no change in this activity was measured in the heart or brain of 25-month-old vs. 5-month-old gerbils [121]. In a separate work, a 50% age-related decline in a single activity (peptidylglutamyl-peptide hydrolase activity) of the hepatic multicatalytic protease was associated with its selective sensitivity, (relative to the multicatalytic protease's other activities), to metal-catalyzed oxidation, suggesting that resistance to oxidants may, (logically), characterize the proteases responsible for degrading damaged proteins [249]. Although the intrinsic protease activity may not decrease with age, there is evidence that repair of oxidized proteins may be less easily induced in response to an oxidative insult in old animals. For instance, exposure of young and old rats to 100% O2 increased the content of protein carbonyls in both groups over a 48 h period. Between 48 and 54 h of exposure, however, alkaline protease activity was induced in young animals, with a corresponding decrease in protein carbonyls to initial levels. In old animals, on the other hand, no increase in activity was observed, and protein carbonyl levels continued to rise throughout the time course [101]. There is circumstantial evidence from mutagenesis studies that either antioxidant defenses or repair of oxidative DNA damage (or both) are less efficient in old mice. The induction of somatic mutations in mice by y-irradiation is from 2.3- to 3.6-fold higher in old rather than in young animals, depending upon the dose [250]. The induction of mutations in young and old animals was reduced by feeding the animals a cocktail of dietary antioxidants, confirming that oxidants played a mutagenic role in these experiments. Therefore, the more pronounced induction of mutations in older mice is indirect evidence of decreased antioxidant defenses and repair [250]. Later experiments employing peripheral lymphocytes from young and old human subjects resulted in similar results [251]. The ability of human peripheral lymphocytes to repair oxidative DNA damage induced by H2O2 has also been found to be less efficient in cells from older donors [252]. Altogether, the results above suggest that older cells may be less able to prevent oxidative damage from occurring, and less effective at removing the damage once it has occurred. There is a clear need for more and better data about agerelated trends in defenses and repair. 4.5 Age-associated trends in oxidant generation The accumulation of oxidative damage could also result from an age-associated increase in the primary generation of oxidants, and some research suggests that this is the case. Generation of H2O2 and 02» by isolated mitochondria and submitochondrial particles from 25-month-old gerbils, for instance, is about

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150-200% that of 5-month-old animals [121], and that of aged rat heart [235] and brain [253] have also been reported to be elevated. On the other hand, a recent study which paid specific attention to maintaining physiological substrate concentrations during in vitro mitochondrial incubations failed to detect an agerelated increase in mitochondria H2O2 output [58]. Recent measurements of oxidant generation in carefully isolated rat hepatocytes from young and old animals, employing an intracellular dye which fluoresces upon oxidation, have confirmed under conditions which preserve cellular integrity that cellular oxidant generation appears to increase [171]. A similar increase in oxidant generation documented in flight muscle mitochondria of houseflies was associated with increases in the activities of every measured component of the electron transport chain except for the content of UQ [254], suggesting that an imbalance in electron transport may be the cause of aberrant reduction of oxygen. In another experiment, a population of chronologically identical 12-day-old flies was separated into phenotypically older "crawlers" and younger "fliers" as described above. Mitochondrial H2O2 generation was twice as high in the crawlers than in the fliers, reflecting their greater phenotypic age. Interestingly, oxidative damage to mitochondrial membranes and proteins has itself been impHcated in enhanced oxidant generation; exposure of isolated mitochondria to the free radical generator 2,2-azobis (2-aminopropane) dihydrochloride or the cross-linking agent glutaraldehyde resulted in mitochondria which were more able to generate H2O2 when fed exogenous substrate [254]. When these in vitro studies are combined with the above evidence of increased oxidative damage, the picture that emerges is a potentially "vicious cycle" of oxidative damage and oxidant generation. 5

INTERSPECIES COMR4RISONS

All of the data discussed in the previous section identify age-related differences within a given species. A complementary, and in some ways more powerful approach, is to compare species that have different MLSR Comparative biochemistry and physiology, inspired by the rate of living hypothesis, has played a key role in estabhshing the free radical theory Several representative studies are described below. 5.1 Oxidative damage and maximum life span potential The accumulation of cardiac lipofuscin in the monkey correlates with its cumulative O2 consumption, starting at sexual maturation [255]. Comparisons between rodents, dogs, and primate species also revealed a close correlation between specific metabolic rate and lipofuscin accumulation [256,257]. The accumulation of the glycooxidation product pentosidine is similar. Short-lived species such as the shrew, (MLSP = 3.5 years), or rat, (MLSP = 3 years), with high specific metaboHc rates, accumulate pentosidine in collagen at a rate of about 0.3—0.5 pmol/


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mg annually. Longer-lived primates such as monkeys and humans accumulate pentosidine at about a tenth as fast [190]. Interspecies comparisons of protein oxidation mirror these results. The protein carbonyl content of 15-day-old individuals of five species of fly correlated negatively with mean life span; the longest-lived species, {Drosophila melanogaster, mean hfe span = 65.5 days), had roughly a third of the level of protein oxidation as shortest-lived species, iPhaenicia sericata, mean life span = 29.5 days) [258]. The content of protein carbonyls was 20% higher in the heart, and about 50% higher in the brain, of 3.5-month-old individuals of Mus musculus (MLSP = 3.5 years) than in 3.5-month-old individuals of Peromyscus leucopus (MLSP = 8 years) [259]. The excretion of the oxo8gua and 8-oxodG in urine is a reflection of whole body oxidative hits to DNA [260]. The validity of its use to measure in vivo oxidative hits is strengthened by a recent study of 33 women, in which both O2 consumption and excretion of 8-oxodG were measured. There was a highly significant positive correlation, (p = 0.00007, r = 0.64), between O2 consumption and excretion of adducts [261]. When the urinary output of the oxidative DNA repair products oxo8gua. thymine glycol and thymidine glycol were compared in mice, rats, and humans, they were found to correlate with species-specific metaboHc rate [260,262]. 5.2 Antioxidant defenses and maximum life span potential One version of the free radical theory proposes that the differences in life span between species are due to species-specific antioxidant capacity. Early work by Cutler and his colleagues tested this association; MLSP correlated positively with SOD but negatively with catalase and GPX [3]. Recently other groups have revisited this hypothesis, with similar results. Comparisons between SOD, catalase, GPX and GSH in brain, liver and heart from mice, rats, guinea pigs, rabbits, pigs, and cows were carried out and revealed that: (i) SOD and catalase activities correlated positively with MLSP; (ii) GSH activity correlated negatively with MLSP; and (iii) GPX correlated positively with MLSP in the brain and negatively in the liver and heart [263]. Interspecies comparisons between more distantly related vertebrate Classes found either no correlation or a negative correlation between antioxidants and MLSP. For instance, in the liver of fish, frogs, birds, and mammals, strongly significant negative correlations were found for catalase, GPX, and GSH, whereas all other measured antioxidants, (SOD, GSH reductase, ascorbate, urate, GSH), failed to correlate with MLSP [264]. When the same antioxidants were measured in brain and lung tissues of the same species, virtually identical results were obtained [265,266]. Comparisons between two very closely related species of mice; the house mouse, {Mus musculus), and the white-footed mousi, {Peromyscus leucopus).

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which Uves twice as long, have revealed higher levels of SOD, catalase, and GPX in brain and heart extracts of the latter, with the differences in GPX being the most dramatic. Also, when brain homogenates of the two species were challenged with X-irradiation, protein carbonyls accumulated at a 3-fold higher rate in shorter-lived M musculus than longer-lived P. leucopus [259]. Altogether, these results suggest that the evolution of longevity has not been associated with a clear-cut increase in antioxidant capacity, at least across the broad sweep of evolution. (The comparisons between M musculus and P. leucopus invites the speculation that the more recent radiation of long-lived species may be associated with the coordinate upregulation of antioxidant defenses). In any case, what emerged from the comparative biochemistry of free radical defenses is a fascinating paradox: birds, which typically have much longer long life spans than rodents, have lower activities of antioxidants and higher metabolic rates. This paradox has inspired the interspecies comparisons of mitochondrial oxidant generation discussed below. 5.3 Generation of reactive oxygen species and maximum life span potential The rate of living hypothesis identified an inverse correlation between metabolic rate and life span, and the free radical theory supphed a convenient mechanistic link: the mitochondrial generation of oxidants. Appealing as it is, one of the problems with the rate of living hypothesis is the conspicuous lack of fit of a few groups, notably birds and primates. Both of these groups live longer than their specific metabohc rate would predict. As a result, the total lifetime oxygen consumption of these groups are quite a bit higher than other groups, (e.g., during their MLSP, pigeons and canaries consume 465 and 1,222 liters of O2 per gram, respectively, whereas mice, rats, guinea pigs, and trout consume 77, 28, 48, and 26 liters of O2, respectively) [240,266]. However, it cannot be assumed that the generation of oxidants is a direct function of metabolic rate, as mitochondria of different species (or even tissues) may exhibit different rates of intrinsic oxidant generation. These lines of reasoning by two laboratories have led to a similar line of experimentation, the results of which suggest that longevity is associated with a lesser capacity for mitochondrial oxidant generation [258,259,267—269]. For example, isolated pigeon mitochondria from brain, liver, or heart consume from 2- to 3-fold as much oxygen as isolated rat mitochondria from the same tissues. However, pigeon mitochondria generate only a third to a half as much H2O2 as rat mitochondria under the same conditions. As a result, the calculated percentage of O2 converted to 02« /H2O2 by mitochondria from lung, liver, and brain is about 10-fold lower in pigeons than in rats. This lower rate of oxidant generation corresponds with the roughly 10-fold longer life span of pigeons [269]. A second, independent comparison of mitochondrial oxidant generation by pigeons and rats found similar results [268]. Detailed respiratory comparisons have shown that generation of oxidants from both complex I and complex III of the mitochondrial electron transport chain is lower in pigeons than rats [270].


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In fact, the earliest interspecies study of mitochondrial oxidant production compared liver mitochondria of five mammalian species and thoracic muscle mitochondria of two species of fly [271], and also found that longer MLSPs were associated with lower mitochondrial oxidant generation. The mitochondria from flies produced from 6-fold more to 300-fold more oxidants than those from mammals. In a more recent comparison of heart tissues of eight diverse mammalian species of widely varying hfe span, sub-mitochondrial particles of the shortlived species were found to generate more 02* than those of long-lived species, a property correlated directly with the concentration of C0Q9, (coenzyme Q possessing nine isoprene units in its isoprenoid tail), and inversely with CoQio, (although experiments intended to demonstrate a direct relationship between CoQ9:CoQio ratio and 02# generation failed to do so) [272]. Interestingly, it appears as if the concept of intrinsic mitochondrial radical generation may also partially explain longevity differences between more closely related species. Measurements of 02» and H2O2 generation by isolated mitochondria from the mouse species M musculus and P. leucopus revealed that the former, which live half as long as the latter, also generate 48—74% more 02# and 300—500% more H2O2. (As is the case with rats and pigeons, long-lived P. leucopus has the higher specific metabohc rate of the two species [259]). Lastly, a comparison of five species of dipteran fly whose mean life spans vary by 2fold also showed that the rate of generation of mitochondrial 02* and H2O2 correlated negatively with life span [258]. Altogether, interspecies comparisons of oxidative damage, antioxidant defenses, and oxidant generation provide some of the most compelling evidence that oxidants are one of the most significant determinants of life span. 6

ENVIRONMENTAL MANIPULATION AND LIFE SPAN

6.1 Dietary restriction Limiting the dietary intake of mammals is a well established way to extend life span [273]. Interestingly, early expectations that DR would lower metabolic rate per se have not been confirmed, and so if DR attenuates oxidative damage, it is not via a simple reduction in oxygen consumption [32,274]. Rather, there is now convincing evidence that dietary restriction (DR) may act by decreasing oxidative stress and increasing antioxidant defenses and repair [275]. For example, mice whose caloric intake was restricted by 40% (DR) compared to ad libitum (AL) fed controls, and who lived 43% longer on average, had a less rapid accumulation of protein carbonyls in brain, heart, and kidney. Moreover, the generation of H2O2 and 02* by mitochondria and sub-mitochondrial particles from DR animals was lower than from AL animals, and also increased less with advancing age. In this study, however, DR did not appear to enhance antioxidant defenses [235]. In another study, 40% DR of rats increased the activities of SOD, catalase, and GPX at older ages; free radical damage, as measured by thiobarbituric

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acid-reactive material and lipofuscin accumulation, was correspondingly lower [276]. In a third study, focusing on mitochondrial oxidant generation and membrane properties of synaptosomal preparations, 40% DR reduced mitochondrial oxidant generation in both young and old samples, and prevented the agedependent decline in membrane fluidity, despite the fact that increases in the cholesterol/phospholipid ratio were common to both groups of animals [277]. Other experiments have been recently reviewed [33,275]. Dietary restriction also delays cancer incidence, which may be a reflection of fewer mutations induced by oxidants in DR animals. Dietary restriction has been shown to strengthen DNA repair, [278], although few studies have focused specifically on repair of oxidative lesions. Not only caloric restriction, but also restriction of protein intake, can forestall the occurrence of cancer in rodent models. Although the mechanism by which protein restriction operates is not clear, enhanced protection against cellular degeneration, including oxidative damage, is a likely candidate. It is, therefore, interesting that the level of protein carbonyls in animals fed a low-protein diet was significantly lower than in animals fed standard lab chow, and that treatment of the animals with y-irradiation induced a greater increase in protein carbonyls in high- than in low-protein animals [279]. 6.2 Activity restriction The metabolic activity of caged houseflies can be reduced by limiting the space available for flight, ("low activity - LA" vs. "high activity = HA"). It is expected that the LA flies should live longer due to lower O2 consumption and generation of oxidants, and this is in fact observed. LA flies exhibit more than a 2-fold increase in both mean life span and MLSR Measurements of protein carbonyls in 14-day-old LA and HA flies, which have not yet begun to die, revealed an elevation of more than 50% in HA flies relative to LA flies in both whole-body extracts [199] and in mitochondria [135]. Under unfavorable conditions, the nematode C elegans may undergo a morphological transformation to form a "dauer larva", a resting stage that does not feed, exhibits altered metabolic activity, and can survive for several times the MLSP of adult nematodes before returning to the adult stage. Interestingly, dauer larvae have also been found to have increase SOD activity [280]. 6.3 Oxygen tension Another experimental manipulation which modulates life span is growth under different O2 tensions. The growth of houseflies in an atmosphere of 100% oxygen, for example, markedly reduces their mean and MLSPs, and also increases the rate of accumulation of protein carbonyls in whole body extracts [199] and in isolated mitochondria [135]. Similarly, elevated atmospheric O2 decreased, and subnormal oxygen increased, the mean and maximum life spans of nematodes

Chapter 2 7: Oxidants and aging

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[281]. Of course, the principal drawbacks of this type of experiment are its inapplicability to mammals, and the fact that elevations or decreases in ambient O2 in such species will be likely to be confound by the overt pathology of hypoor hyperoxia. 7

SUPPLEMENTATION AND LIFE SPAN

7.1 Dietary antioxidants As one of the most intuitive approaches to testing the free radical theory, nutritional supplementation has been attempted with numerous species and compounds, with the result that mean life span has been extended in some instances [282]. In mammals, results have been mixed. To cite recent studies, life-long oral supplementation of rats with UQ was without effect on mortality [283], whereas supplementation of the Senescence Accelerated Mouse strain with "P-CATECHIN", a commercial supplement with antioxidant activities [284,285] resulted in life extension [286]. Negative results from the feeding of antioxidants have been rationalized by arguing that many of the antioxidants fed to mammals interfere with mitochondrial respiration, and so the failure of antioxidant trials to extend MLS? may have been due to their toxicity [287]. Although no large-scale human nutritional intervention trials have been aimed specifically at the study of aging, a few have been undertaken in the hopes that dietary antioxidants might help prevent cancer. Three similar and heavily scrutinized studies are the a-Tocopherol, (3-Carotene (ATBC) Prevention Study [288], the Physicians' Health Study [289] and the (3-Carotene and Retinol Efficacy Trial (CARET) [290], all of which were designed to assess the effect of antioxidants on lung cancer risks. Although the details of these enormous experiments are not important to this discussion, the overall result was instructive: there was either no decrease or a modest increase in lung incidence and mortality with pcarotene administration. These negative results, as well as negative results described above using laboratory rodents, should not be taken as evidence that the free radical theory of aging is flawed. In fact, they prove merely that a complex organism like a human or rodent is unlikely to respond predictably to crude manipulations such as supplementation with one or a small number of compounds [291], as well as that a single endpoint, (such as lung cancer), is not equivalent to aging. Since the time when the results of these human trials were announced, a number of explanations have been forwarded to explain the paradoxical promotion of cancer by p-carotene [292], which indicates that the ultimate value of these trials may be a more precise understanding of this antioxidant. Ultimately, what these experiments prove is that until the biochemistry of dietary and cellular antioxidants is better understood, supplementation trials in laboratory animals or humans in which longevity or the incidence of a single disease is the endpoint will remain unreliable; molecular gerontology should focus on more instructive experiments.

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For example, in a recent short-term feeding experiment, the thiol donor Nacetyl-cysteine, which increases intracellular glutathione concentrations, was found to markedly improve mitochondrial ETC complex activity in rats [293], a parameter of physiological function which has been noted to decrease with age, and short-term feeding with a complex antioxidant-containing plant extract attenuated oxidative damage in rat mitochondria [294]. Similarly, the nutritional supplement (R)-a-lipoic acid, which serves as both a coenzyme and antioxidant in mitochondria in vivo, attenuated an age-related loss of ascorbic acid recycling and increase in sensitivity to oxidant challenge [295]. 7.2 Pharmacological supplements Dramatic antiaging effects have been observed by Floyd and co-workers with the free radical spin trap N-tert-butyl-a-phenylnitrone (PBN). Initial studies used PBN as a spin trap to measure radical generation during ischemia/reperfusion injury of gerbil brain [296]. During reperfusion, the appearance of protein carbonyls correlated well with the loss of glutamine synthetase activity, and the appearance of PBN-dependent nitroxide radical. Interestingly, it was also discovered that pretreatment with PBN attenuated protein oxidation and loss of glutamine synthetase activity, as well as reduced lethality. In a subsequent study which investigated the effect of PBN on spontaneous protein oxidation, it was discovered that twice-daily administration of PBN to old gerbils (32 mg/kg) reversed age-related oxidative damage [297]. For example, during normal aging of gerbils the level of brain protein carbonyls increased 185% and glutamine synthetase activity declined by about 35%. A marked drop in neutral protease activity was also observed. Old animals administered PBN for 2 weeks, however, experienced a reversal of these trends: protein carbonyls dropped, and glutamine synthetase and neutral protease activities were virtually restored. This effect was reversible, as over a 2-week period following cessation of PBN treatment, the oxidation of proteins increased, and the activities of glutamine synthetase and neutral protease fell. Most impressive, however, was the performance of the PBN-treated old gerbils in a test of short-term memory: whereas the old gerbils were twice as likely to make errors as young gerbils, the old animals treated with PBN performed as well as the young [297]. Although these preliminary results held great promise, not only for the study of aging, but also for its therapeutic treatment, subsequent experiments reported that the results were irreproducible [298,299]. Further follow-up work from a third laboratory confirmed the initial findings in gerbil brain, but at the same time found no effect of PBN in lowering the level of protein carbonyls in gerbil heart or mouse brain [300]. The chronic treatment of aged rats with PBN was found to reverse age-related cognitive impairment [301]. Lastly, the administration of PBN from age 20 weeks throughout the life of a short-lived strain of mouse, (the senescence accelerated mouse), increased its life span from 42 to 56 weeks, (an increase of 33%)) [302]. As was above argued to be the case for experi-


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ments with dietary intervention, maximum worth will be derived from pharmacological experiments when they include not only endpoint mortality measurements but more focused studies of cellular effects. 8

SUMMARY

Above, we have outlined a few of the major experimental approaches to testing the free radical theory of aging. In Table 1, the strengths and weaknesses of different modes of experimentation are assessed. How powerful are different types of experiment? Has the free radical theory been supported? Before discussing the merits of individual experiments, it should be stressed that if one is to judge them by their abilities to falsify the free radical theory, they are all bound to fail, for the simple reason that the free radical theory is very hard to falsify. The absence of a predicted outcome, (for instance, life span extension by antioxidant supplementation), may often be explained by (justifiable) ad hoc reasoning. The physiology of oxidative stress, being a complex interaction of endogenous and exogenous factors, generally permits the "explanation" of negative results. How then shall one judge the different types of experiment and the theory itself? In fact, as the results discussed above illustrate, the momentum gathering behind the free radical theory is not due to any single experiment or approach, but rather derives from the extraordinarily multidisciplinary nature of current research. Although no single line of reasoning alone permits definitive conclusions, together they present a compelling case. (This philosophy, that the study of aging should employ a combination of different approaches, has been convincingly espoused before [1]). Age-related oxidative phenomenology continues to provide evidence that senescence is associated with increased oxidant generation, a decline in the robustness of defenses and repair, and an accumulation of end-products of oxidative damage. Although these trends are only correlations, the study of age-related changes has been rewarded with surprises such as the receptor for AGE (RAGE), which turns out to be involved in the etiology of Alzheimer's disease, and evidence that lysosomal lipofuscin may directly disrupt cells in vivo and in vitro. In fact, oxidative phenomenology has focused efforts on understanding the interplay between the components of oxidative stress outlined in Fig. 1. The search for evidence of age-related DNA and protein oxidation has stimulated research into DNA repair, proteolytic salvage pathways, mitochondrial defenses, and so on. Interspecies comparisons have better potential directly to test the free radical theory. As predicted, differences between long- and short-lived species have been documented in all components of oxidative stress. Perhaps most instructive, however, have been results that initially appeared contradictory, such as the fact that birds are an outlier group in rate of living calculations. Attempts to account for the discrepancy have led to promising ad hoc hypotheses about the role of mitochondrial oxidant generation in aging. Unfortunately, relatively few research-

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ers are familiar with or prepared to handle a variety of experimental animals, despite the research potential of a diverse menagerie. In contrast, the model of dietary restriction has appeal precisely because it is so well-established, homogeneous, and relevant to specific human diseases. Although early predictions that DR would result in a lower metabolic rate have proved unfounded [32], the discovery that oxidative defenses are enhanced by DR have strengthened both the free radical theory and the concept of antagonistic pleiotropy [303]. Although currently, results from DR studies are merely consistent with the free radical theory, future experiments may reveal specific pathways whereby DR upregulates defenses and repair. Manipulation of metabolic activity and oxygen concentration are interesting systems, if of somewhat questionable relevance to normal aging. One drawback of such experiments is that they have generally been limited to invertebrates; the manipulation of metabolism in mammals is less straightforward. In the future, transgenic mouse models may allow a specific manipulation of metabolic rate in a consistent genetic background. Attempts to manipulate life span with nutritional and pharmacological antioxidants obviously hold great appeal, as direct and intuitive tests of the theory Although until recently, they have not quite lived up to their promise (at least in mammals), this may have been due to a lack of the appropriate compounds. The serendipitous discovery of PBN's pharmacological properties appears to have opened up new avenues. The answer to the question of whether or not the free radical theory has yet been confirmed depends upon one's conception of what the theory predicts. In its broader sense, ("oxidants contribute significantly to the process of degenerative senescence"), the theory has clearly been validated. In the more strict sense of the theory "oxidants determine MLSP", the data are not yet conclusive, although a large body of consistent data has been generated. 9

PERSPECTIVES

Lastly, we here briefly mention aspects of oxidative physiology which are likely to receive much more attention in years to come, and we end with a discussion of the complex, (and as yet virtually unstudied), ways in which exercise may affect the process of senescence. 9.1 Nitric oxide A topic of great importance, which we have here largely omitted, is the role of nitric oxide (NO#), both as a damaging oxidant in its own right (via its reaction with O2), and as a signaling molecule which is susceptible to oxidative scavenging. NO#, the chemical previously known as endothelium-derived relaxation factor, is generated enzymatically by isozymes of nitric oxide synthase, and is involved in vascular tone, innate immunity, and neuronal signal transduction.


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Moreover, NO reacts with 02# to form peroxynitrite (ONOO ), itself a powerful oxidant. Therefore, not only must nitric oxide synthase be seen as a potential source of damaging oxidants [304,305], but 02* may now be considered physiologically relevant scavengers of the free radical signaling molecule NO* [26]. 9.2 Regulatory roles for oxidants and antioxidants: signal transduction The discovery that NO* could serve as a signaling molecule has foreshadowed a more general recognition that radical species are more than by-products. As mentioned above, the interaction between 02* and NO* indicates that both may be regulatory molecules [26]. In other ways, the relationship between oxidants and signaling is surfacing. For instance, the nitration of tyrosine residues on a synthetic peptide substrate of cell cycle kinases destroyed its ability to be phosphorylated, which illustrates a mechanism for an individual amino acid adduct to exert an amplified effect [306]. At a more physiological level, it has been shown that the sensitivity of hippocampal brain slices to muscarinic acetylcholine receptor agonists, which falls in an age-related fashion, can be restored by various antioxidant treatments, and is potentiated (in old animals) by NO* generating systems [307]. Perhaps most intriguing have been discoveries, such as the potential regulation of aconitases by O2* [106,107] and the realization that cell signaling via the ras family of tyrosine kinases involves oxidants [308], which suggest that oxidants play a role in signal transduction by design. These discoveries strengthen the free radical theory for the following reason. If the regulated, enzymatic generation of oxidants play a role in signaling pathways ("legitimate oxidants"), then even a small increase in oxidant generation by nonregulated pathways ("illegitimate oxidants") may have an exaggerated impact, by mimicking the "legitimate" process. The arrival of "redox regulation" as a viable field of inquiry, as evidenced by numerous topical recent reviews [309—311], may usher in a period when oxidants are seen as molecules of central physiological importance, in much the same way as are (for instance) lipid metabolites. As far as acceptance of the free radical theory is concerned, such a paradigm shift will probably lead to de facto acceptance of its broader conception, (the idea that oxidants play a role in the process of aging). After all, if it turns out that oxidants and oxidative reactions are central to physiology, then how could they not be central to aging as well? If the free radical theory once lacked appeal because oxidants appeared to be stoichiometrically minor by-products, those days may soon be over. On the other hand, in its more narrow conception ("oxidants define MLSP"), the free radical theory still lacks experimental confirmation. A hypothetical example will illustrate this point. Even if mitogenic stimulation by ras, for instance, were to involve oxidant generation in mice and humans, and the disregulation of ras signaling by "illegitimate" oxidants in aged individuals were shown to be important in degenerative senescence, the different life spans of these organisms still beg an explanation; it still would remain to be explained why the

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process of oxidative disregulation takes so much longer to occur in humans than in mice. In conclusion, despite the growing consensus that the oxidants and oxygen free radicals are involved in degenerative senescence, there remain countless mechanistic questions to be uncovered, as well as a central outstanding unknown: do oxidants determine maximum life span, in humans and other organisms? 9.3 Oxidants and apoptosis Another topic we have decided to omit, because of space limitations and because this is an extremely fast-moving and frequently reviewed area, is the role of oxidants and mitochondria in apoptosis. Suffice it to mention that in the previous year or two there have appeared dozens of experiments documenting either the loss of mitochondrial membrane potential [312], the release of mitochondrial cytochrome c [313], (or an "apoptosis inducing factor" [312]), into the cytosol, or the generation of oxidants by mitochondria during apoptosis [312]. Combined with the mitochondrial localization of the Bcl-2 family of anti-apoptosis proteins, and copious evidence that oxidant challenges can induce apoptosis [314—316], these experiments have shown that mitochondria are central to the process of cell suicide [312,313]. Despite this general consensus, however, there exist a number of disagreements about the details of the mitochondrion's role in the process [317], and even greater uncertainty about the role of oxidants. An equally complex problem will be defining the role of apoptosis itself in aging [148]. For instance, by eliminating neuronal cells, apoptosis may contribute to neurodegeneration, yet effective apoptosis is also clearly critical in preventing (age-related) tumorigenesis. In short, both the machinery and impact of apoptosis are still incompletely understood; conclusions about the interplay between oxidants, apoptosis and aging will have to await further experimentation [148]. 9.4 Exercise and life span Although the predictive power of the free radical theory is far from established, one can still speculate about the way in which exercise-related oxygen toxicity might affect the process of aging, and life span itself In our minds, this question centers around the interactions outlined in Fig. 1. In other words, exercise induces tremendous cellular adaptation, often in response to cellular damage, (e.g., as in the case of muscle building). Presumably, oxygen toxicity from exercise could, therefore, cause either long-term oxidative damage, (from increased oxidant generation by mitochondrial respiration) or, due to the upregulation of inducible antioxidant defenses, it could protect cells from the endogenous oxidative insults of ordinary metabolism. Discovering whether, and in what direction, exercise affects the basic processes of senescence will be an interesting question to attack. (Of course, for the average modern human, who runs a fairly high risk of dying from diseases linked to a sedentary lifestyle, the prescription for a longer and healthier life is almost certainly to exercise more!).


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10 ACKNOWLEDGEMENTS We would like to thank Caleb Finch for critically reading the manuscript, and Irwin Fridovich for oflFering numerous detailed and useful comments. This chapter is a modified version of an article which appeared in Physiological Reviews 1998;78:547-581. B.N.A. is indebted to the National Cancer Institute (Outstanding Investigator Grant CA39910) and the National Institute of Environmental Health Sciences (Grant ES01896). 11 ABBREVIATIONS A(3: AGE: ATP: C0Q9: CoQio: DOPA: DR: ETC: GPX: GSH: G6PD: HA: LA: MLSP: mSOD: mtDNA: NADH: NADPH: 8-oxodG: 8 - oxogua: PBN: RAGE: ROOH: SOD: TEARS:

amyloid P-peptide advanced glycation end-product adenosine triphosphate coenzyme Q (chain length = 9) coenzyme Q (chain length =10) 3,4-dihydroxyphenylalanine dietary restriction electron transport chain glutathione peroxidase glutathione glucose-6-phosphate dehydrogenase high-activity low-activity maximum life span potential mitochondrial superoxide dismutase mitochondrial DNA nicotinamide adenine dinucleotide, reduced form nicotinamide adenine dinucleotide phosphate, reduced form 8-oxodeoxyguanosine 8 - oxoguanine N-tert-butyl-a-phenylnitrone receptor for advanced glycation end-products alkyl hydroperoxide superoxide dismutase thiobarbituric acid-reactive substances

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299. Cao G, Cutler RG. Arch Biochem Biophys 1995;320:195-201. 300. Edamatsu R, Mori A, Packer L. Biochem Biophys Res Commun 1995;211:847-849. 301. Socci DJ, Crandall BM, Arendash GW Brain Res 1995;693:88-94. 302. Dubey A, Forster MJ, Sohal RS. Arch Biochem Biophys 1995;324:249-254. 303. Kenyon C. Cell 1996;84:501-504. 304. Poderoso JJ, Carreras MC, Lisdero C, Riobo N et al. Arch Biochem Biophys 1996;328:85-92. 305. Schulz JB, Matthews RT, Henshaw DR, Beal MR Neuroscience 1996;71:1043-1048. 306. Kong SK,Yim MB, Stadtman ER, Chock PB. Proc Natl Acad Sci USA 1996;93:3377-3382. 307. Joseph JA, Villalobos-Molina R, Denisova N, Erat S et al. Ann N Y Acad Sci 1996;786: 112-119. 308. Irani K, X i a ^ Zweier JL, Sollott SJ et al. Science 1997;275:1649-1652. 309. Suzuki YJ, Forman HJ, Sevanian A. Free Radic Biol Med 1997;22:269-285. 310. Demple B. Gene 1996;179:53-57. 311. Sen CK, Packer L. FASEB J 1996;10:709-720. 312. Zamzami N, HirschT, Dallaporta B, Petit PX et al. J Bioenerg Biomemb 1997;29:185-193. 313. Reed JC. Cell 1997;91:559-562. 314. Dypbukt JM, Ankarcrona M, Burkitt M, Sjoholm A et al. J Biol Chem 1994;269:30553-30560. 315. Johnson TM, Yu ZX, Ferrans VJ, Lowenstein RA et al. Proc Natl Acad Sci USA 1996;93: 11848-11852. 316. Slater AF, Nobel CS, Orrenius S. Biochim Biophys Acta 1995;1271:59-62. 317. Jacobson MD. Trends Biochem Sci 1996;83-86.

i,r'2000 Elsevier Science B.V. All rights reserved. Handbook of Oxidants and Antioxidants in Exercise. C.K. Sen, L. Packer and O. Hanninen, editors.

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Caloric restriction, exercise and aging Roger J.M. McCarter Department of Physiology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284, USA. Fax: +1-210-567-4410.

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INTRODUCTION 1.1 Aging processes 1.2 Caloric restriction (CR) 1.3 Physical activity 1.4 Inter-relationships between caloric restriction and exercise 1.5 Questions to be addressed CALORIC RESTRICTION AND AGING 2.1 Different regimens 2.2 Physiologic and metabolic effects 2.3 Effects of CR on pathology 2.4 Mechanisms of action EXERCISE AND AGING 3.1 Types of exercise 3.2 Physiologic and metabolic effects 3.3 Effects on longevity 3.4 Mechanisms of action CR, EXERCISE AND AGING 4.1 Overview 4.2 Experimental results 4.3 Insights from combined effects of CR and exercise SUMMARY 5.1 CR and aging 5.2 Exercise and aging 5.3 CR, exercise and aging PERSPECTIVES 6.1 Hormesis 6.2 CR, exercise and hormesis ABBREVIATIONS REFERENCES

INTRODUCTION

1.1 Aging processes Despite more than a century of very active investigation, identification of mechanisms underlying the aging of muWcellular organisms remains elusive [1,2]. Recent insight from evolutionary biologists indicates one reason for this surprising deficiency: a variety of different mechanisms may be involved in limiting the life span of different organisms. For example, animals not limited to terrestrial living such as birds and flying lemurs are protected from many predators and stresses. Such

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animals may have evolved greater longevity and an array of mechanisms of aging different from their earth-bound counterparts of a similar size [3]. Another reason for the lack of understanding is the scarcity of the experimental techniques with which the rate of aging, and in turn longevity, may be manipulated reproducibly In particular, it should be noted that manipulations which shorten the life span are not especially helpful for gaining insight into aging processes. Such manipulations may simply shorten life by damaging organs and/or promoting disease processes, rather than by modulating aging processes. In this regard caloric restriction (CR) and exercise are of special importance for probing the aging processes. This is because these two manipulations represent strategies which have been demonstrated to improve function and to extend life expectancy in a variety of species. Since extreme CR and intense physical exertion are known to be harmful, it is important to establish the characteristics of restriction and exercise which are beneficial. Also of importance, is to establish criteria for determining whether or not the beneficial effects of these manipulations are the result of slowing aging or due to changes unrelated to aging. Since mechanisms of aging have not yet been precisely identified there is little current agreement regarding definitions in this area. Medawar [4] pointed out that aging is used to describe almost all changes with time in biological properties. These changes are usually assumed to be deleterious although many properties either do not change with age or the changes which occur may not be detrimental to survival. Due to these difficulties most gerontologists focus on the survival of cohorts born at similar times and, in particular, on the exponential increase in rate of mortality which occurs with advancing postmaturational life. The survival time at which 50% mortality occurs, the median life span, is termed the lifeexpectancy of the group. This quantity is influenced by many factors, including protection from the environment and the presence or absence of disease. For example, the life-expectancy of men and women in the USA has increased dramatically during the present century, in association with decreased infant mortality and improved public health and sanitation, among other factors, as shown in Fig. 1 [5]. In contrast, the maximum life span (or time of survival of the 10% longest-lived members of the cohort) is a more robust quantity and less susceptible to change. For example, there is no evidence that the maximum human life span (about 120 years) has changed over recorded time. Extending the maximum life span, therefore, is one criterion used by gerontologists as evidence that a given manipulation has retarded aging processes [6]. Finch et al. [7] point out that the maximum life span is determined by few data points in often small cohort sizes. These authors suggest the rate of mortality doubling following attainment of sexual maturity (the mortality rate doubling time (MRDT)) may be a more rehable indicator of the rate of aging. Even this criterion is complicated by data showing that the rate of mortality does not continue to increase exponentially with age at advanced ages in cohorts of large numbers [8]. Due to these compHcations there is no single generally agreed upon test for establishing rates of aging. However, there is general agreement that functional decline and increased pathology are

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characteristic features of aging [9]. On this basis, therefore, many gerontologists accept that manipulations retarding aging processes should satisfy at least three criteria. They should: 1) increase the MRDTor some measure of maximum life span, 2) slow the age-related decline of physiological function, and 3) reduce the severity or prevent the incidence of age-related pathology 1.2 Caloric restriction (CR) Early studies by McCay and colleagues [10,11] estabhshed that feeding rats only enough food to permit maintenance of weight or small increases in weight resulted in increased longevity compared with similar rats fed ad libitum. Subsequent studies by others demonstrated decreased incidence of age-related diseases associated with reduced intake of food [12]. Systematic investigation and characterization of this effect have more recently been carried out by Walford and colleagues in mice [13], by Masoro and colleagues in rats [14], and by many others in a wide variety of species [13]. These studies have consistently demonstrated increased median and maximum life span, increased MRDT, decreased rates of functional decline and reduced age-related pathology in animals fed less food than those fed ad libitum. Thus, the CR (or food restriction, or diet restriction) paradigm is widely accepted as one which acts to retard aging processes. Mechanisms responsible for this effect are under intense investigation. Attempts to uncover the underlying mechanisms have thus far been unsuccessful. A major reason for this failure is the fact that this simple nutritional manipulation is associated with a very large number of molecular, organ and systemic changes. One such change, which has been well documented in rodents, is a surprising increase in physical activity in animals eating less food, when compared with those fed ad libitum.

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1.3 Physical activity Regular physical activity is widely regarded as an effective strategy to maintain psychological and physiological status with advancing age [15]. As noted above for CR, exercise training results in a very large number of compositional and physiological changes, many of which are in a direction counter to those associated with advancing age. Increased life-expectancy has also been found in both rodent and human studies associated with lifelong physical activity Of the many different possible types of physical activity, only aerobic or endurance-type exercise has been systematically studied with respect to survival [16]. It should be noted, however, that resistance or strength training is widely believed to also exert beneficial effects, especially with respect to maintenance of muscle function with age [17]. In contrast, lengthening of active muscles in so-called eccentric exercise is known to damage muscle fibers and may have more widespread systemic effects [18]. For any given type of exercise there exist age-related levels of intensity which are necessary to induce training effects. The intensity of endurance exercise is most often assessed by measuring the oxygen consumption required to sustain the exercise as a fraction of maximum oxygen consumption (^02max)- ^^ contrast, intensity of resistance exercise is assessed by measuring the load lifted as a fraction of the maximum load which can be lifted once only (IRM, or one repetition maximum). Lifelong effects of habitual physical activity would then be expected to be a function of both the duration and intensity of exercise, as well as the type of exercise undertaken. 1.4 Inter-relationships between caloric restriction and exercise Both of these manipulations are known to induce a large number of changes in body composition and function. Many of the changes result in the organism exhibiting a physiologically more youthful status at a given age. It is especially of note that both of these manipulations result in the organism having greater ability to maintain homeostasis in the face of most, but not all, challenges. Since reduced ability to respond to challenge is a characteristic feature of advancing age, it is tempting to explore the possibility that sustained physical activity is part of the mechanism by which CR retards aging processes. Alternatively, since both CR and exercise constitute metabolic challenges, perhaps the beneficial effects of these regimens may share a common basis in providing a chronic low level stress capable of inducing sustained mobilization of cellular defense systems. The more modest effects on longevity of regular exercise in comparison with those of CR may then be viewed in terms of the more specific nature of the metabolic challenge presented by exercise to some, but by no means all tissues. Both of these manipulations result in a reapportioning of available energy supplies away from growth, so that CR and exercising animals would be expected to exhibit lower body weights and altered body composition, if food intake is not altered.

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1.5 Questions to be addressed The primary goal of this work is to evaluate the use of CR and exercise as strategies to gain insight into mechanisms of aging. What insights may be gained from determining the shared effects of CR and habitual physical activity? Secondly, since CR results in increased physical activity, does increased physical activity play a role in the anti-aging effects of CR? Finally, both of these manipulations represent metabolic challenges to the organism. Possibly an altered metabolic rate is a factor in the retardation of aging by CR and does the increased metabolic rate of exercise reduce the otherwise beneficial effects of CR? These questions will be addressed by examining data obtained mainly in laboratory rodents, but some information is available in species ranging from fruit flies to non-human primates and in aging men and women. Discussions will cover CR and exercise separately, then in combination. 2

CALORIC RESTRICTION AND AGING

2.1 Different regimens As noted previously there is widespread agreement that CR extends longevity by retarding aging processes. The effect is indeed robust since increased longevity with CR has been demonstrated in species ranging from worms to rats, in diets of variable nutrient composition and using a variety of different regimens [13]. Studies are also currently underway to determine the validity of the effect in non-human primates and in men and women [19—21]. It should be noted that CR initiated in adulthood might not be beneficial in humans. The results of Lee and Paflfenbarger [22] demonstrate increased risk of all cause mortality for individuals experiencing weight loss of even 1 kg during a 10-year period. In contrast, the data of Bergman [23], analyzed by Jones [24], suggest no difference in the rate of aging of Australian prisoners of war held in concentration camps during World War II, in comparison with that of Australian civilians of similar age. The ubiquitous presence of the anti-aging effect of CR suggests that it acts by altering fundamental aging processes. The possibility exists that this effect may have evolved in response to sporadic food shortages in nature [25]. Thus, the effect of CR on longevity may be different or may not occur in species which have evolved under conditions of plentiful supplies of food. Such conditions would exist for example in moist tropic latitudes for plant-eating animals [25]. However, for almost all species examined to date under laboratory conditions, restriction of caloric intake has resulted in retardation of aging [1] as demonstrated in Fig. 2. The power of this simple nutritional strategy to affect aging processes may be assessed from the variety of different regimens under which it has been observed. The effect has been most systematically studied in rodents, so subsequent discussion focuses mainly on results obtained using laboratory rodents. Another important factor is the health of the animals. Conventional

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Age, Days

Fig. 2. Survival curves for male Fischer 344 rats fed ad libitum (group A, n = 115) and fed 40% less than ad libitum (group R, n = 115). Reproduced with permission from the Gerontological Society of America from [27].

rodent facilities are often compromised by the spread of infectious agents amongst animal colonies. Analysis of such facilities vs. barrier-type facilities indicates improved health and longevity in rodents maintained under specific pathogen free (SPF) conditions [26]. In order to eliminate the possibility that CR increases longevity simply by protecting against the effects of infectious pathogens, rather than by modulating aging processes, most modern studies have been conducted with rodents that were maintained under barrier protected SPF conditions [27]. In many of these studies restriction of food intake was initiated immediately after weaning and then extended over the life span. Significant extension of life has also been demonstrated to occur even when restriction was initiated in adulthood, or when restriction was carried out only during the phase of rapid growth, i.e., from 6 weeks of age to 6 months of age in Fischer 344 rats. These effects are shown in Fig. 3, from the work of Yu et al. [28]. Restriction of food was always by 40% of ad libitum intake in these rats. Degree of extension of median life span depended upon the duration of restriction (i.e., group 2 > group 4 > group 3). However, it is of interest that maximum life spans of groups 2 and 4 rats were similar (1,226 and 1,177 days, respectively), i.e., restriction of food from 6 months of age onwards was as effective as restriction of food from 6 weeks of age in extending maximum life span. Similar effects were found in two long-lived mouse strains (B10C3Fi and C57BL/6J) by Weindruch and Walford [29], who initiated restriction (about 20% below ad libitum feeding) at 12 months of age. Widely different degrees of CR have been used by different investigators, ranging from a mild restriction of 10% to severe restriction of 70% less than ad libitum levels [30]. Many studies are compromised by the absence of complete survival data or by the use of non-SPF animals and the short life span of control animals, suggesting the possibility of unhealthy conditions. However, most stud-


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Fig. 3. Survival of male SPF Fischer 344 rats with caloric restriction at different times. Group 1 was fed ad libitum. Group 2 rats were fed 40% less food than group 1 from 6 weeks of age. Group 3 rats were fed 40% less food than group 1 from 6 weeks to 6 months of age. Group 4 rats were fed ad libitum to 6 months of age, then fed 40% less food than group 1 rats from that point onwards. Reproduced with permission from Gerontological Society of America from [28].

ies suggest a direct relationship between degree of restriction and the extension of hfe. Data from four separate studies in rats and mice with varying degrees of restriction from 25 to 65% of ad hbitum intake are shown in Fig. 4. Such results indicate that retardation of aging processes depends upon the level of restriction of food intake. Taken together with the previous discussion of duration of restriction, the data suggest that both intensity and duration are important factors in the action of CR on aging, at least in laboratory rodents. In contrast, several other variables appear to be unimportant in this action, e.g., the method of restricting calories appears uncritical, since several studies involving every-other-day (EOD) feeding, meal feeding or even periodic fasting, found increased longevity [13]. Variations in composition of the diet, such as by altering protein, fat and mineral levels similarly did not affect the anti-aging action [9]. The conclusion to be drawn from a large body of data from different laboratories using various rodent strains and diets is that it is the reduced intake of calories which is the critical factor in the anti-aging action. The important question then is how the aging processes are coupled to caloric intake. Identification of this coupling would be expected to yield valuable insights regarding mechanisms of aging. The search for this coupling was initiated by identifying physiological processes and pathologies which are affected by the reduced input of calories.

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have greater FEVi.o than do sedentary individuals [83]. This ability of exercise to reset the status, or set-point, of a physiological parameter is strikingly similar to some changes in function observed with CR. Examples and implications of such resettings of physiological status with age have been explored by Richardson and McCarter [84]. In addition to slowing the decline of physiologic and metabolic parameters with age, exercise has also been viewed as an effective strategy for the prevention and treatment of age-associated diseases such as non-insulin-dependent (type 2) diabetes mellitus, coronary heart disease, colon cancer, hypertension and osteoporosis [85,86]. The conclusion is that exercise modulates many deleterious aspects of aging, including deterioration of function and increased incidence and severity of disease [86]. On this basis, exercise is a potential agent for retarding aging processes. The remaining criterion is, however, a critical factor: evidence is required that exercise modulates life span. This will now be examined in detail. 3.3 Effects on longevity The earliest studies correlating physical activity and longevity were those documenting rates of mortality associated with professions and competitive sports [87,88]. In general, the data suggested lower risk of mortality with increasing levels of physical activity Systematic investigation of the effects of exercise on longevity were first undertaken by Paflfenbarger and colleagues in an extended series of studies, summarized by Lee et al. [86]. Perhaps the best known of these, the Harvard Alumni Health Study, involved 16,936 male alumni whose activities


815

were followed from 1962 and 1966 until 1978 [89]. The age range at baseline was 35—74 years and all subjects were free of physician-diagnosed coronary heart disease. Estimates were made of the amounts of energy expended weekly on all types of physical activity, from walking to competitive sports, and mortality was then followed. In all age groups those expending up to 3,500 kcal/week on physical activity exhibited decreased risk of mortality than those expending less than 500 kcal/week. The inverse relation between risk of mortality and level of physical activity was present regardless of the presence of other risk factors such as cigarette smoking, high body mass index, hypertension, etc. The investigators concluded that the risk of inactivity was only slightly less than that associated with cigarette smoking for these individuals. The data suggested that vigorous physical activity delayed mortality by about 2 years [89]. Other studies in different, mostly male populations, suggest that the benefit of exercise is in delaying mortality rather than in extending the maximum life span [90,91]. Emerging data suggest similar effects in women and this benefit can be realized even when exercise is initiated late in life (65 years and older) [86]. Thus, a large number of studies provides evidence that regular, vigorous exercise increases life expectancy in humans but may not extend maximum life span. Extension of maximum life span has been used as a criterion for establishing that aging processes are retarded by a given manipulation. In order to establish whether or not the beneficial effects of exercise involve retardation of aging, it is essential to measure effects on maximum life span. This is difficult to establish in men and women. Investigations involving laboratory animals have been used for this purpose. The use of animal models includes both advantages and disadvantages. The age of onset of exercise and type of exercise can be controlled and precise measurement of effects on survival can be determined. However, the relevance of the outcomes to theories of aging and to effects in men and women must be carefully evaluated. For example, early animal studies frequently involved conditions inducing stress, such as in forced treadmill running or in forced swimming of rodents during daylight hours, when these nocturnal animals would usually be asleep [16]. Studies in flies have involved removing wings and/or housing conditions which prevented usual physical activity [92]. Housing conditions of rodent colonies may have involved the presence of infectious disease in many animals [72]. Interpretation of data from earlier studies is thus, difficult because of the presence of factors known to influence survival, in addition to the possible effects of exercise. Systematic studies relating survival to exercise in laboratory animals have been conducted using endurance-type training. The important variables in these studies are: 1) the use of forced or voluntary exercise, such as in treadmill running and swimming vs. voluntary wheel running; 2) the age of onset of exercise, usually following weaning, during adulthood or in late middle-age; 3) the intensity of exercise, since physiological adaptations to exercise are known to involve threshold levels of activity [93];

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4) gender, since female and male rats respond differently to exercise, with females increasing food intake but males exhibiting no change in food intake when exercising [76]; and 5) strain, not just species of animal, since different rat strains exhibit large differences in willingness or desire to exercise [16]. The earliest published work in this area apparently is that of Slonaker [70], who demonstrated decreased longevity in rats with voluntary wheel running. This result was supported by subsequent studies involving forced treadmill exercise or running in drums, by McCay et al. [56] and by Benedict and Sherman [71]. The results were viewed as confirming the Rate of Living theory of aging, in that the presumed increased daily metabolic rate associated with exercise would lead to a more rapid consumption of the fixed lifetime metabolic energy potential. In contrast, later studies by Retzlaflfet al. [94] found that 10 min of daily walking (11.5 m/min) increased both median and maximum life span of Sprague-Dawley rats. As pointed out by Holloszy and Khort [72], data of this study are unusual in many respects, such as in exercising rats being heavier than sedentary control rats and, importantly, because the sedentary control animals were remarkably short-lived. Similar results were obtained by Edington et al. [95], who demonstrated increased longevity in rats executing treadmill walking for 20 min/day at 10 m/min. Again, however, variable survival of control rats raised questions regarding the possible presence of disease in these animals. This possibility was also suggested by the finding of increased mortality when the exercise was initiated in old rather than in young rats. Most of these earlier studies, therefore, provide conflicting data and are compromised by questions related to experimental design and to the health of the animals. The issue was more directly addressed by Goodrick and colleagues in a series of studies involving male and female Wistar rats, running voluntarily in cages with wheels attached from 6 weeks of age. The data demonstrated increased median and maximum life span of running vs. sedentary rats [96,97]. The conclusion drawn was that, as in the case of CR, exercise acted as a retardation of growth and so extended life, as originally postulated by McCay et al. [10]. When exercise was initiated at later ages (10.5 or 18 months of age) there were no effects on survival [97]. In this case, the authors suggested the data were consistent with the existence of an "age threshold" for beneficial effects of exercise, as originally postulated by Edington et al. [95]. However, an alternative explanation is that the low levels of running activity of the older rats were not sufficient to induce physiological adaptations, regardless of age [72]. A similar explanation may attend the absence of longevity effects following long-term swimming activity in rats, as found by Beauchenne et al. [98]. The first studies utilizing animals known to be free of infectious disease (using SPF rats) were conducted by Holloszy and colleagues. These studies also provided the first data on pathology present at death so that an assessment could be made of whether or not the exercise modulated age-related disease. Holloszy et al. [99] used SPF male Long-Evans rats. Exercise was initiated at 6 months of age and the rats initially ran vigorously


817

— about 7 km per day. After 3—6 months running activity declined but the authors found that decreasing food intake by about 8% below ad libitum consumption resulted in sustained lifetime running. A control group of rats fed 8% less than ad libitum demonstrated no longevity effects due to this mild CR. There was a significant increase in median life span of running rats in comparison with that of sedentary rats (1,012 ± 138 days vs. 923 ± 160 days, respectively). However, there was no significant effect of running activity on maximum life span, in contrast to that found by Goodrick [96]. Moreover, histopathological analysis of tissues at death revealed no significant differences in the causes of death between exercising and sedentary rats, in contrast to food-restricted, paired weight controls, which exhibited significantly reduced neoplasms [99]. Similar results were later found in the case of female Long-Evans rats [100] for running activity initiated at 4 months of age: significant increase in median but not maximum life span in runners vs. sedentary rats. In these rats, the complication of decreased caloric intake to sustain running activity of the previous study [98] was avoided. The extensive and declining running with the age of the female Long-Evans rats is shown in Fig. 10. Holloszy et al. [99] suggested the difference between their results and those of Goodrick [96] might be due to the use of SPF vs. nonbarrier protected rats, i.e., that the control Wistar rats of Goodrick's study may have had increased mortality because of the presence of infectious disease and that exercise protected the running rats, resulting in the apparent extension of both median and maximum life span. This conclusion is strongly supported by subsequent studies involving both lifelong exercise and CR. 3.4 Mechanisms of action Existing literature provides evidence for all possible outcomes of the effects of exercise on longevity, ranging from no effect to the extension of both median and maximum life span. It is important to bear in mind that these studies deal only with endurance-type exercise and that physiological adaptations to this type of exercise are known to involve threshold levels of intensity [93]. Lack of effects on longevity may thus be a consequence of insufficient levels of exercise. Conversely, strenuous exercise is known to be deleterious in the presence of disease, so shortened life span may be related to the presence of multiple stresses (disease and exercise) rather than to aging effects. These issues, therefore, complicate interpretation of existing data. However, in the most rigorously controlled studies [99,100] consistent results were obtained. The studies suggest that beneficial physiological adaptations resulting from voluntary wheel running afford protection from disease and early death but do not slow the rate of aging. This results in an increased median but not a maximum life span, i.e., it seems that endurance-type exercise may not retard aging processes, despite its CR-like action in decreasing the fraction of consumed energy available for growth, proliferation and/or the maintenance of homeostasis. A further test of this conclusion comes from studies combining CR and voluntary wheel running.

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Part X: Aging CR, EXERCISE AND AGING

4.1 Overview The initial metabolic response to reduced food intake appears entirely appropriate to enable the animal to conserve limited energy resources, i.e., there is an acute reduction in resting and daily energy expenditure and a reduction in the thermal effect of food [36,101]. On the other hand, several studies demonstrate that CR does not result in reduced spontaneous activity of rats in their cages [28,101,102] as might be expected to further conserve energy resources. On the contrary, providing rodents (v^hose food intake has been reduced) with running v^heels results in a substantial increases in voluntary vs^heel running wîth age [102,103]. If CR and exercise separately exert beneficial effects, it may be possible then for additive benefits to be realized by combining these two manipulations. Alternatively, if increased physical activity plays a role in the beneficial effects of CR, then increasing levels of exercise might further extend the beneficial effects of CR. Several investigators have tested these hypotheses. 4.2 Experimental results This was first done by McCay et al. [56] using middle-aged "albino" rats (200—450 days old) running 2 h per day in barrels and eating a restricted diet, such that weights of sedentary rats were 10% less than those of sedentary rats eating ad libitum. Median life span was greatest in restricted running rats, but maximum life span of these rats was less than that of restricted sedentary rats. Goodrick et al. [97] initiated voluntary wheel running at 6 weeks of age in male Wistar rats fed ad libitum or fed every other day (EOD). Using this regimen, body weights of sedentary EOD rats were 20% less than those of sedentary ad libitum fed rats. Both ad libitum fed and EOD rats ran voluntarily throughout life (about 2 km/day). Voluntary running increased maximum life span in ad libitum fed rats (10^^ percentile survivors) from 88 to 115 weeks for sedentary vs. running rats respectively. In contrast, running activity decreased maximum life span in EOD rats from 158 to 145 weeks. Thus, both of these early studies suggested deleterious effects of exercise on age-related benefits of CR. However, the studies did not employ SPF animals and the data may be interpreted as an effect of exercise in possibly exacerbating effects of chronic disease in these conventionally housed animals. The first use of SPF rats was the study of Holloszy and Schechtman [103]. These authors initiated voluntary wheel running in male Long-Evans rats at 3 months of age and utilized CR of about 30% less than ad libitum. Both ad libitum fed and CR rats ran extensively throughout life, with CR rats exhibiting higher levels of daily activity, averaging about 3 km/day. Running activity decreased median life span in CR rats but there was no significant effect on maximum life span when compared with sedentary CR rats. The increased mortality between 20 and 30 months of age in the restricted runners


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suggests that effects of exercise might diminish benefits of CR, as suggested by the work of Skahcl^ et al. [104]. However, subsequent work by Holloszy [72] resulted in no early deaths in restricted runners. These effects have been recently investigated in the author's laboratory using male SPF Fischer 344 rats with CR of 40% and voluntary wheel running initiated at 6 weeks of age [102]. There were four groups of rats: A, sedentary, fed ad libitum; AE, fed ad libitum and provided with wheels in their cages; B, sedentary, fed 40% less food than A rats; BE, fed same amount of food as B rats and provided with running wheels in cages. Figure 12 illustrates the profound effect of CR on voluntary wheel running: there were immediate and large difference in running activity of AE vs. BE rats (about 1 vs. 6 km/day, respectively). AE rats greatly reduced their running activity with age. Remarkably, BE rats exhibited high, sustained running with age such that these rats were running almost 5 km/day at a time when all AE and A rats had died. Running activity significantly reduced body weights of BE rats and even the low activity levels of AE rats reduced their peak body weights (Fig. 13) and decreased oxidative stress in cardiac and liver tissues [105,106]. Despite the intense wheel running, the spontaneous movement of BE rats around their cages was not less than that of sedentary B rats or sedentary A rats. Figure 14 demonstrates that the low level of running had no effect on the survival of ad libitum fed animals (AE vs. A rats). In contrast, the high levels of running of BE rats did increase median life span but did not increase maximum life span (BE vs. B rats). No effect of intense running activity on early mortality was found, in contrast to the earlier results of Holloszy and Schechtman in Long-Evans rats [103]. Pathology at death was measured in all rats (40 rats in each of the four groups). Even low levels of exercise significantly reduced the incidence of severe nephropathy in ad libitum fed rats (AE vs. A rats) but had no effect on the incidence of pituitary tumors and lymphomas. In BE rats, exercise resulted in a sig70006000 5000 H

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Part X: Aging

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nificant increase in cardiomyopathy and did not affect the incidence of other tissue pathologies. In this study, daily metabolic rates were also measured over 24 h, with rats in their cages and under usual living conditions. Metabolic rate was expressed relative to metabohc mass (body weight to the power 0.75), since lean mass could not be determined because of the extensive tissue pathology

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48

Age (months)

Fig. 14. Survival with age in male F344 rats. Open squares: group A rats, sedentary and fed ad libitum; solid squares: group AE rats, fed ad libitum with running wheels in cages; open circles: group B rats, sedentary, fed 40% less than group A rats; solid circles: group BE rats, fed 40% less food than group A rats with running wheels in cages. There were 40 rats in each group at the start of the experiment. Reproduced with permission from Editrice Kurtis s.r.l. from [102].


821

assays conducted on all rats. Over the life span the specific metabohc rate of BE rats was significantly greater than that of other rats. It is of some interest that these rats also exhibited the greatest median life span and had maximum life span similar to that of B rats. A more recent study utilized male SPF Fischer 344 rats restricted by only 10% less than ad libitum (unpubhshed data). Running activity of these rats was sustained over most of the life span, as shown in Fig. 15, and falls between the extensive activity of the 40% CR group (BE) and the very low activity of the ad libitum fed rats (AE). There was a remarkable effect on longevity of this combined intermediate running and mild CR. Median life span (but not maximum life span) of these rats was similar to that of sedentary, 40% restricted rats. Maximum hfe span was significantly greater than that of ad libitum fed rats, but less than that of 40% restricted rats. However, studies recently concluded by my colleague Dr H. Bertrand (personal communication) demonstrate that this effect is attributable to the 10% restriction of food, rather than to the concomitant moderate running activity. It seems, therefore, that a reduction in food intake of even 10% in male Fischer 344 rats is sufficient to induce significant effects on longevity, in contrast to the Long-Evans rats utilized by Holloszy and colleagues [99,103].

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Age (Months)

Fig. 15. Variation of wheel running with age and different levels of restriction in male F344 rats. SoHd diamonds: rats fed ad libitum; open squares: rats fed 10% less than ad libitum; solid circles: rats fed 40% less than ad libitum. Values are mean ± SEM, n = 40, with wheel running expressed as a percentage of maximum running activity of BE rats. Reproduced with permission from Editrice Kurtis s.r.l. from [102].

822

Part X: Aging

4.3 Insights from combined effects of CR and exercise Studies using conventionally housed rats [56,97] demonstrated increased mortality when CR and exercise were combined. In contrast, more recent studies employing barrier maintained SPF rats found no change in maximum life span when these two manipulations were combined [102,103]. Since the rats of the later studies were known to be free of infectious disease, the data suggest that aging processes are not further retarded by combining CR with lifelong running activity. Rather, the addition of running activity to possibly unhealthy rats provided sufficient stress to promote mortality in the earlier studies. It should be pointed out that the combination of CR and exercise did result in reduction of oxidative stress in some tissues (cardiac mitochondria and liver microsomes [105,106]) but also increased the incidence of cardiomyopathy present at death [102]. Thus, lifelong intense exercise in the BE Fischer 344 rats produced both beneficial and adverse effects. Also, the small size (Fig. 13) and high levels of exercise (Fig. 12) of these BE rats resulted in the highest metabolic rate per unit mass of all rats. Despite having significantly higher specific metabolic rates over the life span than other rats, these BE rats also exhibited the highest average survival rate (Fig. 14). Thus, the life-prolonging actions of CR were not reduced by increased specific metabolic rate. These data, together with those of earlier studies [34], therefore, argue strongly against the role of metabohc rate in the action of CR on aging. The increased median longevity of BE rats over B rats (Fig. 14) is consistent with the results of many studies in exercising men and women [86], i.e., sustained exercise increases life expectancy. This result is in disagreement with those of eariier rodent studies [97,103], but suggests that when exercise of sufficient intensity is sustained over a life span in healthy animals and in healthy men and women, life expectancy, but not maximum life span is increased. The impressive extension of median and maximum life span produced by only 10% restriction of calories is of interest. This result in Fischer 344 rats is in contrast to the absence of longevity effects of an 8% restriction in the study of Holloszy and Schechtman [103]. The difference may be related to intensity of running or to different rat strains — Fischer 344 vs. Long-Evans. If the latter, this would be consistent with the suggestion of Masoro and Austad [25] that differences in evolutionary history might change the physiological response to CR, with some genetic types being more sensitive to this nutritional challenge than others. The protective effects of exercise against early mortality may be viewed in terms of protection against disease without retardation of aging. The results also suggest that increased physical activity is not an important component of the mechanism by which aging is retarded in CR. The broadly protective effects of CR apparently are not additive with the limited protection provided by sustained exercise. The data suggest that mechanisms related to the mobilization of cellular defenses against stress merit investigation, since it seems possible that both CR and exercise may act by mobilizing processes protective of cellular homeostasis. In particular, CR and exercise may act by initiating hormetic effects [107] as first sug-


823

gested by Lindell [108] and extended by Masoro [109]. These possibilities will be explored in the final section. 5

SUMMARY

5.1 CR and aging There are currently no, generally, agreed upon definitions of aging and the molecular basis of aging remains undetermined. However, there is acceptance of criteria for establishing that a given manipulation has slowed the aging processes, whatever these processes may be. These criteria include experimental demonstration of: 1) increased maximum life span or increased MRDT, 2) decreased rate of functional decUne, and 3) decreased incidence or severity of pathology. CR is widely viewed as the only manipulation known to reliably retard aging processes in mammals because it satisfies all three of these criteria. The work of many investigators demonstrates that the anti-aging effects of CR depend on the restriction of energy intake rather than on restriction of any particular dietary component. Also, this action is not related to developmental effects, since it is successful even when initiated in adulthood. Identification of mechanisms underlying this action have been unsuccessful, in part due to the very large number of functional changes which occur in long-term CR. Despite this, current research has enabled many suggested mechanisms of action to be rendered unlikely. These mechanisms include: the reduction of metabolic rate, retarded growth and reduction of adiposity A focus point of the present discussion is the possibility that increased lifelong physical activity plays an important role in the anti-aging action of CR. 5.2 Exercise and aging There is considerable evidence that long-term endurance exercise and resistance exercise result in adaptations that are counter to many of the usual deleterious consequences of aging. These include maintenance of muscle mass, decreased adiposity and improved cardiovascular and pulmonary function. Habitual exercise also decreases the risks of developing cardiovascular disease, type 2 diabetes and osteoporosis. There is evidence that intensity as well as duration of exercise is important in causing physiological adaptations. Thresholds of intensity may exist for producing particular beneficial effects, such as in maintenance of muscle mass, and effects on longevity. However, even low intensities of exercise have been shown to induce beneficial metabolic changes in animal studies. Also, epidemiological studies demonstrate that inactivity, or a sedentary life style, is by far the greatest modifiable risk factor for many age-associated diseases. Regarding effects on longevity, there is evidence in studies of men and women

824

Part X: Aging

that life-expectancy or median life span may be extended by lifelong exercise of moderate intensity Animal studies have produced equivocal results. However, in many of these studies it is not clear that the animals were healthy and free of disease. More recent work using SPF rats known to be free of infectious disease has produced consistent results: long-term voluntary wheel running of moderate to high intensity was associated with extension of median, but not maximum life span. These results have been interpreted to indicate that such exercise does not alter aging processes, but rather exerts protective effects, which enables a larger fraction of the population to live longer. 5.3 CR, exercise and aging Animal studies employing CR and exercise in combination have also produced equivocal results. Some of these studies indeed indicate detrimental effects of exercise on benefits derived from CR. The same caveats described previously regarding the importance of the animal's health apply to these results. More recent work using SPF rats has consistently demonstrated no significant effect (positive or negative) of exercise on the extension of maximum life span by CR. The absence of an effect on maximum life span is particularly striking in view of the intense running of the rats (about 4 km/24 h) over most of the life span. Data from this study [102] suggest that both physical activity and specific metabolic rate are not important components of the anti-aging action of CR. This conclusion is strengthened by recent data showing even mild (10%) CR can exert significant anti-aging effects with or without simultaneous voluntary running. In summary, studies to date suggest that both CR and exercise may act to mobilize cellular protective actions. The different characteristics of mobilization of protective mechanisms induced by CR and exercise may provide valuable insight into the anti-aging mechanism of CR and into aging processes themselves. 6

PERSPECTIVES

6.1 Hormesis Several authors have suggested that CR may exert beneficial effects because it imposes a metabolic stress on the organism resulting in the mobilization of protective mechanisms [108—110]. There is indeed an extensive literature documenting the fact that organisms across wide phylogenetic lines respond to low levels of stress by improving their functional status. Organisms subjected to such low levels of stress exhibit enhanced function, less susceptibility to environmental challenge and increased longevity, whereas high levels of the same stress result in death [107]. The phenomenon was first identified and defined as hormesis by Southam and Ehrlich in studies of naturally occurring inhibitors of fungal growth [111]. Later studies in laboratory animals extended the concept to effects of radiation and chemical agents. These were shown to induce beneficial effects.


825

including life span extension at low doses but were lethal at higher doseages [112]. It is now generally accepted that this phenomenon may be present in all cells and has been conserved throughout evolutionary development [107]. Masoro [109] has pointed out that severe reduction of caloric intake causes damage and probable death, whereas low to moderate levels of CR induce a wide range of beneficial effects including life span extension. As such, CR may represent another example of hormesis. Similar reasoning can be apphed to increased levels of physical activity Moderate levels of exercise are known to induce beneficial effects, including increased life expectancy. Both CR and exercise represent metabolic challenges to the organism and, therefore, might be expected to result in adaptive, possibly protective, mechanisms. To what extent are the life prolonging effects of CR and exercise examples of hormesis? 6.2 CR, exercise and hormesis This discussion will focus on the increased longevity associated with hormesis. The extent to which known effects of CR and exercise are consistent with the large body of data available from studies of laboratory animals exposed to low doses of radiation and chemical agents will be examined. Neafsey [113] has pointed out that increased longevity in hormesis is unrelated to decreased caloric intake, in that food intake and weight gain are often normal or increased under these conditions. Analysis of survival curves by Sacher [6] indicates that hormesis in the absence of toxic effects would not alter the rate of aging, i.e., in a plot of the logarithm of mortality rate vs. age there is no change of slope (designated oc by Sacher) and, therefore, no change in the MRDT Rather, extension of life by hormetic effects results from a reduction in the "vulnerability" parameter, a measure of the initial vulnerability of the population to causes of disease and mortality. This parameter (designated Go by Sacher) represents the intercept on the mortality rate axis of the regression of the logarithm of mortality rate vs. age. Analysis of data from several studies of CR demonstrates the effect of CR on survival is to decrease oc (decreased slope, increased MRDT) without affecting Go, in such plots [113]. Thus, Sacher's analysis suggests hormetic effects provide protection by reducing initial vulnerability without altering aging processes. CR, on the other hand exerts protective actions by slowing aging processes, without altering the initial vulnerability A further important point is that even relatively brief periods of CR result in life span extension [28] whereas termination of exposure to an hormetic agent does not result in longevity benefits for surviving organisms [113]. Further study is, therefore, needed before equating the effects of CR and hormesis on longevity. In particular it remains possible that the protective effects of these two phenomena arise from different molecular mechanisms. Effects of exercise apparently are in better agreement with hormetic phenomena, since exercise is viewed as extending life by mechanisms not related to aging processes, i.e., exercise prolongs life by reducing vulnerability (Go) but does not reduce the aging rate constant (a). It should be noted that functional

826

Part X: Aging

benefits of exercise (such as on pulmonary function) are not fully preserved after the termination of exercise [83], consistent with the absence of benefits extending beyond termination of low level stress in hormesis [113]. Molecular mechanisms associated with cellular protection and life extension in CR have been attributed to daily periods of moderate hyperadrenocorticism [109], to enhanced synthesis of mRNA [108], to decreased oxidative and glycation stress [66], and to enhanced immune function, amongst other possibilities [13]. The beneficial effects of exercise on increased longevity are not clearly related to events at the molecular level in all cells, other than by increasing rates of protein turnover in working muscle cells and in cells subserving the increased energy needs of the exercise. However, the net result is a decreased risk of mortality from all causes whatever the molecular mechanisms involved may be [74]. Molecular mechanisms of life extension in hormesis are likewise not known. Suggestions include upregulation of the heat shock (or stress) response, enhanced prostaglandin synthesis and/or increased cell proliferation, amongst other possibilities [113]. Identification of molecular mechanisms underlying the different protective effects exerted by CR and exercise promises to yield valuable insights into aging processes. As suggested by Masoro [109], one approach to achieving this end may be to investigate the involvement of hormesis in these effects. 7

ABBREVIATIONS

CR: EOD: FEV: MRDT: RM: SPF: '**'02„ax-

8

caloric restriction every-other-day forced expiratory volume mortality rate doubling time repetition maximum specific pathogen free maximal oxygen consumption

REFERENCES

1. Finch CE. Longevity, Senescence and the Genome. Chicago: University of Chicago Press, 1990; 1-662. 2. Comfort A. The Biology of Senescence. New York: Elsevier, 1979. 3. Austad SN, Fischer KE. J Gerontol 1991;46:B47-B53. 4. Medawar PB. An Unsolved Problem of Biology. London: HK Lewis Publisher, 1952. 5. Fries JF, Crapo LM. Vitality and Aging. San Francisco: WH Freeman Publishers, 1981. 6. Sacher GA. Life table modification and life prolongation. In: Finch C, Hayflick L (eds) Handbook of the Biology of Aging. New York: Van Nostrand Reinhold, 1977:582—638. 7. Finch CE, Pike QWhitten M. Science 1990;249:902-903. 8. Carey JR, Liedo P, Orozco D,Vaupel J. Science 1992;258:457-458. 9. Masoro EJ. J Gerontol 1988;43:B59-B64. 10. McCay CM, Cromwell MF, Maynard LA. J Nutr 1935;10:63~79. 11. McCay CM, Maynard LA, Sperling G, Barnes L. J Nutr 1939;18:1-13. 12. Saxton JA. Biol Symp 1945;11:177-196.


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13. Weindruch R, Walford RL. The Retardation of Aging and Disease by Dietary Restriction. Springfield, IL: Charles C.Thomas, 1988;l-337. 14. Yu BR Rev Biol Res Aging 1990;4:349-371. 15. McAuley E, Cournea KS, Lettunich J. Gerontologist 1991;31:534-539. 16. McCarter R. Ann Rev. Gerontol Geriatr 1995;15:187-228. 17. Evans WJ. Nutr Rev 1996;54:535-539. 18. Zerba E, Komorowski TE, Faulkner JA. Am J Physiol 1990;258:C429-C435. 19. Kemnitz J, Roecker EB, Weindruch R, Olsen DP, Baum ST, Bergman RN. Am J Physiol 1994; 266:E540-E547. 20. Lane MA, Ingram DK, Cutler RG, Knapka J, Barnard DE, Roth GS. NY Acad Sci 1992;673: 36-45. 21. Hass, SB, Lewis SM, Duffy PH, Erschler W, Feuers RJ et al. Mech Ageing Devel 1996;91: 79-94. 22. Lee IM, Paffenbarger R. J Am Med Assoc 1992;268:2045-2049. 23. Bergman RN. J Gerontol 1948;3:14-20. 24. Jones HB. The relation of human health to age, place and time. In: Birren JE (ed) Handbook of Aging and the Individual. Chicago: University Chicago Press, 1959:336—363. 25. Masoro EJ, Austad SN. J Gerontol 1996;51A:B387-B391. 26. Weisbroth SH. Exp Gerontol 1972;7:417-425. 27. Yu BP, Masoro EJ, Murata I, Bertrand H, Lynd FT J Gerontol 1982;37:130-141. 28. Yu BP, Masoro EJ, McMahan CA. J Gerontol 1985;40:657-670. 29. Weindruch RH,Walford RL. Science 1982;215:1415-1418. 30. McCarter R. Clin Geriatr Med 1995;11:553-565. 31. Yu BP Proc Soc Exp Biol Med 1994;205:97-105. 32. Liepa GU, Masoro EJ, Bertrand HJ,Yu BP Am J Physiol 1980;238:E253-E254. 33. McCarter R, Palmer J. Am J Physiol 1992;263:E448-E452. 34. McCarter R. Energy Utilization, In: Masoro EJ (ed) Handbook of Physiology, vol 2, Aging. New York: Oxford University Press, 1995:95-118. 35. Garrow JS. Energy Balance and Obesity in Man. London: Elsevier-North Holland, 1978: 1-195. 36. McCarter R, McGee J. Am J Physiol 1989;254:E175-E179. 37. Masoro EJ,Yu BP, Bertrand HA. Proc Natl Acad Sci USA 1982;79:4239-4241. 38. Duffy PH, Fevers RJ, Leakey JA et al. Mech Ageing Dev 1989;48:117-133. 39. Pearl R. The Rate of Living. New York: Alfred Knopf, 1928;1-185. 40. Rubner M. Das problem der Lebensdauer und seine Beziehung-en zum Wachstum under Ehrnarung. Munich: Oldenburg, 1908:1-204. 41. Yu BP Physiol Rev 1994;74:139-163. 42. Chipalkatti S, De A, Aiyer AS. J Nutr 1983;113:944-950. 43. Koizumi A,Weindruch R,Walford R. J Nutr 1987;117:361-367. 44. Yu BP Free Radic Biol Med 1996;21:651-668. 45. Matsuo M, Gomi F, Kuramoto K, Sagal M. Gerontology 1993;48:133-138. 46. Laganiere S,Yu BP Mech Ageing Devel 1989;48:221-230. 47. Masoro EJ, McCarter R. Ageing 1991;3:117-128. 48. Maeda H, Gleiser CA, Masoro EJ, Murata I, MacMahan CA, Yu BP J Gerontol 1985;40: 671-688. 49. Iwasaki K, Gleiser CA, Masoro EJ, McMahan CA, Seo E,Yu BP J Gerontol 1988;43:B5-B12. 50. Cheney KE, Liu RK, Smith GS, Meredith P, Mickey MR, Walford R. J Gerontol 1983;38: 420-430. 51. Fernandes G, Friend P, Yunis E, Good RA. Proc Natl Acad Sci USA 1978;75:1500-1504. 52. Loyd T Life Sci 1982;34:625-635. 53. Shimokawa I, Higami T Effect of dietary restriction on pathological processes. In: Yu BP (ed) Modulation of Aging Processes by Dietary Restriction. Boca Raton: CRC Press, 1994:247—266.

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90. Pekkanen J, Marti B, Nissimen A, Tuomilehto J, Punsar S, Karvonen M. Lancet 1987; 1: 1473-1477. 91. Lindsted KD,Tonstad S, Kuzma JW J Clin Epidemiol 1991;44:355-364. 92. Sohal RS, Buchan PB. Exp Gerontol 1981;16:157-162. 93. Fitts RH, Booth FW, Winder WW, Holloszy JO. Am J Physiol 1975;228:1029-1033. 94. Retzlaff EJ, Fontaine J, FuturaW Geriatrics 1966;21:171-177. 95. Edington DW, Cosmas A, McCafferty WB. J Gerontol 1972;27:341-343. 96. Goodrick CL. Gerontology 1980;26:22-23. 97. Goodrick CL, Ingram DK, Reynolds J, Freeman R, Cider NL. J Gerontol 1983;38:36-45. 98. Beauchenne RE, Dellwo W, Darabian P, Haley-Zitlin V, Wright DL. Abstr of Biological Effects of Dietary Restriction, An International Conference, Washington, DC, 1990. 99. Holloszy JO, Smith EK,Vining M, Adams S. J Appl Physiol 1985;59:826-831. 100. Holloszy JO. J Gerontol 1993;48:B97-B100. 101. Boyle PC, Storlien LH, Harper AE, Keesey RE. Am J Physiol 1982;241:R392-R397. 102. McCarter R, Shimokawa I, IkenoY, Higami Y, Hubbard G,Yu BP, McMahan, CA. Ageing 1997; 9:73-79. 103. Holloszy JO, Schechtman KB. J Appl Physiol 1991;70:1529-1535. 104. Skalicky MG, Hofecker G, Kment G, Niedermuller H. Mech Ageing Develop 1980; 14: 361-377. 105. Kim JD,Yu BP, McCarter RJ, Lee S^ Herlihy JT. Free Radic Biol Med 1996;20:83-88. 106. Kim JD, McCarter RJ,Yu BP Ageing 1996;8:123-129. 107. Furst A. Health Phys 1987;52:527-530. 108. Lindell A. Life Sci 1982;31:625-630. 109. Masoro EJ. Exp Gerontol 1998;33:61-66. 110. Totter JR. Health Phys 1987;52:549-551. 111. Southam JM, Ehrlich J. Phytopathology 1943;33:517-524. 112. Calabrese EJ, McCarthy ME, Kenyon E. Health Phys 1987;52:531-541. 113. Neafsey PJ. Mech Ageing Devel 1990;51:1 - 31.


(^'2000 Elsevier Science B.V. All rights reserved. Handbook of Oxidants and Antioxidants in Exercise. C.K. Sen, L. Packer and O. Hanninen, editors.

r^^^ OJ I


Oxidative stress and the pathogenesis of sarcopenia M.E. Lopez^T.A. Zainal^, S.S. Chung\ J.M. Aiken^ and R.Weindruch^'^ Departments of Ânimal Health and Biomedical Sciences, ^Nutritional Sciences, and ^Medicine, University of Wisconsin, Madison, WI53706, USA. "^ Wisconsin Regional Primate Research Center, University of Wisconsin and Geriatric Research, Education and Clinical Center, Wm. Middleton VA Medical Center (GRECC-llG), 2500 Overlook Terrace, University of Wisconsin at Madison, Madison, WI 53705, USA. E-mail: [email protected]

1 INTRODUCTION 2 CHANGES IN SKELETAL MUSCLES WITH AGING 2.1 Morphological changes 2.2 Changes in muscle function 2.3 Changes in metabolic capacity 3 POSSIBLE CAUSES OF SARCOPENIA 3.1 Denervation-reinnervation process 3.2 Contraction-induced injury 3.3 Satellite cell changes 4 OXIDATIVE STRESS AND SARCOPENIA 4.1 Hypothesis 4.2 Mitochondria, oxygen radicals and aging 4.3 Mitochondrial myopathies and encephalomyopathies 4.4 Age-associated abnormalities of mtDNA and the electron transport system 4.5 Oxidative damage 4.6 Antioxidant status 5 INTERVENTIONS 5.1 Exercise 5.2 Antioxidant supplementation 5.3 Caloric restriction 6 SUMMARY 7 PERSPECTIVES 8 ACKNOWLEDGEMENTS 9 ABBREVIATIONS 10 REFERENCES

1

INTRODUCTION

A loss of motor ability occurs with senescence in mammals and in other classes of animals. In humans, physical frailty increases markedly with old age leading to a myriad of medical and social problems such as injuries, lack of independence and institutionahzation [1,2]. The annual costs imposed by physical frailty in the USA in 1990 were estimated at nearly US$80 billion and, by 2030, this could exceed US$130 billion (in 1990 dollars) unless interventions are found [3].

Part X: Aging

832

Although deleterious changes with age in the nervous and cardiovascular systems, among others, contribute to physical frailty, endogenous alterations occurring in skeletal muscle may underlie muscle loss and dysfunction. The loss of skeletal muscle mass during aging, often referred to as sarcopenia, is reflected by decreases of 25—50% in the cross-sectional area of several limb muscles due to muscle fiber atrophy and loss [4] (Fig. 1). Muscle mass appears to be the main determinant of the loss of strength with aging [5]. Because of the enormity and universality of sarcopenia, it is important to understand the underlying mechanisms so that interventions can be rationally designed. Several hypotheses have been proposed to explain the causes of sarcopenia, including cumulative effects of contraction-induced injuries [6], decline in the recruitment of satellite cells to replace damaged muscle fibers [7], decline in the number of motor units [8] and increased oxidative stress/damage [9]. This latter hypothesis is one of the main focuses of this article. The essential feature of the oxidative stress hypothesis of aging is that under normal physiological conditions, the use of oxygen by cells generates toxic reactive oxygen species (ROS) and their metabolites (ROM), which cause macromolecular damage [10,11] (Fig. 2). Some oxidative damage is irreversible, progressively accumulating with age, and is postulated to be the main cause for the decline in physiological vigor with aging. Besides causing molecular damage, ROM also influence normal cellular functions (e.g., transcriptional control, signal transduction and apoptosis [12,13,14]). Therefore, increases in oxidative stress with aging may alter key pathways which control cellular functions. Although the oxidative stress hypothesis has garnered some support, its validity has not been broadly tested, especially in relation to functional losses such as those occurring in skeletal muscle. Skeletal muscle, brain and heart, which are largely composed of postmitotic cells dependent on oxidative energy metabolism, are logical sites where oxidative damage may accrue, irreversibly damaging cells and thereby being especially problematic. o

CM

E E, 5000n

SlO-

H2O2

Secondary ROS Hydroxy!, peroxyl, peroxynitrite, etc.

T ^ Macromolecular oxidative damage_ (DNA, RNA, proteins, lipids)

y Repair or turnover

Alteration of redox-sensitive cellular functions (signaling pathways, apoptosis, etc.)

Irreversible molecular damage Physiological attrition Aging, disease and death Fig. 2. Proposed involvement of oxidative stress and damage in aging. An X indicates the interdiction by nonenzymatic antioxidant defense mechanisms. SOD = superoxide dismutase. (Modified from Weindruch and Sohal [234].)

If the oxidative stress hypothesis is vahd, it should explain the mechanism of the decreased stamina and strength associated with aging. Stamina depends on the steady supply of adenosine triphosphate (ATP), the main source of which is the mitochondrion. Mitochondria are not only the main producers of ATP, but also the main consumers of oxygen and generators of ROM. Accordingly, mitochondria may also be the victims of the ROM that they generate. Self-inflicted damage to mitochondria may represent the primary factor for loss in muscle mass and be the hallmark of senescence. This chapter first provides a noncomprehensive overview of sarcopenia by summarizing age-associated morphological, functional and metabolic changes which occur in skeletal muscles. Next, the possible causes of sarcopenia are discussed. This is followed by a more detailed appraisal of one suggested cause: oxidative stress of mitochondrial origin. The development of interventions to

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Part X: Aging

attenuate the progression of sarcopenia is an important area of inquiry and the final topic surveyed. 2

CHANGES IN SKELETAL MUSCLE WITH AGING

2.1 Morphological changes A gradual decrease in muscle fiber diameter, degeneration of sarcoplasm and replacement of muscle fibers by fat and connective tissue are some of the changes that occur with age in skeletal muscles. The result is an age-associated decrease in the percentage of fat-free mass and an increase in the percentage of body fat [15—17]. Three prominent age-associated morphological changes which contribute to this phenotype are discussed herein: muscle atrophy, changes in fiber type and denervation. 2.1,1 Muscle atrophy Both muscle fiber loss and atrophy may contribute to sarcopenia [18]. A consequence of senile muscle atrophy is reduced muscle strength, which lowers the ability of older persons to engage in physical activity. In humans, muscle atrophy has been evaluated by measuring the cross-sectional area (CSA) of the muscle using ultrasound, computerized transaxial tomography or magnetic resonance. CSA and volume of plantar flexor muscle were, 12 and 18% lower in 58- to 68year-old persons compared to 21- to 33-year-olds, respectively, [15]. When whole vastus lateralis (VL) cross-sections were studied, both the muscle area and the total fiber number decreased by '^ 40% in people from 20 to 80 years of age [18] (Fig. 1). It was concluded that muscle CSA loss begins as early as 25 years, leading to about a 10% decrease by age 50 and then continues at an accelerated rate. As capillary beds provide oxygen and energy substrates to muscle fibers and remove the byproducts of muscle activity (e.g., lactate), vascularization might influence the functional performance of muscle fibers. However, age-related changes in the number of capillaries around a fiber have not been reported in old rats for the soleus, flexor digitorum longus (FDL) and extensor digitorum longus (EDL) [19—21]. In humans, no differences with age occurred (but only up to 65 years) in the capillary ratio of the VL [17]. These data suggest that decreased capillarity is not a major contributor to the deleterious changes observed in skeletal muscle with aging. One advantage of animal models (e.g., laboratory rodents), is that whole muscles from healthy old animals can be obtained, weighed and fiber numbers and types determined. There exist several reports describing age-associated decreases in muscle weight in old rats, three of which are summarized in Table 1 and Fig. 3. These data show muscle-specific decreases in mass of 11—38% in rats at variable stages of senescence. Findings on the influence of age on muscle fiber num-

Chapter 29: Oxidative stress and the pathogenesis of sarcopenia

835

Table 1. Decreases in muscle weight in senescent rats. Ages (months)

Muscle

% Decrease

Reference

10,29

Gastrocnemius Plantaris Quadriceps Soleus Gastrocnemius Plantaris Soleus EDL Gastrocnemius Plantaris EDL Soleus

27 27 27 11 38 37 18 16 25 22 15 13

[36]

6,36

9,31

[24]

[232]

ber in rats are somewhat contradictory. We observed a 26% drop in VL fiber number in 4- vs. 32-month-old male Wistar rats [22]. When studying rat soleus, Larsson and Edstrom [23] found a 13% decrease in fiber number in marginally old rats (~ 22 months), when compared to young (6 months). However, several other investigators have reported no age-related changes (Table 2). Thus, the effects of aging may be muscle-specific and depend on whether the muscle is weight-bearing. For example, soleus is a weight-bearing muscle while EDL is not. Only limited attention has been given to studying sarcopenia in very old rats and mice (> 30 months old). Thus, laboratory rodents appear underutilized as models of sarcopenia in very old (> 80 years) people. IMice and rats from longlived strains at 20 and 30 months of age are, respectively, 50% and 75% through the ^ 40-month maximum life span typically attained. Thus, based on mortality characteristics, they would most closely resemble people less than 80 years of C\J

3000

A.

2500 X

o LU

2000-1

I

5000-

^ <
30 months) can proliferate in vitro [90], albeit at a lower proliferative potential [91]. The decline in proliferative potential was most pronounced during the first weeks of life and continued at a lower rate thereafter. The reduced number and proliferative potential of satellite cells are hypothesized to explain the low regenerative potential of muscle with age. Several studies have examined muscle recovery after denervation (e.g., [92, 93]). In the muscles which are reinnervated, the degree of recovery depends on the ability of the nerve to support the fibers, as well as the ability of the fibers to respond to the nerve. Muscle mass has been shown to decrease more than 50% within one month of denervation [92], such that the time to reinnervation also affects the degree of recovery. Immediately after denervation, there is an increase in the number of satellite cells, presumably in response to the muscle injury; however, after about 2 months, the number of satellite cells decreases [89,93]. A significant question raised by Carlson [7], is whether satellite cells from aged muscles are in sufficient numbers and have the regenerative capacity to restore the cytoplasmic volume of the muscle fibers. If not, satellite cell changes with age may play an important role in the fiber loss and atrophy. 4

OXIDATIVE STRESS AND SARCOPENIA

Although the above hypotheses provide compelling evidence for important roles of neurons, satellite cells and injury in sarcopenia, they do not exclude a causal role of oxidative stress in muscle loss. For example, what molecular events lead to age-associated motor neuron loss and sarcomere weakening? What causes satellite cell number decline and reduced in vitro proliferative capacities with age? Could these age-associated changes result from oxidative damage over time? Consistent with this scenario is the observation by Zerba et al. [81] that the secondary injury after repeated muscle contractions was diminished by SOD treatment, implicating oxidative damage as mediating the injury The possible contributions of oxidative stress/damage to the three hypotheses of sarcopenia just considered have not been closely evaluated. There exists, however, evidence which argues for independent contributions of oxidative stress/damage in sarcopenia.

846

Part X: Aging

4.1 Hypothesis We are investigating the contributions of oxidative stress of mitochondrial origin to sarcopenia (Fig. 4). Our hypothesis is that oxidative stress, originating principally from normal cellular respiration, damages mitochondria, the sites of the electron transport system (ETS). The ROS and ROM, which are generated as abnormal byproducts of the ETS, damage diverse macromolecules (lipids, proteins and nucleic acids). This damage results in a less efficient, "leaky" ETS which produces less ATP and more ROS, further damaging mitochondria. With age, cells would have an accumulation of oxidatively damaged mitochondria which cannot generate sufficient ATR The ultimate consequence may be cellular death. Since skeletal muscle and nervous tissues are mostly postmitotic and therefore, have limited regenerative capacities, they would appear to be quite vulnerable to such damage. Accordingly, we hypothesize that the loss of skeletal muscle fibers and perhaps motor neurons may result, in part, from mitochondrial ROS and ROM generation. There are reasons to believe that this scenario may contribute to the development of sarcopenia. Free radicals have been proposed as contributing to other situations of muscle damage, such as that occurring after exercise or in muscular dystrophy, malignant hyperthermia, ischemia/reperfusion damage and alcoholic and inflammatory myopathies [94]. Skeletal muscle accounts for a large share of the body's total oxygen consumption at rest, due to its large mass, and most of the oxygen consumption during vigorous physical activity Also, as noted, skeletal muscle, as a postmitotic tissue, has low repair capacities that occur in more mitotically active tissues. Here we consider the contributions of mitochondria and oxidative stress/damage to the development of sarcopenia. 4.2 Mitochondria, oxygen radicals and aging The mitochondrion is unique to mammalian cellular organelles in that it contains its own ^16.5 kb double-stranded circular DNA genome. It encodes 22 tRNAs, 2 rRNAs and 13 polypeptides of the ETS (Fig. 5). The genome is abundant in every nucleated cell with two to 10 copies per mitochondrion [95] and 10s to 100s of mitochondria per cell, depending on the cells energy requirements [96]. The enzyme complexes of the ETS are encoded by both nuclear and mitochondrial genes (Fig. 6). Complex I (NADH dehydrogenase) and complex IV (COX) contain the largest number of mitochondrial subunits, with mtDNA encoding seven of approximately 40 subunits of complex I and three of the 13 A '^'^'^ 1 oxidative^ mtDNA stress ^ deletion

->

ETS defects

1 oxidative>^ ETS abnormal fiber stress ^

-^

fiber atrophy and loss

Fig. 4. Hypothesized mechanism for the involvement of oxidative stress in skeletal muscle fiber atrophy and loss during aging. Whether or not oxidative damage causes mtDNA deletions is uncertain.


847

ATPaseS K ' ATPaseS

Fig. 5. Organization of mammalian mitochondrial genome. Letters indicate tRNA genes. OH and OL are heavy and light strand origins of replication, respectively.

Inter membrane space

TRANSLOCATOR ATP

MtDNA 7 Nuclear 32 Fig. 6. Complexes of the ETS and oxidative phosphorylation in the mitochondrial inner membrane. Complex numbers are circled. The number of subunits contributed by either the mitochondrial or nuclear genome is indicated below each complex. (Adapted from Wallace [235].)

848

Part X: Aging

subunits of complex IV. Complex II (SDH) is entirely nuclear encoded while complex III (ubiquinol cytochrome c reductase) has one of its 10 peptide subunits encoded by mtDNA. Age-associated damage to the mtDNA encoding these subunits, therefore, may reduce the enzymatic activities of the complexes [97]. The mitochondrion, as the site of cellular oxidative respiration, has an interior rich in oxygen. It has been estimated that ^ 90% of cellular oxygen is consumed in mitochondria and -^ 2% of that oxygen is converted to superoxide free radicals by complexes of the ETS in the mitochondrial inner membrane [98]. High levels of ROM may damage mitochondrial (and nonmitochondrial) macromolecules such as lipids, proteins and DNA. Accordingly, mitochondria represent potential hotspots for damage and, therefore, several investigators have postulated a mitochondrial role in aging. Harman [99] originally proposed the free radical theory of aging which focused on cellular damage via lipid peroxidation. Soon thereafter, Szilard [100] hypothesized a somatic mutational theory of aging, arguing that the aging process is the result of an accumulation of mutational hits in the nuclear DNA over time. The theory has subsequently been extended to include the idea that, compared to nuclear genome, mtDNA is more susceptible to oxidative damage due to the presence of higher levels of ROS, a lack of protective histones and less efficient DNA repair [11,101,102]. 4.3 Mitochondrial myopathies and encephalomyopathies Support for the mitochondrial/oxidative stress hypothesis of sarcopenia derives from the occurrence of mtDNA abnormalities in human neuromuscular diseases. Histological evaluation of mitochondrial enzymatic activities has revealed abnormalities in a broad class of neuromuscular disorders known as mitochondrial myopathies and encephalomyopathies (reviewed in [97,103,104]. The disorders include myoclonic epilepsy and ragged red fiber (RRF) disease, mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), Kearns-Sayre syndrome and Leber's hereditary optic neuropathy (LHON). These diseases are, clinically and biochemically, a diverse group of disorders, affecting primarily those tissues having the highest energy demands: brain, skeletal muscle and heart. The mitochondrial abnormalities associated with these diseases range from point mutations in the case of LHON [105] and MELAS [106] to large mtDNA deletions in the mitochondrial encephalomyopathies (reviewed in [103,104]). RRFs, so named because of their appearance when stained with Gomori trichrome [107], occur at high levels in skeletal muscle from mitochondrial myopathy patients. RRFs result from an abnormal accumulation of mitochondria causing hyperreactive staining for the ETS's entirely nuclear-encoded complex II (designated SDH"^"^ fibers). Fibers negative for COX activity (designated COX fibers) also occur in myopathy patients. These two enzymatic defects can co-localize within the same fiber [104, 108—110]. Mitochondrial DNA abnormalities have been linked to these oxida-


849

tive defects in skeletal muscle from myopathy patients [109,111,112]. For these studies, in situ hybridization, using probes to normal and mutant mtDNA, was combined with histochemical techniques for the localization of COX and SDH activities. These studies have shown that most of the affected fibers contain increased levels of mutant mtDNA, with a corresponding decrease in levels of wild type mtDNA. These results indicate that mtDNA abnormalities can localize in diseased muscle fibers where they may cause oxidative defects. 4.4 Age-associated abnormalities of mtDNA and ETS Mitochondria may be causally involved in the aging process [10,113,114]. The number of mitochondrial genomes containing large deletions increases with age in several tissues [115—118]. These age-associated mtDNA deletions assume multiple forms in humans [115,116,119—121]. Studies of age-associated mtDNA deletions have also been performed in animal models, with multiple deletions detected in skeletal muscles of rhesus monkeys [122,123], as well as in several tissues of mice [124—127] and rats [22,128,129]. Most of these studies detected the highest deletion levels in nervous tissue, heart and skeletal muscle. The physiological importance of the age-associated occurrence of mtDNA deletions remains unclear. Analysis of skeletal muscle homogenates has detected very low levels of these age-associated mtDNA deletions (typically .: F 't k

W'y

iL-

'..".t^

•''.

853

^îm^^''

' . .1

Fig. 8. Fiber atrophy occurs in COX /SDH^^ fibers. COX (A,F,G,J) and SDH (B-E,H,I,K,L) stains from a 25-year-old (A-F) and 32-year-old (G-L) rhesus monkey Arrows indicate a representative atrophic fiber in pictures A-F and another in pictures G-L. Total fiber length examined from A to F is 624 |im and from G to L is 448 fim. (From Lee et al. [135].)

P450 electron transport in the endoplasmic reticulum [154] are all fundamental and continuous sources of ROS. As we discuss elsewhere, mitochondria are presumed to be the primary cellular sites of ROS production and oxidative damage incurred during aging. We now review evidence supporting the accumulation of oxidative damage in mammalian skeletal muscle lipid, DNA and protein during aging.

Part X: Aging

854 Table 5. Oxidative damage increaseswith age in skeletal muscle Observation

Species

Muscle

Ages

Reference

Lipofuscin

Rat Human Rat Rat Rat Rat Rat

Soleus VL Tibialis Gastrocnemius VL Soleus Soleus VL Hindlimb VL VL Hindlimb Hindlimb VL Hindlimb Hindlimb VL Hindlimb Hindlimb Hindlimb Hindlimb

29 months 17—76 years 2, 11, 30 months 12, 24 months 4,26, 31 months 4, 24 months 5, 15, 27 months

[162] [55] [163] [156] [157] [158] [159]

3, 31 months 25—93 years 6—27 years 5, 16, 25 months 8, 27 months 25—93 years 5, 28 months 3—31 months 25—93 years 5, 28 months 3—31 months 4, 14, 30 months 5, 28 months

[160] [161] [122] [124] [169] [161] [177] [160] [161] [177] [160] [179] [189]

TEARS

MtDNA deletions 8-OHdG Carbonyls

Sulfhydryls Dityrosine 3NT

Mouse Human Monkey Mouse Mouse Human Rat Mouse Human Rat Mouse Mouse Rat

4,5.1 Lipid peroxidation Many studies have examined the effect of aging on various measures of hpid peroxidation in mammaUan tissues. Most of these studies have measured the tissue content of thiobarbituric acid reactive substances (TEARS) as a marker of endogenous Hpid peroxidation, and the resuhs have been contradictory [155]. Unhke other tissues, ample evidence shows that TEARS content increases with age in skeletal muscle [156—161]. Comparing gastrocnemius muscle from 24vs. 12-month-old male Fischer 344 rats, Starnes et al. [156] reported that TEARS content increased with aging. Formation of TEARS, measured as malondialdehyde (MDA), was found to be elevated in both skeletal muscle crude homogenate and mitochondrial fractions from rats aged 26 and 31 months vs. 4-month-old animals [157] and in soleus muscle, but not in gastrocnemius muscle, when comparing 24- with 4-month-old rats [158]. Furthermore, MDA concentrations increased in rat soleus and VL muscles at 15 and 27 months vs. 5 months of age [159]. Recently, studying mitochondria isolated from upper hindlimb skeletal muscle. Lass et al. [160] demonstrated that TEARS content increases markedly with age in male C57EL/6Nia mice between 3 and 31 rather than 30 months of age. Comparing vastus medialis or lateraHs muscle from 66 people aged 25—93 years, there was a significant age-dependent increase in levels of MDA [161]. In addition to TEARS content, several investigators [55,162—165] have exam-

Chapter 29: Oxidative stress and the pathogenesis ofsarcopenia

855

ined age-related alterations in skeletal muscle levels of lipofuscin, a byproduct of the oxidative polymerization of lipids. Following a report by Gutmann et al. [162] describing accumulation of lipofuscin with aging in rat skeletal muscle, Orlander et al. [55] measured lipofuscin in skeletal muscle from 16- to 76-yearold men. Lipofuscin was found more frequently in biopsies from older men. However, comparative fine-structural analysis of lipofuscin in skeletal muscle of rats of different ages (2, 11 and 30 months of age) revealed no significant ageassociated changes in the presence of lipofuscin [163]. More recently, Beregi et al. [164] utilized electron microscopy to examine skeletal muscle cells isolated from humans and CBA mice and reported that the presence of lipofuscin in mitochondria was greater in older people and mice. Lastly, no age-related increase in lipofuscin was observed in skeletal muscle from male NMRI mice at 20 months vs. 1 month of age [165]. 4.5.2 DNA oxidation Although numerous oxidative lesions occur in DNA, 8-hydroxylation of the guanine base is the most abundant [102]. A major mutagenic lesion, 8-hydroxydeoxyguanosine (8-OHdG) produces predominantly G ^ T transversion mutations [166]. This type of alteration has previously been shown to generate point mutations in mitochondrial DNA (mtDNA) due to mispairing [103]. Additionally, 8OHdG accumulates in DNA exposed to ROS [167]. Taken together, these studies support the notion of 8-OHdG as a useful marker for estimating age-induced DNA damage. For example, Fraga et al. [168] reported an age-related increase in the 8-OHdG concentration in the intestine, kidney and liver of Fischer rats between 1 and 24 months of age. However, the role of DNA oxidation in aging skeletal muscle is less well estabhshed. Also using 8-OHdG as an indicator of DNA oxidative damage, Sohal et al. [169] demonstrated that the concentration of 8-OHdG in skeletal muscle was greater in 27-month-old rather than 8month-old C57BL/6 male mice, suggesting an age-related increase. Comparing vastus medialis or lateralis skeletal muscle from 66 subjects aged 25—93 years, there was a significant age-dependent increase in levels of 8-OHdG [161]. Lastly, as discussed elsewhere in this review, multiple mtDNA deletions have been reported to increase with aging in skeletal muscle [122,124]; however, the etiology of these deletions is unclear but could involve DNA oxidative damage [125]. 4.5.3 Protein oxidation Typically, protein oxidation results in the generation of various amino acid residue modifications. For example, site-specific, metal-catalyzed oxidation of arginine, lysine, proline and threonine amino acid residues results in the production of carbonyl derivatives [170,171]. In addition, carbonyl groups may be introduced into proteins by reactions with aldehydes, such as MDA and 4-hydroxy-2-nonenal (HNE), produced during lipid peroxidation [172,173]. Therefore, carbonyl de-

856

Part X: Aging

rivatives provide a reliable marker of oxidative damage. Cysteine and methionine residues are particularly sensitive to oxidation by almost all forms of ROS. Cysteine residues are converted to disulfides while methionine residues are oxidized to methionine sulfoxide; however, repair systems can convert the oxidized forms of cysteine and methionine residues back to their normal state. Accordingly, a decrease in the amount of protein sulfhydryl group provides a useful marker of cysteine oxidation. Another protein oxidative modification that may be used as an indicator of oxidative damage involves formation of dityrosine from tyrosine [174,175]. Since dityrosine is not typically found in proteins, it provides an excellent marker for measuring protein oxidative damage in skeletal muscle with aging. Protein oxidative damage is particularly detrimental with aging because of the vital role of proteins in biology. Protein carbonyl content has been shown to increase with age in mouse heart, liver and kidney [176]. However, investigations of age-related changes in protein carbonyl content in mammalian skeletal muscle is sparse. Using mitochondria isolated from upper hindlimb skeletal muscle. Lass et al. [160] demonstrated that protein carbonyl content increases 2-fold with age when comparing male C57BL/6 mice between 3 and 31 rather than 30 months of age, suggesting that mitochondria in skeletal muscles accumulate significant oxidative damage with aging. In addition, it should be noted that caloric restriction (CR) prevented the age-related increase in carbonyl content [160]. Comparing vastus medialis or lateralis skeletal muscle from 66 subjects aged 25—93 years, there was a significant age-dependent increase in protein carbonyl levels [161]. Viner et al. [177] reported that levels of carbonyl groups per mole of Ca-ATPase in sarcoplasmic reticulum vesicles isolated from hindlimb muscles of male Fischer 344 rats aged 5 or 28 months slightly increased with aging. Investigations into age-related changes in skeletal muscle protein sulfhydryl content is lacking. A comparison of total sulfhydryl content in sarcoplasmic reticulum vesicles isolated from hindlimb muscles of male Fischer 344 rats aged 5 or 28 months, revealed an age-related decrease [177]. In addition, Viner et al. [177] reported that sarcoplasmic reticulum Ca-ATPase from old as compared to young rats demonstrated a lower content of sulfhydryl groups. Specifically, based on a total of 24 cysteine residues, the difference in protein thiols corresponds to a loss of 1.5 mol cysteine/mol Ca-ATPase during aging. It should be noted that an age-related modification of cysteine residues in skeletal muscle has also been established for other proteins such as phosphoglycerate kinase [178]. Using mitochondria isolated from upper hindlimb skeletal muscle. Lass et al. [160] demonstrated that protein sulfhydryl content decreased with age when comparing male C57BL/6 mice between 3 and 31 rather than 30 months of age. Again, CR prevented the age-related loss of protein sulfhydryl content [160]. These studies indicate an age-related loss of protein sulfhydryl content in skeletal muscle. Tyrosyl radicals, which are generated by peroxidases and other heme proteins, can react with each other to form dityrosine, a stable end product. Since these residues are not typically found in proteins, their detection in skeletal muscle


857

with aging would suggest increased tyrosyl radical-induced oxidative damage with age. Similar to protein sulfhydryl content, a gap currently exists in the literature concerning formation of dityrosine radicals with age. In a study comparing hindlimb skeletal muscle from three groups of male C57BL/6 mice (4, 14 and 30 months of age), levels of dityrosine were increased at 30 months compared to 4 months of age when using an isotope dilution gas chromatography-mass spectrometry (GC/MS) method [179]. These investigators suggest that the accumulation of dityrosine with age in skeletal muscle is consistent with the idea that tyrosyl radical plays a role in age-related protein oxidation. Interestingly, CR prevented the increase in dityrosine levels in skeletal muscle with age [179]. Nitric oxide (NO) is a free radical generated enzymatically by nitric oxide synthase (NOS), an enzyme that exists in three isoforms [180]. These isoforms include type I NOS (neuronal constitutive or nc-NOS), type II NOS (inducible or i-NOS) and type III NOS (endothelial constitutive or ec-NOS). Nitric oxide is synthesized by each isoform of NOS as it catalyzes the oxidation of L -arginine to L-citrulline, a reaction requiring molecular oxygen and nicotinamide adenine dinucleotide phosphate (NADPH) as cosubstrates and producing citrulline as a by-product. Essential co-factors include flavin adenine nucleotide (FAD), flavin mononucleotide (FMN), tetrahydrobiopterin and calmodulin. Synthesis of NO by nc-NOS and ec-NOS, two constitutive isoforms of NOS, is regulated by calcium-dependent calmodulin binding; however, i-NOS is transcriptionally regulated. Production of NO in skeletal muscle is functionally important for vascular control, muscle metabohsm and contractile function [181]. Type I NOS is expressed by muscle fibers and by neuronal axons in the muscle tissue. Immunohistochemical staining has shown that type I NOS is localized to the sarcolemma of fasttwitch fibers [182,183]. Interestingly, immunohistochemical labeling has demonstrated that type III NOS co-localizes with SDH [184]. Calcium-dependent NOS activity is enriched in the mitochondrial fraction of muscle homogenates [184], further supporting an association of type III NOS with mitochondria. These findings are significant to aging research since mitochondria are hypothesized to be the most important cellular sites of age-associated oxidative stress/ damage. Immunohistochemical analysis demonstrated that type II NOS, in response to an inflammatory challenge, could be upregulated in skeletal muscle myocytes, as well as endothelial cells and macrophages within the muscle [185]. Nitric oxide reacts with superoxide anions to generate peroxynitrite (ONOO ), one of the most reactive free radical species in biological systems and a recognized mediator of oxidative injury [186]. Methionine and cysteine protein residues are very susceptible to oxidation by peroxynitrite, while tryptophan and tyrosine are selective targets for peroxynitrite-dependent nitration. Peroxynitrite, along with other reactive nitrogen species (RNS) generated as a result of the interaction between oxygen and NO, catalyzes the nitration of protein tyrosine residues to form 3-nitrotyrosine. The nitration of tyrosine residues by these RNS inhibits cyclic interconversion of protein tyrosine residues between phosphorylated and

858

Part X: Aging

unphosphorylated forms [187]. Therefore, peroxynitrite-induced nitration compromises various cell regulation and signal transduction pathways mediated by phosphorylation within the cell. The accumulation of nitrotyrosine-modified proteins as detected by immunohistochemical staining with antisera against 3nitrotyrosine has been shown to be a convenient marker of peroxynitrite-induced oxidative damage [188]. Therefore, techniques designed to measure the accumulation of 3-nitrotyrosine with age in skeletal muscle may provide a tool to better understand the role of oxidative stress and damage in the etiology of sarcopenia. Unlike the situation for ROS, investigation of the involvement of RNS in sarcopenia has been limited [189,190]. 3-Nitrotyrosine may provide a good marker of oxidative damage because it is not normally found in proteins. In order to test the hypothesis that proteins damaged by RNS accumulate in skeletal muscle with age, Leeuwenburgh et al. [190] utilized GC-MS techniques to measure 3nitrotyrosine in skeletal muscle from 9- and 24-month-old female Long-Evans/ Wistar hybrid rats. They observed no influence of age on levels of protein-bound 3-nitrotyrosine in VL. However, a recent study has demonstrated that certain proteins are oxidized selectively in vivo. After isolating sarcoplasmic reticulum vesicles from hindlimb muscles of male Fischer 344 rats,Viner et al. [189] reported that the level of nitration of the SERCA2 a slow-twitch isoform of Ca-ATPase in these vesicles was greater in aged (28 months) than adult (5 months) rats. Specifically, these investigators found approximately one and four nitrotyrosine residues per young and old Ca-ATPase, respectively Further, they reported that nitration was undetectable in a closely related form of Ca-ATPase, strongly suggesting that certain isoforms are selectively modified by RNS. Therefore, analysis of 3-nitrotyrosine in whole tissue homogenates, as performed by Leeuwenburgh et al. [190], may fail to detect a selective increase in the oxidation of specific proteins by RNS. These studies suggest that, as a result of their interaction with RNS, only certain proteins accumulate oxidative damage during aging. 4.6 Antioxidant status In considering the oxidative stress/damage hypothesis of sarcopenia, it is important to examine the contributions that cellular antioxidant defenses may play in attenuating the process. Elaborate cellular defense mechanisms involving both enzymatic and nonenzymatic antioxidant systems are present in mammals to contend with oxidative stress. For example, primary enzymatic antioxidants in the cell include SOD, catalase (CAT) and glutathione peroxidase (GPX) [98]. Glutathione (GSH) and a-tocopherol (vitamin E) are considered to be the major cellular nonenzymatic antioxidants [191]. Investigations into whether or not these enzymatic and nonenzymatic antioxidant systems change with age in skeletal muscle, as described in Table 6, may help elucidate the etiology of sarcopenia. Skeletal muscle has been widely studied for the effects of aging on the enzymatic antioxidant system [61,157-160,165,192-199] and on GSH status [159,199]. In


859

Table 6. Antioxidant enzyme activities increase with age in skeletal muscle. Enzyme

Species

Muscle

Ages (months)

Reference

SOD

Rat

Biceps brachii Extensor Gastrocnemius Plantaris Sternomastoideus VL VL Soleus Hindlimb Gastrocnemius VL VL Hindlimb Soleus Hindlimb Gastrocnemius VL Diaphragm Gastrocnemius Soleus Soleus VL Hindlimb Soleus

3, 19,23

[192]

4,26,31 5, 15,27 4,24 2,26 4, 15,27 4,26,31 5, 15,27 11,26,34 4,24 1,7,20 4, 15,27 4,26,31 4,24 4,24

[157] [179] [197] [198] [193] [157] [159] [196] [197] [165] [193] [157] [62] [158]

5, 15,27

[159]

11,26,34 4,24

[196] [197]

CAT

GPX

Rat Rat Rat Mouse Rat Rat Rat Rat Rat Mouse Rat Rat Rat Rat Rat Rat Rat

general, an age-associated increase in the level of the enzymatic and nonenzymatic antioxidant systems occurs in aging skeletal muscle. Ji et al. [157] proposed that increased antioxidant enzyme activity levels in older animals may be a response to elevated cellular production of free radicals during the aging process. Oh-Ishi et al. [197] have suggested that the changes observed in antioxidant enzymatic activity with age are directly correlated with the oxidative potential of the muscle. Compared to their glycolytic fast-twitch muscle counterparts, oxidative slow-twitch muscles may have increased susceptibility to age-related oxidative stress and damage as a result of their greater oxidative capacity. 4.6J Superoxide dismutase Superoxide dismutase, which catalyzes the conversion of superoxide to oxygen and hydrogen peroxide, is one of the most important enzymes in the antioxidant enzymatic system found in skeletal muscle. There are two major forms of SOD, a copper and zinc containing enzyme (CuZnSOD), localized in the cell nucleus and cytoplasm [200], as well as a manganese containing enzyme (MnSOD) found specifically in mitochondria [201]. Despite considerable investigation into the influence of age on total SOD,

860

Part X: Aging

CuZnSOD and MnSOD activity levels [157-160,192-198], definitive conclusions are difficult to reach because the data show either no change or increased activity with age. This discrepancy may derive from differences in muscle fiber type, assay methods, diet and/or animal sex and strain. In the first report on the SOD status in aging skeletal muscle, total SOD, CuZnSOD and MnSOD activities were determined in various skeletal muscles from male rats between 3 and 23 months of age [192]. In this study, total SOD activity increased progressively with age in nearly all muscles sampled. The activity levels of CuZnSOD and MnSOD were found by others [157] to be greater in VL from old male Wistar rats (26 and 31 months) than in 4-month-old rats. An age-related increase in total SOD activity in both soleus and VL muscles was observed when two groups of male Fischer 344 rats, 5 and 27 months of age, were compared [159]. In addition to greater activity and content of CuZnSOD in both soleus and EDL muscles of older male Fischer rats, Oh-Ishi et al. [197] saw an age-related increase in MnSOD activity normalized to CS activity in soleus muscle. These investigators also found no influence of age on the expression of mRNAs for SOD isoenzymes. Furthermore, CuZnSOD and MnSOD content, as well as CuZnSOD, but not MnSOD activity, were increased with aging when comparing diaphragms from mice aged 2 months and 26 months [198]. Unlike these studies demonstrating increased SOD activity with age, Vertechy et al. [193] found no change in either CuZnSOD and MnSOD activity or content with age in gastrocnemius muscle from male albino rats aged 4, 15, or 27 months. Ji et al. [194] demonstrated no age-related change in total SOD activity in VL muscle when comparing rats 5 and 27.5 months of age. Furthermore, Lawler et al. [158] confirmed that total SOD activity in both gastrocnemius and soleus muscles did not change with age when comparing 4- and 24-month-old female Fischer 344 rats. These investigators also showed that SOD activity in diaphragm was unaffected by aging in these rats whilst Luhtala et al. [196] reported that total and MnSOD activities were unaltered in rat hindlimb skeletal muscle between 11 and 34 months of age. Also using mitochondria isolated from upper hindlimb skeletal muscle. Lass et al. [160] demonstrated that SOD activity remained unaltered with age when comparing male C57BL/6Nia mice between 3 and 31 rather than 30 months of age. These reports suggest that age-related changes in SOD activity require further investigation using parallel studies in order to avoid such variables as muscle fiber type, assay methods and/or animal's sex and strain. Clarification of possible age-related changes in SOD activity is necessary since increased SOD activity has been shown to be accompanied by increased formation of hydrogen peroxide [202]. In response to elevated hydrogen peroxide levels, CAT and GPX activity would be expected to increase with aging. 4.6,2 Catalase Catalase plays a major role in the cell's enzymatic antioxidant defense system by


861

converting hydrogen peroxide to water and oxygen. Catalase is localized to peroxisomes and cytoplasm [203]. Several studies have shown that an age-dependent increase in CAT activity occurs in skeletal muscle [157,159,193,196,197] although, as with SOD, evidence exists which disputes this notion [160,194,198]. Vertechy et al. [193] found that CAT activity increased in gastrocnemius muscle when comparing 15- or 27month-old male rats with animals aged 4 months. Furthermore, CAT activity was doubled in rat VL at 31 months compared with 4 months of age [157] and in rat soleus and VL muscles at 15 and 27 months but not at 5 months of age [159]. Interestingly, Luhtala et al. [196] reported that CAT activity in mitochondria- and peroxisome-enriched fractions from rat skeletal muscle homogenates increased progressively with age between 11 and 34 months of age, whereas the cytosolic fractions showed no significant alteration. Furthermore, the increase in CAT activity was attenuated by CR [196]. Catalase activity increased in rat soleus muscle with aging, but not in EDL muscle when comparing two groups of rats 4 and 24 months of age [197]. However, no age-related change in CAT activity was observed in rat VL muscle between 5 and 27.5 months of age [194] and in diaphragm when comparing two groups of mice 2 and 26 months of age [198]. Employing mitochondria isolated from upper hindlimb skeletal muscle. Lass et al. [160] demonstrated that catalase activity was unaltered with age when comparing male C57BL/6 mice aged 3—31 rather than 30 months. Therefore, data describing age-dependent changes in catalase have been inconsistent. 4,6,3 Glutathione system Glutathione is one of the major nonenzymatic antioxidant defenses in the cell and is one of the most effective scavengers of oxygen radicals. Glutathione peroxidase utilizes GSH to reduce peroxides. Glutathione is oxidized to glutathione disulfide (GSSG) as a result of these cellular functions and is regenerated by the enzyme glutathione reductase (GR). The reducing power of NADPH from the hexose monophosphate shunt is required for the reduction of GSSG back to GSH. The rate-limiting enzyme for the hexose monophosphate shunt is glucose-6-phosphate dehydrogenase (G-6-PDH). Glutathione is also utilized by glutathione S-transferase (GST) which catalyzes the conjugation of GSH with a variety of organic peroxides in order to increase their water solubility and promote excretion. Lastly, transport of GSH across cell membranes requires that GSH be cleaved into its constitutive amino acids, whose transport across the membrane requires the enzyme y-glutamyltranspeptidase (GGT). In the first report to investigate the effect of aging on GPX in skeletal muscle, an age-related increase in GPX activity was detected in skeletal muscle from male NMRI-mice at 20 months of age vs. 1-month-old animals [165]. Following this first study, substantial evidence has confirmed this age-dependent increase in GPX activity in skeletal muscle [61,157-159,193,196,197]. However, a few studies have reported no age-related change in GPX activity [160,195,198] or

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decreased GPX activity with age [194]. Vertechy et al. [193] reported that GPX activity was greater in rat gastrocnemius muscle at 15 and 27 months of age compared with 4 months. Glutathione peroxidase activity was found to be elevated in both cytosolic and mitochondrial fractions from skeletal muscle of rats aged 26 and 31 months vs. 4-month-old animals [157] and in rat soleus and VL muscles when comparing 15- and 27-month-old animals with rats aged 5 months [159]. Increased GPX activity with age was also reported in rat diaphragm [61] and in gastrocnemius and soleus muscles [158] in 24- but not 4-month-old female Fischer 344 rats. However, Luhtala et al. [196] reported that GPX activity in mitochondria and peroxisome enriched fractions of rat skeletal muscle homogenates increased progressively with age between 11 and 34 months of age, whereas the cytosolic fractions demonstrated no significant alteration. As was the case for catalase, the increase in GPX activity with aging was prevented by CR. GPX activity was also found to increase in rat soleus muscle with aging, but not in the EDL, when comparing rats at 4 and 24 months of age [197], suggesting that the increased oxidative capacity of the slow-twitch soleus muscle may underlie this difference. No age-related change in GPX activity was observed in rat [195] or mouse diaphragm [198]. Using mitochondria isolated from upper hindlimb skeletal muscle. Lass et al. [160] demonstrated that GPX activity remained unaltered with age when comparing male C57BL/6 mice between 3 and 31 rather than 30 months of age. In contrast, decreased GPX activity was observed in rat VL at 27.5 months of age when compared to animals 5 months of age [194]. As with GPX activity, GR activity is increased in rat gastrocnemius muscle when comparing 27-month-old rats with those aged 4 months [193]. Much information on the glutathione system in aging rat muscle has been provided by Ji's laboratory They first reported [157] that GR, GST and G-6-PDH activities were increased in VL muscle of rats at 26 and 31 months of age compared with 4 months. Increased activity of GR and GST were observed at 15 and 27 months in both soleus and VL muscles of male Fischer 344 rats when compared to 5-month-old animals [159]. Investigation of GST activity in rat VL muscle revealed increased activity in 27.5-month-old compared to 5-month-old animals [194]. Leeuwenburgh et al. [159] also demonstrated that the activity of GGT, a key enzyme for the uptake of GSH by skeletal muscle, increased with aging in VL but not soleus muscle. Leeuwenburgh et al. [159] have shown that the concentration of GSH, as well as the GSH/GSSG ratio in soleus muscle increased significantly with age in male rats at 15 and 27 months of age compared with 5 months, however, these parameters did not change in VL muscle. Since VL muscle is composed predominantly of fast-twitch fibers while soleus muscle is a slow-twitch oxidative muscle, these data support the hypothesis that the functional deterioration of mitochondria with age contributes to the increased production of free radicals observed during aging. Ohkuwa et al. [199] also reported that the concentration of GSH and the GSH/GSSG ratio in gastrocnemius and soleus muscle of female Wistar


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rats increases at 25 weeks compared to 8 weeks of age. However, as found also by Leeuwenburgh et al. [159], GSSG levels did not demonstrate an age-related change. In contrast to these studies, Noy et al. [204] reported that decreased skeletal muscle concentrations of GSH, NADH and NADPH concentrations in rats between 4 and 6 months and 28 and 32 months of age were accompanied by corresponding increases in their oxidized forms. 4,6.4 Vitamin E Vitamin E is considered to be an important nonenzymatic defense against lipid peroxidation. This molecule is embedded within membranes and clearly has an antioxidant role [205]. Comparing gastrocnemius muscle from 24-month vs. 12month-old male Fischer 344 rats, Starnes et al. [156] reported that levels of atocopherol did not change with age. However, these same investigators revealed that levels of oc-tocopherol quinone, the oxidized form of a-tocopherol, increased in the older rats. Since oxidation of a-tocopherol precedes lipid peroxidation, detection of a-tocopherol quinone is considered to be a sensitive estimate of cellular oxidant stress. These increased quinone levels with age suggest that older animals must oxidize more vitamin E than younger animals to prevent lipid peroxidation. 5

INTERVENTIONS

Sarcopenia is a natural, age-associated phenomenon which compromises the independence and well being of older individuals. However, several lines of evidence suggest that the progression of sarcopenia can be attenuated or partially reversed by one or more candidate interventions. These include exercise training, antioxidant supplementation and CR. 5.1 Exercise Both resistance and endurance training have beneficial effects on skeletal muscle in older persons. Resistance training can lead to increased muscle mass and strength, mainly by increasing fiber CSA. Endurance training has a positive effect on oxidative enzyme activities. In addition, it increases the level of activity of older individuals. Taken together, exercise training, primarily strength training, has positive effects in reversing or preventing the age-associated loss of muscle mass.

5,1,1 Strength training There is solid evidence that healthy older people respond favorably to strengthtraining programs, defined as training in which the resistance against the muscle is progressively increased over time (reviewed in [43,206,207]). Exercise designed

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to increase strength emphasizes high force development with relatively short durations of exercise. Resistance training intensity is generally reported as a percentage of one repetition maximum (1 RM), the maximum amount of force that a muscle group can generate with one single contraction. Studies employing high training intensities in older subjects result in very substantial improvements in strength. A high-intensity (80% of 1 RM) strength training program performed by men between 60 and 72 years of age for 12 weeks resulted in an average increase in knee flexor and extensor strength of 227% and 107%, respectively [208]. When older women (average 72 years) were subjected to strength training for 6 months, a 50% increase in strength of quadriceps was reported [209]. In a similar study, older subjects (average 75 years) showed an increase in strength in all muscles except those of the ankle region [210]. Resistance training can lead to increased muscle mass and strength in old humans, primarily by increasing fiber CSA. Frontera et al. [208] reported that strength training led to an 11% increase in midthigh CSA in 60- to 72-year-olds, as assessed by computed tomography. This increase may have been due to a 34%o increase in type I and 28% increase in type II fiber area. Furthermore, a 4%) increase in quadriceps CSA was found in a group of elderly women (average 72 years) when subjected to strength training for 6 months [209]. Strength training programs can be beneficially applied to frail older persons. When Fiatarone et al. [211] subjected very old individuals (86—96 years) to a strength training program for 8 weeks, a 174% increase in strength and \\% increase in CSA of the quadriceps resulted. In another study of frail, older (72—98 years) persons on strength training for 10 weeks, increases in muscle strength (113%) and CSA (3%) were obtained [212]. A third study of frail, (determined by being unable to descend stairs step over step without holding the railing), older people (average 78 years) trained for 10 weeks led to increases in strength of 9-16% [213]. Although strength training has the potential to increase functional independence of and to decrease injury risk in older individuals, it appears necessary to continue the training as long as possible. For example, a group of elderly women (average 83 years) who had been subjected to a strength training program for 8 weeks were followed up a year thereafter [214]. Quadriceps isometric strength had dropped ^^25% compared to the subjects' post-training values and ^ 10% from the pretraining values. Also, the functional mobility improvements gained from this resistance exercise had also been lost. Increases in strength and muscle mass obtained with strength training programs also increase the ability to perform activities of daily life (e.g., stair climbing, walking, chair rising) [209,213]. These exercise programs also reduce the risk of falling. Buchner et al. [210] found a decrease in the fall rate in the 18month period of follow-up after a 6-month resistance training protocol applied to old subjects (average 75 years). Furthermore, an increase in maximal aerobic capacity (VO2 max) was reported after strength training in older men, suggesting that increased muscle mass can increase maximal aerobic power [215].


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5A.2 Endurance training Several studies show that older persons favorably respond to aerobic exercise (reviewed in [43]). Unlike resistance training, endurance training requires long sessions repeated at least three times per week and involves high repetition muscle contractions. Endurance training leads to minimal strength gains, however, it results in increased maximal aerobic capacity (V02max). A 6-week endurance training program resulted in an increase in V02max in a group of older men (average 64 years) [216]. Elderly subjects (average 75 years) who participated in an endurance training program for 6 months showed a 9% increase in aerobic capacity (V02max) [210]. For endurance training to be beneficial, it has to be performed on a regular basis. For example, older women and men (average 62 years) who had exhibited a 15% increase in V02max after a 4-month endurance training program were studied. After 4 months of detraining, V02max returned to its original pretraining value [217]. Some adaptations may occur in skeletal muscle due to prolonged endurance training. Endurance-trained older men show a higher percentage of type I fibers and a lower percentage of type lib fibers than normal, fit individuals. In addition, fiber CSA of type I fibers in the VL muscle was 16% greater in trained vs. untrained subjects [17]. However, Freyssenet et al. [216] did not find any changes in the distribution and CSA of type I, Ila and lib fibers in the VL after older subjects (average 64 years) were subjected to a 6-week endurance training program. Other changes have been described in the muscles from endurance-trained older persons. An increase in capillary density in skeletal muscle is one of the first changes to occur during endurance training. Proctor et al. [17] found 25—30% higher values in the VL of trained vs. untrained in both young and old subjects. Also studying the VL, Freyssenet et al. [216] reported an increase in the number of capillaries/fiber, especially of those in contact with type I fibers. Endurancetrained older individuals (from 50 to 65 years) displayed 37% higher CS activity in the VL when compared to untrained [17]. In addition, CS activity is higher in the gastrocnemius of trained older people when compared to untrained [15]. Taken together, these studies indicate that, in healthy older persons, exercise programs can substantially improve strength and endurance. 5.2 Antioxidant supplementation The use of antioxidants has been shown to reduce oxidative stress in skeletal muscle (Table 7). As discussed earlier, the injury caused by lengthening the EDL muscles of mice was lessened by treatment with PEG-SOD [81]. Vitamin E supplementation decreased levels of carbon-centered and hydrogen radicals in homogenates of rat soleus [218]. Vitamin E (30 mg/kg) injected into immobilized, atrophying soleus muscles from young rats was found to reduce levels of TEARS in [219]. Also, vitamin E treatment can inhibit exercise-induced protein oxidation in rats [220] and lipid peroxidation in people [221].

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Table 7. Lowered oxidative stress in skeletal muscles after antioxidant supplementation. Situation

Antioxidant

Species

Muscle

Reference

Force generation (PQ) (postinjury) Free radical levels

SOD

Mouse

EDL

[81]

Vitamin E (in vitro) Vitamin E

Rat

Soleus

[218]

Rat

Soleus

[219]

Vitamin E

Rat

Several

[220]

Vitamin E

Human

VL

[221]

Lipid peroxidation (immobilization atrophy) Protein oxidation (postexercise) Lipid peroxidation (postexercise)

Not all data support the efficacy of antioxidants in lowering oxidative damage in skeletal muscles. For example, when 5 month old rats were fed a diet supplemented with ascorbic acid, a-tocopherol, P-carotene and a synthetic antioxidant, no influence of diet was seen on levels of oxidative damage (o-tyrosine and 3-nitrotyrosine) in the VL were observed at 9 and 24 months of age [190]. Together, these data support the necessity of testing the influence of long-term administration of antioxidant nutrients or enzymes on the progression of sarcopenia. 5.3 Caloric restriction The restriction of calorie intake when conducted without essential nutrient deficiency extends maximum life span and retards both the rate of biological aging and the development of late-life diseases in mice and rats [222,223]. Caloric restriction also extends life span in water fleas, rotifers, spiders and fish [222]. Two long-term studies are ongoing to determine if CR retards diseases and aging in a primate species (rhesus monkeys) [224,225]. One hypothesis to explain the beneficial effects of CR is that it lowers oxidative stress/damage [10,222,226,227]. Several lines of evidence support this hypothesis. In laboratory rodents, CR decreases the steady-state concentrations of the products of oxidative damage to protein, lipids and DNA in several tissues including skeletal muscles [169,176,228,229]. This effect may involve decreased mitochondrial production of ROS and ROM [176]. As summarized in Table 6, old rodents show consistent increases in antioxidant enzyme activities in skeletal muscle. We measured muscle mass and antioxidant enzyme activities in hindlimb skeletal muscle samples from male BrownNorway X Fischer 344Fi rats of various ages fed normally or subjected to CR at 14 weeks of age [196]. We found that the total muscle mass recoverable from the upper hindlimb muscles of CR rats was ^ 30% less than that of ad libitum fed controls at 11 months of age. However, CR countered the 50% loss of muscle mass observed in very old (34 months) ad libitum fed rats. These data on muscle


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mass loss partially agree with those of Yu et al. [230] who studied the eflfect of CR on the weight of the gastrocnemius in male Fischer 344 rats. They found that muscle mass of the CR rats averaged 60% that of controls until 18 months of age when the ad libitum rats began to lose mass. At 25 months of age, muscle mass did not differ between groups but the CR group progressively lost mass thereafter. Our data differ from these in showing no loss of muscle mass between 11 and 34 months in rats on CR; however, this disparity may reflect longevity differences between the two rat strains. As for the antioxidant enzyme activities in our study [196], there was no influence of age or CR on SOD activity, but clear age-associated increases in the activities of GPX and CAT were observed in the 1,000 x g supernatant and 12,000 X g fraction (which is enriched in mitochondria) in ad libitum fed rats and these increases were attenuated by CR. A logical speculation is that antioxidant enzyme induction in muscle from old ad libitum fed rats is driven by an agerelated increase in ROS as was suggested by Ji et al. [157]. A recent collaborative study with Sohal's laboratory focused on the age-associated accrual of oxidative damage to mitochondrial macromolecules in mouse hindlimb skeletal muscles [160]. The influences of CR initiated at 4 months of age on these measures were also evaluated. The level of mitochondrial protein carbonyls was 150% greater in 29-month-old when compared to 7-month-old mice. When mitochondrial protein sulfhydryl content was analyzed, 18-monthold mice had 50% lower levels when compared to 3-month-old mice. In addition, TEARS levels were increased 2.5-fold in mice 12—14 months old when compared to young. Furthermore, the rate of superoxide radical generation was 41% higher in older mice when compared to young. None of these age-associated changes were seen in mice subjected to CR. In ad libitum fed mice, there was no influence of age on activities of antioxidant enzymes such as SOD, CAT and GPX; however, CR mice showed an age-associated increase in CAT activity. Taken together, these data demonstrate an increase in oxidative damage to mitochondrial macromolecules with age which is attenuated by CR. The previous study focused on the effects of early-onset CR. Additional data from our laboratory indicate that CR does not have to be started early in life to attenuate the development of sarcopenia. We studied skeletal muscles in rats subjected to CR in late middle age (at 17 months) [22]. A decrease in the number of muscle fibers in the control-fed animals (3—4 months compared to 30—32 months) was found with age in the VL. However, late-onset CR prevented this age-associated loss of muscle fibers and attenuated the loss in type I fibers. The increase with aging in mtDNA deletion products in soleus and ADL was lessened by CR. Furthermore, when histological studies were performed, fewer ETS abnormal fibers were found in CR rats. The data from this latter study [22] suggest that CR started in late middle age can ameliorate age-associated mitochondrial abnormalities associated with sarcopenia. Accordingly, CR can be viewed as an intervention that may potentially be applied later on in life to retard the progression of sarcopenia. It is also pos-

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sible that the way by which CR attenuates the development of sarcopenia can be discovered and pharmacologically mimicked. An optimistic scenario would involve treating an individual with the "CR mimetic" which retards sarcopenia's progression in the face of a normal caloric intake. 6

SUMMARY

1. Sarcopenia, an important contributor to senile physical frailty, is characterized by age-dependent changes in muscle mass, fiber type composition and innervation. 2. Changes in muscle function, such as decreased strength and endurance and metabolic capacity also occur in aging skeletal muscle. 3. Hypothesized causes of sarcopenia include denervation-reinnervation, contraction-induced injury, satellite cell changes and oxidative stress/damage. 4. Oxidative stress of mitochondrial origin may contribute to the age-dependent development of sarcopenia in a focal way 5. Understanding the mechanism by which caloric restriction attenuates the development of sarcopenia may result in effective clinical therapy 7

PERSPECTIVES

Sarcopenia is an important topic because it universally afflicts older persons and causes physical frailty Accordingly, studies designed to elucidate the etiology and pathogenesis of sarcopenia are important to pursue as they will provide insights into preventive strategies and therapies. Recent advances in stem cell biology [231], which have suddenly provided new opportunities for the replacement of dead and atrophic muscle fibers, are worthy of vigorous pursuit. Optimistically, these lines of research will eventually produce sarcopenia-specific interventions which will contribute to an increased span of robust health for humans. 8

ACKNOWLEDGEMENTS

Our research is supported by NIH grants from the National Institute on Aging (POl AG11915, ROl AG11604 and T32 AG00213), the National Center for Research Resources (Primate Research Center Program RR00167). The authors gratefully acknowledge the assistance of Ms. Jennifer Christensen in preparing this article. This is publication #99—08 from the Madison Geriatric Research, Education and Clinical Center and #38—031 from the Wisconsin Regional Primate Research Center.


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ABBREVIATIONS 8-OHdG: ATP: Ca-ATPase: CAT: COX: CR: CS: CSA: CuZnSOD: EDL: ETS: FAD: FDL: FG: FMN: FOG: G-6-PDH: GC/MS: GGT: GPX: GR: GSH: GSSG: GST: HNE: LDH: LHON: MDA: MELAS:

8-hydroxydeoxyguanosine adenosine triphosphate calcium-ATPase catalase cytochrome C oxidase caloric restriction citrate synthase cross-sectional area Copper, ZincSOD extensor digitorum longus electron transport system flavin adenine nucleotide flexor digitorum longus fast glycolytic flavin mononucleotide fast oxidative glycolytic glucose-6-phosphate dehydrogenase gas chromatography-mass spectrometry y-glutamyl transpeptidase glutathione peroxidase glutathione reductase glutathione glutathione disulfide glutathione S-transferase 4-hydroxy-2-nonenal lactate dehydrogenase Leber's hereditary optic neuropathy malondialdehyde mitochondrial encephalopathy with lactic acidosis and stoke-like episodes MHC: major histocompatibility complex MnSOD: manganese-containing enzyme mtDNA: mitochondrial DNA NADH: nicotinamide adenine dinucleotide NADPH: nicotinamide adenine dinucleotide phosphate NO: nitric oxide NOS: nitric oxide synthase ONOO : peroxynitrite PEG: polyethylene glycol PEG-SOD: polyethylene glycol adsorbed superoxide dismutase RM: repetition maximum RNS: reactive nitrogen species

870 ROM: ROS: RRFs: SDH: SERCA2: SOD: SO: TBARS: VL: V02max:

Part X: Aging reactive oxygen metabolites reactive oxygen species ragged red fibers succinate dehydrogenase sarcoplasmic endoplasmic reticular calcium ATPase 2 superoxide dismutase Slow Oxidative thiobarbituric acid reactive substances vastus lateralis maximal O2 consumption

10 REFERENCES 1. Weindruch R (ed). Reducing Frailty and Falls in Older Persons. Springfield, IL: CC Thomas, 1991. 2. Fried LR Frailty. In: Hazzard WR (ed) Principles of Geriatric Medicine and Gerontology, 3rd edn. New York: McGraw-Hill Inc., 1994;1149-1156. 3. NIH. Physical Frailty, A Reducible Barrier to Independence for Older Americans. NIH Report to Congress, September 1991;91-397. 4. Lexell J. Human aging, muscle mass, and fiber type composition. J Gerontol Biol Sci 1995;50: 11-16. 5. Evans WJ. Exercise, nutrition and aging. Clin Geriatr Med 1995;11:725—734. 6. Faulkner JA, Brooks SV, Zerba E. Muscle atrophy and weakness with aging: contractioninduced injury as an underlying mechanism. J Gerontol Biol Sci 1995;50A:124—129. 7. Carlson BM. Factors; Influencing the repair and adaptation of muscles in aged individuals: satellite cells and innervation. J Gerontol Biol Sci 1995;50A:96-100. 8. Larsson L. Motor units: remodeling in aged animals. J Gerontol Biol Sci 1995;50A:91—95. 9. Weindruch R. Interventions based on the possibility that oxidative stress contributes to sarcopenia. J Gerontol Biol Sci 1995;50A:157-161. 10. Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science 1996;273: 59-63. 11. Beckman KB, Ames BN. The free radical theory of aging matures. Physiol Rev 1998;78: 547-581. 12. Sen CK, Packer L. Antioxidant and redox regulation of gene transcription. FASEB J 1996; 10: 709-720. 13. SunY, Oberley LW Redox regulation of transcriptional activators. Free Radic Biol Med 1996; 21:335-348. 14. Kroemer G, Zamzami N, Susin SA. Mitochondrial control of apoptosis. Immunol Today 1997; 18:44-51. 15. Coggan AR, AbduljaHl AM, Swanson SC, Earle MS, Farris JW, Mendenhal LA et al. Muscle metabolism during exercise in young and older untrained and endurance-trained men. J Appl Physiol 1993;75:2125-2133. 16. Baumgartner RN, Stauber PM, McHugh D, Koehler KM, Garry PJ. Cross-sectional age differences in body composition in persons 60+ years of age. J Gerontol Biol Sci 1995;50:M307—316. 17. Proctor DN, Sinning WE, Walro JM, Sieck GC, Lemon PW Oxidative capacity of human muscle fiber types: effects of age and training status. J Appl Physiol 1995;78:2033—2038. 18. Lexell J, Taylor CC, Sjostrom M. What is the cause of the ageing atrophy? Total number, size and proportion of different fiber types studied in whole vastus lateralis muscle from 15- to 83-yearold men. J Neurol Sci 1988;84:275-294.


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their role in enzyme-aging. Mech Ageing Dev 1985;29:63—69. 205. Horton AA, Fairhurst S. Lipid peroxidation and mechanisms of toxicity. Crit Rev Toxicol 1987; 18:27-79. 206. Fielding RA. The role of progressive resistance training and nutrition in the preservation of lean body mass in the elderly J Am Coll Nutr 1995;14:587-594. 207. Frischknecht R. Effect of training on muscle strength and motor function in the elderly. Reprod Nutr Dev 1998;38:167-174. 208.FronteraWR, Meredith CN, O'Reilly KP, Knuttgen HG, Evans WJ. Strength conditioning in older men: skeletal muscle hypertrophy and function. J Appl Physiol 1988;64:1038—1044. 209. Welsh L, Rutherford OM. Effects of isometric strength training on quadriceps muscle properties in over 55-year-olds. Eur J Appl Physiol Occ Physiol 1996;72:219—223. 210. Buchner DM, Cress ME, de Lateur BJ, Esselman PC, Margherita A J, Price R et al. The effect of strength and endurance training on gait, balance, fall risk, and health services use in community-living older adults. J Gerontol Biol Sci 1997;52:M218—224. 211. Fiatarone MA, Marks EC, Ryan ND, Meredith CN, Lipsitz LA, Evans WJ. High-intensity strength training in nonagenarians. Effects on skeletal muscle. JAMA 1990;263:3029—3034. 212. Fiatarone MA, O'Neill EF, Ryan ND, Clements KM, Solares GR, Nelson ME et al. Exercise training and nutritional supplementation for physical frailty in very elderly people. N Engl J Med 1994;330:1769-1775. 213. Chandler JM, Duncan PW, Kochersberger G, Studenski S. Is lower extremity strength gain associated with improvement in physical performance and disability in frail, communitydwelling elders? Arch Phys Med Rehab 1998;79:24-30. 214. Connelly DM,Vandervoort AA. Effects of detraining on knee extensor strength and functional mobility in a group of elderly women. J Orthop Sports PhysTher 1997;26:340—346. 215.Frontera WR, Meredith CN, O'Reilly KP, Evans WJ. Strength training and determinants of VOsmax in older men. J Appl Physiol 1990;68:329-333. 216. Freyssenet D, Berthon P, Denis C, Barthelemy JC, Guezennec CY, Chatard JC. Effect of a 6week endurance training programme and branched-chain amino acid supplementation on histomorphometric characteristics of aged human muscle. Arch Physiol Biochem 1996; 104: 157-162. 217. Pickering GP, Fellmann N, Morio B, Ritz P, Amonchot A, Vermorel M et al. Effects of endurance training on the cardiovascular system and water compartments in elderly subjects. J Appl Physiol 1997;83:1300-1306. 218. Hiramatsu M,Velasco RD, Packer L. Decreased carbon centered and hydrogen radicals in skeletal muscle of vitamin E supplemented rats. Biochem Biophys Res Commun 1991;179:859—864. 219. Kondo H, Miura M, ItokawaY. Oxidative stress in skeletal muscle atrophied by immobilization. Acta Physiol Scand 1991;142:527-528. 220. Reznick AZ,Witt E, Matsumoto M, Packer L. Vitamin E inhibits protein oxidation in skeletal muscle of resting and exercised rats. Biochem Biophys Res Commun 1992; 189:801—806. 221. Meydani M, Evans WJ, Handelman G et al. Protective effect of vitamin E on exercise-induced oxidative damage in young and older adults. Am J Physiol 1993;264:R992—R998. 222. Weindruch R, Walford RL. The Retardation of Aging and Disease by Dietary Restriction. Springfield, IL: CC Thomas, 1988. 223. Yu BP (ed). Modulation of Aging Processes by Dietary Restriction. Boca Raton, FL: CRC Press, 1994. 224. Ingram DK, Cutler RG, Weindruch R, Renquist DM, Knapka JJ, April M et al. Dietary restriction and aging: The initiation of a primate study J Gerontol Biol Sci 1990;45:B148—B163. 225. Kemnitz JW, Weindruch R, Roecker EB, Crawford K, Kaufman PL, Ershler WB. Dietary restriction of adult male rhesus monkeys: Design, methodology, and preliminary findings from the first year of study J Gerontol Biol Sci 1993;48:B17-B26. 226. Weindruch R, Cheung MK, Verity MA, Walford RL. Modification of mitochondrial respiration by aging and dietary restriction. Mech Ageing Dev 1980; 12:375—392.

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221. Yu BP, Langaniere S, Kim J-W. Influence of life-prolonging food restriction on membrane lipoperoxidation and antioxidant states. In: Simic MG, Taylor KA, Ward JF, von Sonntag T (eds) Oxygen Radicals in Biology and Medicine. New York: Plenum, 1989; 1067—1073. 228. Laganiere S,Yu BP. Antilipoperoxidation action of food restriction. Biochem Biophys Res Commun 1987;145:1185-1191. 229. Chung MH, Kasai H, Nishimura S, Yu BP. Protection of DNA damage by dietary restriction. Free Radic Biol Med 1992;12:523-525. 230. Yu BP, Masoro EJ, Murata I, Bertrand HA, Lynd FT. Life span study of SPF Fischer 344 male rats fed ad libitum or restricted diets: longevity, growth, lean body mass and disease. J Gerontol 1982;37:130-141. 231. Solter D, Gearhart J. Putting stem cells to work. Science 1999;283:1468-1470. 232. Cartee GD, Bohn EE, Gibson BT, Farrar RP Growth hormone supplementation increases skeletal muscle mass of old male Fischer 344/Brown Norway rats. J Gerontol Biol Sci 1996;51A: B214-219. 233. McCarter RJM, Masoro EJ,Yu BP. Rat muscle structure and metabolism in relation to age and food intake. Am J Physiol 1982;242:R89-93. 234. Weindruch R, Sohal RS. Caloric intake and aging. N Engl J Med 1997;337:986-994. 235. Wallace DC. Mitochondrial DNA in aging and disease. Sci Am 1997;277:40-47.

C'2000 Elsevier Science B.V. All rights reserved. Handbook of Oxidants and Antioxidants in Exercise. C.K. Sen, L. Packer and O. Hanninen, editors.


Molecular mechanisms of oxidative stress in aging: free radicals, aging, antioxidants and disease Michael Pollack and Christiaan Leeuwenburgh University of Florida, Aging Biochemistry Laboratory Center for Exercise Science, College of Health and Human Performance, P.O. Box 118206, Gainesville, Florida 32611, USA. Tel: +1-352-392-0584. E-mail: cleeuwen @ufl. edu

1 INTRODUCTION 2 SOURCES OF OXIDANTS 2.1 Mitochondria 2.1.1 Mitochondria and aging 2.2 Phagocytes 2.2.1 Phagocytes and aging 3 AGING AND ANTIOXIDANT DEFENSES 4 PROTEIN OXIDATION AND AGING 5 REPAIR AND TURNOVER OF OXIDATIVELY DAMAGED PROTEINS 6 PHYSIOLOGICAL RELEVANCE OF OXIDATION 7 LIFE-PROLONGING INTERVENTIONS 7.1 Dietary restriction 72 Chronic adapted exercise 73 Antioxidant therapy 73.1 Natural and synthetic antioxidants 8 ANTIOXIDANTS IN HUMAN HEALTH AND DISEASE 8.1 Cardiovascular disease 8.2 Neurodegenerative diseases 8.2.1 Alzheimer's 8.2.2 Parkinson's 9 SUMMARY 10 PERSPECTIVES 11 ACKNOWLEDGEMENTS 12 ABBREVIATIONS 13 REFERENCES

1

INTRODUCTION

The aging process has been shown to result in an accelerated functional decline. The exact mechanisms that cause this functional decline are unclear. The free radical theory of aging, however, has gained strong support because it is able to explain some of the processes that occur with aging and the degenerative diseases of aging. This theory proposes that an increase in oxygen radical production with age by mitochondria produce an increase in cellular damage [1—4]. Indeed, researchers have shown that oxygen utilization by mitochondria of aerobic organisms can generate several reactive radicals, such as superoxide (O2 ), hydrogen peroxide (H2O2), and possibly hydroxyl radical (HO*; [5—7]).

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In addition, nitric oxide (NO) is also produced by mitochondria [8] and may have imphcations for the aging process and several disease states associated with aging. Phagocytes are another potent source of oxidant production, and they produce O2 , H2O2, HO-, NO-, and hypochlorous acid (HOCl; [7,9-10]). HOCl is an inflammatory mediator and a strongly oxidizing and chlorinating compound that can form other reactive metabolites, such as nitryl chloride (NO2CI) and nitrogen dioxide (NO2), in the presence of nitrite [11]. Recently, scientists have shown that activated human polymorphonuclear neutrophils convert nitrite into NO2CI and N02' metabolites, which can significantly contribute to the formation of potentially harmful compounds [12]. The potentially deleterious effects of reactive oxygen, reactive nitrogen, and chlorinating species — for simplicity, referred to as oxidants or radicals — can affect the aging process. Aerobic organisms are well-protected against oxidative challenges by sophisticated antioxidant defense systems. However, it appears that during the aging process an imbalance between oxidants and antioxidants balance may occur, referred to as oxidative stress. Oxidative stress induced by oxidant species occurs under conditions when antioxidant defenses are depleted or when the rate constants of the radical reactions are greater than the antioxidant defense mechanisms [13]. As we age the defense mechanisms preventing oxidation may decline in specific tissues, and accelerated oxidative damage could, therefore, trigger a deterioration in physiological function. We will critically look at this interrelationship. Oxidative damage of biomolecules increases with age and is postulated to be a major causal factor of cellular biochemical senescence [14—20]. There is much support for this hypothesis, including the following observations: Studies by Sohal and coworkers using transgenic Drosophila in which the antioxidant enzymes, superoxide dismutase (SOD) and catalase (Cat) — two enzymes that scavenge the highly reactive oxidants, superoxide and hydrogen peroxide, respectively — are overexpressed this showed increases in the average and maximum life span and a reduction in oxidative damage [21,22]. Also, several aged species produce a significantly greater amount of reactive oxygen species compared to their younger counterparts [19,20,23]. Moreover, caloric restriction intervention, — that is, a restriction of 60% of the caloric intake of the ad libitum regimen without malnutrition — extends the life span of rodents by 40% [24,25]. In addition, other species such as invertebrates and fish also show remarkable increases in life span with caloric restriction [25—28]. The postulated mechanism is that a reduction in oxygen consumption, with a concomitant reduction in metabolic rate and body temperature in caloric restricted animals, lowered the chronic oxidative stress with age. In addition, caloric restriction also retards a variety of age-related deleterious biochemical and physiological changes, such as the development of cancer and diabetes [25,28]. Furthermore, caloric restriction attenuates protein oxidation, DNA damage, and lipid peroxidation in several housefly and animal models [21-30]. Other evidence that links oxidant generation and aging is provided by the strong inverse correlation between the rate of mitochondrial oxidant generation

Chapter 30: Molecular mechanisms of oxidative stress in aging

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and the maximum life span among diflferent species, i.e., animals with high mitochondrial metabolism have a short life span since they have higher oxygen consumption and therefore higher oxidant production [27,28]. In addition, there is a strong correlation between maximum life span of a species and its SOD activity (the first defense against reactive oxygen species). For example, the activity of human SOD is approximately 16-fold greater than that of mice. The increase in SOD activity may provide more protection against superoxide radicals. This would strongly suggest that life span may, in part, depend on the activity of this enzyme [27,31]. We will review several potent sources of oxidant formation and the potentially deleterious effect of oxygen, nitrogen, and chlorinating species on cellular biomolecules. The mitochondria and phagocytes will be highlighted in relationship to biological aging and selected diseases of aging. 2

SOURCES OF OXIDANTS

The oxygen molecule in its diatomic ground state (Êg O2) is essential for the production of energy. By itself it qualifies as a radical species since it has two unpaired electrons, each of which is located in a different n antibonding orbital. These two electrons also have parallel spins, i.e., they both share the same spin quantum number. Consequently, ground state oxygen is sparingly reactive: so far to oxidize O2 another molecule by accepting an electron pair, both electrons would have to possess antiparallel spins relative to the unpaired electrons in O2, according to Pauli's exclusion principle. This criterion is seldom met in a typical electron pair, so oxygen tends to accept electrons one at a time. In vivo, multielectron reduction of O2 is carried out by a coordinated series of enzymes that reduce O2 1-electron at a time. If O2 accepts a single electron, the electron must enter an antibonding orbital producing the superoxide radical O2 . Two-electron reduction of O2, with the addition of 2H'^, generates hydrogen peroxide (H2O2). Most O2 is metabolized by SOD to H2O2 and oxygen. In reactions catalyzed by free transition metals such as Fe^"^, O2 and H2O2 can generate the extremely reactive hydroxyl radical (HO). This molecule is believed to be the major cause of damage to proteins, lipids, and DNA. Oxidative damage to these biomolecules seems to depend on hydrogen peroxide and a reduced transition metal. Therefore, molecules that contain transition metals, such as aconitase (a Krebs cycle enzyme), are likely to undergo oxidative damage [17,18,32-34]. 2.1 Mitochondria The main function of mitochondria is energy production. During oxidative phosphorylation, however, highly reactive oxygen radicals are generated. It has been estimated that the release of reactive intermediates accounts for about 2% of the oxygen consumed during respiration. One major site of oxidant production

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occurs in the mitochondrial electron transport chain in which O2 is reduced to H2O (Fig. 1). In this process, electrons from NADH are donated to complex I (NADH dehydrogenase complex), and electrons from succinate are donated to complex II (succinate dehydrogenase complex). Ubiquinone, also known as coenzyme Q or UQ, accepts electrons from both complexes and is sequentially reduced, one electron at a time, to ubisemiquinone and ubiquinol. Ultimately, electrons are transferred through complex III (UQ-cytochrome c reductase), cytochrome c, and complex IV (cytochrome-c oxidase) with the result of reducing O2 to H2O. Mitochondrial electron transport, however, is imperfect and results in the production of O2 ~ from the one-electron reduction of O2. Some speculate that this electron comes from the free radical ubisemiquinone: instead of accepting another electron and proton to form ubiquinol, one of the electrons may leak from ubisemiquinone and reduce O2 to O2 . Enzymatic dismutation of O2 ~ then leads immediately to H2O2, another important biological oxidant [7]. How much O2 and H2O2 is generated during mitochondrial respiration? In state four respiration, which is characterized by a high degree of reduction of the electron carriers and a limiting supply of ADP, H2O2 production is at its maximum. It has been estimated that under these conditions the release of reactive intermediates accounts for 2% of the oxygen consumed during respiration [5]. Ultimately how much of this mitochondrial H2O2 is derived from dismutation of O2 ~ in the electron transport chain? Measurement of O2 ~ production cannot be accomplished in intact mitochondria because it is dismutated by mitochondrial SOD (mSOD). However, by isolating submitochondrial fractions and by removing mSOD by sonication and washing, it is possible to detect electron transport chain O2 production. In experiments done in the 1970s, it was found that submitochondrial particles produce from 4- to 7- nmol O2 ~/min per mg of protein, resulting in 02~/H202 ratios of 1.5—2.1 [6]. Since two 02^ anions dis-

Fig. 1. Mitochondrial oxidant production; MnSOD: manganese superoxide dismutase; MtNOS: mitochondrial nitric oxide synthase.


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mutate to form one H2O2 molecule, these results indicate that O2 is almost certainly a precursor of mitochondrial H2O2 (Fig. 1). The enzyme nitric oxide synthase (NOS), which produces the free radical gas NO from L -arginine, has been primarily investigated in endothelial cells. However, recent experiments have been performed which indicate that NOS in mitochondria produce NO- (Fig. 1). First, several studies using immunohistochemical techniques showed that skeletal muscle expresses endothelial-type nitric oxide synthase (ec-NOS). These studies also show that there is a strong correlation of ec-NOS expression to mitochondrial content visualized histochemically by succinate dehydrogenase [35]. In addition Bates et al. [36,37] have demonstrated localization of NO- synthase in mitochondria isolated from heart, skeletal muscle, kidney, and brain using a monoclonal antibody against the ec-NOS. Recently, mitochondria have been unambiguously identified as sources of NO-, using electron paramagnetic resonance with spin-trapping techniques. Giulivi et al. isolated NOS from Percoll-purified rat liver mitochondria [38]. Several different mitochondrial preparations, such as toluene-permeabilized mitochondria, mitochondrial homogenates, and a crude preparation of NOS, were incubated with the spin trap A^-methyl-D-glucamine-dithiocarbamate-Fe II which produced a signal ascribed to the NO- spin adduct [8]. The intensity of the signal increased with time, protein concentration, and L-arginine, and decreased with the addition of the NOS inhibitor N^-monomethyl-L-arginine. Kinetic parameters, molecular weight, requirement of cofactors, and cross-reactivity to monoclonal antibodies against macrophage NOS suggest similarities to the inducible form. However, the constitutive expression of this NOS enzyme and its membrane localization may indicate a distinctive isoform [8]. These findings suggest that mitochondrial NOS and NO- production may not only have an important role as a cellular transmitter, messenger, or regulator, but also as an active player in oxidative metabohsm [8,34—38]. Since NO- and O2 react to produce another very reactive oxidant, peroxynitrite (ONOO ), it is clear that mitochondria are a major source of free radicals and oxidants. 2,1.1 Mitochondria and Aging Several studies have investigated if there is an age-associated increase in the generation of oxidants by mitochondria. An experiment using mongolian gerbils (Meriones unguiculatus) found that O2 and H2O2 production increased with age, especially in isolated mitochondria and submitochondrial particles from the aged heart [39]. In another study using whole hepatocytes of older rats, it was found that older hepatocytes had a decrease in the mitochondrial membrane potential of 30% and an increase in mitochondrial hydrogen peroxide generation of 23%. H2O2 levels within the liver cells were also increased [40]. Furthermore, another study on intact, isolated rat hepatocytes reached the same conclusion: cellular oxidant generation by mitochondria increases with age [41]. Similarly, experiments using intact muscle mitochondria from house flies has

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shown that the rate of H2O2 generation progressively increases 2-fold as the house fly ages [42]. On the other hand, an experiment which attempted to maintain proper physiological substrate concentrations during in vitro mitochondrial incubations detected no difference in H2O2 generation by rat heart mitochondria when comparing 24-month-old rats (senescent adults) and 6-month-old rats (young adults) [43]. The enhanced generation of oxidants by older mitochondria may itself be caused by oxidative damage to mitochondrial membranes and proteins. In one experiment, when isolated mitochondria were exposed to oxidant generators like glutaraldehyde (an intermolecular cross-linking agent) or 2,2-azobis-di-hydrochloride, the mitochondria generated H2O2 at an increased rate [42]. This evidence, along with the above studies, paints a vicious cycle of mitochondrial oxidant damage and oxidant generation. In connection with such findings, Miquel and his colleagues [44,45] have widely promulgated the mitochondrial mutation theory of aging. In this theory, senescence is linked to mutations of mitochondrial DNA (mtDNA) in differentiated cells. As discussed earlier, isolated mitochondria from aged animals have an increased production of reactive oxygen species compared to young animals. This observation may be of key importance because it is likely to increase the rate at which proteins and DNA are oxidized. Since these mutations occur in postmitotic cells and mtDNA lacks excision and recombination repair mechanisms, it has been postulated that these mutations would lead to problems in replication, leading to a decline in physiological performance and the pathogenesis of many age-related diseases [44,45]. In addition, mtDNA is not protected by histones or DNA-binding proteins and, therefore, is directly exposed to a high steady-state level of reactive oxygen and nitrogen species. Thus, oxidative modification and mutation of mtDNA may occur with great ease. Mutations in mtDNA can lead to the production of less functional respiratory chain proteins, resulting in increased free radical production and possibly more mtDNA mutations. This vicious cycle operates in different tissues at variable rates and leads to differential accumulation of oxidatively modified mtDNA. This may ultimately reduce energy output and contribute to the common signs of normal aging. In this context, one study by Yakes et al. [46] compared whether mtDNA accumulates more oxidative DNA damage relative to nuclear DNA; it also investigated whether there were differences in repair between nuclear and mtDNA. Specifically, the researchers investigated the formation and repair of H202"induced DNA damage in a 16.2 kb mitochondrial fragment and a 17.7 kb fragment flanking the P-globin gene. Fibroblasts treated with a relatively high dose of hydrogen peroxide exhibited 3-fold greater damage to the mitochondrial fragment compared with the nuclear fragment. Furthermore, damage to the nuclear fragment was completely repaired within 1.5 h; whereas, no DNA repair in the mitochondrial fragment was observed [46]. These data suggest that mtDNA is more susceptible to oxidants, and mitochondrial repair mechanisms may be nonexistent or less efficient.


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2.2 Phagocytes Phagocytic cells are another major source of oxidants. Neutrophils and other phagocytes attack pathogens using a mixture of oxidants - for example, O2 , NO-, H2O2, and HOCl. The process of phagocytosis begins when the neutrophil travels to a site of infection as directed by certain chemotactic signals that are generated at the infection site. When foreign microbes, which may be bound by serum-derived glycoproteins (i.e., opsonized), perturb the plasma membrane of a neutrophil, a dormant pyridine-nucleotide-dependent oxidase is activated. This pyridine nucleotide oxidase is believed to be a reduced nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) or a reduced nicotinamide adenine dinucleotide oxidase (NADH oxidase), whose action might involve a b type cytochrome [7,10]. This process initiates a "respiratory burst", which lasts for 15 to 20 min and reduces O2 to O2 (a one-electron reduced product) and H2O2 (a two-electron reduced product). It also serves as the first source of oxidant production in phagocytes. Simultaneously, the plasma membrane of the phagocyte invaginates around the foreign particle, surrounding it and subsequently pinching off to become a phagosome. Also during this time period, degranulation occurs: granular lysosomes migrate toward the phagosome, fuse with it, and empty their granular contents into it; after fusion with lysosomes, the phagosome becomes a phagolysosome. The contents of the lysosomes primarily include digestive enzymes like acid hydrolases, neutral proteases, and alkaline phosphatases. These lysosomes also contain cationic proteins, lipopolysaccharides, lactoferrin, myeloperoxidase (MPO), and biopolymers that might be involved in bactericidal reactions. This entire process occurs extremely rapidly taking no longer than a few minutes [7,10]. Inside the phagolysosome, the heme containing enzyme MPO forms an enzyme-substrate complex with H2O2 that can then catalyze the two-electron oxidation of halides hke CI , Br , and I" by H2O2. The oxidation of the halides, particularly C I , forms a toxic agent with potent antimicrobial properties. In the case of chloride, the oxidation product is HOCl, which subsequently kills the ingested microorganism. The H2O2 needed for this process is generated in the respiratory burst and can be detected in the phagosome [9]. Furthermore, a recent study on the MPO-hydrogen-peroxide-chloride system showed that MPO also generates CI2 gas and that human neutrophils employ chlorine gas as an oxidant during phagocytosis [47]. 2.2,1 Phagocytes and aging Currently it is unclear if there are age-associated trends in oxidant production by phagocytes. Age-dependent oxidant generation is relevant because it is very common for older individuals to suffer from inflammatory-related conditions such as arthritis. Some studies have indicated that oxidant production is a function of

Part X: Aging

age; in fact, several experiments have shown that older macrophages produce a decreased amount of O2 , H2O2, NO- and other oxidants [48—50]. In a study on rat peritoneal macrophages by Alvarez et al., production of O2 was decreased by 50% and H2O2 by 75% in older macrophages [49]. NO- production was also reduced to 40% with age [48]. In a study done on human monocytes by Alvarez et al., they found that superoxide production was age and sex dependent. It decreased 45% in men and 70% in women during aging [51]. Other studies have shown that oxidant production is age-dependent, but in these studies the generation of reactive oxygen species appeared to increase with age. In a study by Lavie et al., they found a 2-fold increase in oxidant production by senescent peritoneal macrophages after being stimulated by latex and zymosan [52]. Also, a study on patients with obliterative atherosclerosis of the lower legs showed an increase in superoxide anion production by polymorphonuclear leukocytes (PMNLs) under basal conditions. After stimulating these PMNLs with formyl-methionin-leucyl-phenylalanine and calcium ionophore, the researchers found that the PMNLs of the atherosclerotic patients had an increased ability to release myeloperoxidase and elastase than PMNLs of healthy, middle-aged subjects [53]. Moreover, in a recent study there was an increased concentration of hydrogen peroxide, following stimulation by formyl peptide, in individual neutrophils from older volunteers (ages 65 and older) compared to neutrophils from younger volunteers (aged 21—34 years). By analyzing enzyme kinetics, the researchers concluded that the age-associated accumulation of H2O2 in stimulated neutrophils could be accounted for by impaired glutathione peroxidase (GPX) [54]. Further studies are warranted to investigate whether age causes an increase or decrease in oxidant production by PMNLs and whether aging-associated human diseases significantly affect oxidant production. 3

AGING AND ANTIOXIDANT DEFENSES

The possibility that oxidative stress and aging is mainly due to a decline in antioxidant defenses is still not clear. In fact, most researchers would argue that antioxidant defenses are, in general, not different between young and old animals. We will briefly discuss the general trends of several major organs, the effects of age on each of the major antioxidant systems, and the effect of selected antioxidants on age. This topic has been extensively reviewed by Matsuo [55], and we will discuss some of the general findings on the antioxidant enzymes — SOD, Cat, and GPX. We will also discuss the major trends involving the changes, if any, in water soluble and lipid soluble antioxidants, mainly focusing on vitamin C, GSH, and vitamin E, realizing that there are a multitude of other known and possibly unknown antioxidant defenses which may change with age. SOD is a cytosolic (copper/zinc) and a mitochondrial (manganese) isoenzyme, which dismutates the superoxide radical to oxygen and hydrogen peroxide. The majority of the studies, which investigated total SOD activity in the brains of rats and mice, have found little change in the enzyme's activity with age [55].


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(One study, in fact, actually found an increase in mitochondrial SOD activity in the brain of rats with age, which is not what one would expect if decreased antioxidant enzymes were a major factor in aging) [56]. Furthermore, the majority of the studies investigating the liver of mice also showed no change in total SOD. In contrast, many studies in rats showed a decrease in liver SOD activity [55]. This discrepancy is unclear, but could give an indication of variable adaptations between species. Most studies show that SOD activity in the heart of both mice and rats does not change with age [55]. Skeletal muscle SOD, however, does increase with age in old rats compared to either young or middle-aged rats (Table 1, [57—60]). Some of the inductions of SOD with age could reflect a chronic adaptation to the increase in superoxide production. Gluthathione peroxidase is the major hydrogen peroxide scavenger enzyme, and it is found in both the cytosol and mitochondria. Together with its substrate GSH (discussed later), they form a formidable defense against hydrogen peroxides and lipid peroxides. Rat brain shows no change with age in this important reactive oxygen species scavenging enzyme [55]. Both the liver and the heart GPX show about an equal number of studies reporting either no change or a decrease in enzyme activity; thus, there is no consistent pattern of change with this enzyme [55]. Again, skeletal muscle GPX activity increased with age in rats, indicating an adaptive change to reduce oxidative stress in this tissue (Table 1 [57]). Another H2O2 metabolizing enzyme is Cat. It metabolizes hydrogen peroxide to oxygen and water. This enzyme is primarily found in the peroxisomes, but it is also located, less abundantly, in mitochondria. The literature shows no consistent trend in changes of Cat activity in brain tissues with aging; an equal amount of studies show an increase or a decrease in activity [55]. This may reflect difficulties in the measurement of Cat activity. Cat activity in the liver seems to decline consistently in aging rats. In striking contrast. Cat activity increases in the heart and skeletal muscle of aging rats [55,57]. For example, Leeuwenbugh et al. found that skeletal muscle Cat activity increased by a large margin of 150% comparing 4.5-month-old rats with 26.5-month-old rats, and there was a 40% increase comTable 1. Activities of antioxidant enzymesin deep vastus lateralis (DVL) skeletal muscle of young and old rats [56].

Young Adult Old

GPX DVL

CAT DVL

SOD DVL

4.25 ± 0.29 5.70 ± 0.34^ 5.88 ±0.36^

15.8 ±2.2 29.4 ± 3.8^ 39.6 ± 4.2*^

1960 ± 143 2190 ± 118 2510 ± 120^

Values are means ± SE; GPX: glutathione peroxidase; SOD: superoxide dismutase (units/g wet weight); CAT: catalase (K x 10^/g wet weight); GSH: glutathione jimol/g wet weight; Young = 4.5 months; Adult = 14.5 months; Old = 26.5 months. ^p 30 g ethanol

1.36 1.82 1.40 0.80 2.34 1.21 2.02

1.07-1.73 1.16—2.84 1.04-1.86 0.44-1.45 1.17-4.68 0.80-1.23 1.07-3.80

Âsbestos nonexposed; Âsbestos-exposed.

2 x 2 factorial trial of P-carotene (50 mg on alternate days), aspirin (325 mg on alternate days), both, or placebo. The aspirin component was stopped after 6 years because of favorable results in the prevention of myocardial infarction. The |3-carotene component was followed for 12 years. No modification of risk was observed in this trial. 5.2.3.6 Interpretation of intervention studies. The reasons for a lack of effect might lie in the fact that antioxidants are only markers of certain foods and that such foods contain the effective components. Plants contain many compounds likely to protect against cancers (glucosinolates, isothiocyanates, S-methyl cysteine, allyl sulfurs, phytates, phyto-estrogens) some of which demonstrate antioxidant properties (phenolic compounds, flavonoids). Interest focused on them when it became obvious that the protective effect of vegetables and fruit intakes was larger than that of the nutrients usually evaluated as components of the ingested fruit or vegetables. The failure of intervention studies with P-carotene and vitamin E enhanced this trend. Witte et al. [113] demonstrated it elegantly by further adjusting for potentially anticarcinogenic constituents of fruit and vegetables (namely antioxidants and fiber) the OR showing a decreased risk of colorectal adenomas in persons with a high intake of fruit and vegetables. None of the associations was decreased by adjusting for the relevant antioxidant nutrients, indicating that protective effects might reflect unmeasured constituents in these foods, at least for colorectal adenomas (only risk reduction with whole grain was decreased by adjusting for fiber, indicating that this particular reduction was fiber-dependent). Knowledge of the precise content of these compounds in food and of their metabolism and physiological role is scarce, however, especially with regard to adsorption through the intestinal barrier and the chemistry of their derived metabolites in human and animal organisms. The study conducted by Hertog et al. [114] is of special interest because it was preceded by analytical work on several foods to identify and quantitate the main flavonoids present in the diet of the surveyed population. The intake of quercetine and kaempferol was not associated with cancer incidence (whereas it was shown to be protective against cardiovas-

942

Pcirt XI: Disease processes

cular diseases). A more recent prospective study using the same database as Hertog et al. [114] showed a reduction of risk for cancer [115]. The follow-up of 9,959 Finnish men and women over 24 years showed a reduction for all sites (0.80, CI: 0.67—0.96), mainly reflecting the strong effect on lung cancer (0.54, CI: 0.34-0.87). Experimental studies using such nonnutrient plant components are essentially conducted in vitro thus, avoiding the question of absorption. What is more, they generally pertain to properties other than antioxidant ones, such as the effect on the cell cycle [116,117]. Although it is possible that P-carotene is only a marker of other beneficial plant components, this does not explain the increased risk observed with supplementation. One essential reason for the increased risk appears to be the situation of the subjects with regard to the natural history of cancer. In the ATBC [4] and CARET [5,111] studies, the particular local conditions may be responsible for implementing the pro-oxidant capacities of (3-carotene: NO2, which is known to interfere with P-carotene, is provided by current smoking, and molecular oxygen at atmospheric pressure is provided by breathing. The CARET study permits further interpretation, because the asbestos- and tobacco-exposed subgroups have different point estimates for relative lung cancer risk (though with overlapping confidence intervals). Serum (J-carotene above the median at baseline does not reduce lung cancer risk in asbestos-exposed subjects (RR = 0.95; 95% CI: 0.60, 1.49 vs. RR = 0.62; 95% CI: 0.46, 0.82 in heavy smokers). Besides, in asbestos-exposed subjects there is no difference in relative lung cancer risk between former and current smokers (Table 2). It is, therefore, likely that NO2 does not play a part in the oxidative stress to which asbestos workers are subject. The NO2 pathway transforming (3-carotene into pro-oxidant described above thus, does not operate in this situation. P-Carotene can, therefore, react with another pro-oxidant, the anion superoxide of the inflammatory reaction caused by asbestos, enhancing carcinogenesis. Or it can act by another mechanism, the high level of antioxidant, protecting proliferation of transformed cells. However, the p-carotene plasma levels for the supplemented group were no higher in the lung cancer cases than in the cancer-free subjects. 6

SUMMARY

6.1 Mechanisms The study of the mechanisms at work during the natural history of cancer strongly suggests that antioxidants can oppose transformation of normal cells by mutagenic oxidative stress. They probably also play a part at the promotion stage as reactive oxygen species act as second messengers in transcellular signaling systems, although antioxidant effects have been ambiguous in some models. This ambiguity might result from two situations, either:

Chapter 31: Oxidative stress, antioxidants and cancer

943

1) antioxidants become pro-oxidants because of particular conditions (P-carotene, and atmospheric pressure of oxygen and NO2 stress), or because of lack of regeneration (deficit in vitamin C to regenerate vitamin E); or 2) antioxidants allow promotion by reactive oxygen species because they protect the DNA replication process in the cells from oxidative stress. The shift of the oxidant-antioxidant balance toward the antioxidant side observed in several tumor cells might also take place at the stage of tumor growth and facilitate carcinogenesis. But the shift is more probably the result of genetic instability in the regulation of apoptosis and of overexpression of antioxidant enzymes, rather than of exogenous antioxidants (except perhaps in the case of very high dosage supplementation). The natural history of cancer and the chemistry of antioxidants suggest factors or mechanisms which might explain the increased risk of lung cancer in the population at risk covered by the ATBC and the CARET studies, although these are still speculative at present: 1) the presence of initiated cells in the populations at risk; 2) antioxidants possibly becoming antioxidants in specific situations; 3) high dosages of p-carotene; and 4) a shift in the pro-oxidant/antioxidant balance. 6.2 Epidemiological findings Findings of epidemiological studies (prospective and retrospective case-control studies) support the protective role of antioxidants in the early stages of carcinogenesis. This is not true of all cancers, however: the only cancers which are consistently inversely associated with antioxidants are epithelial cancers in which carcinogens come into direct contact with the target cells: tobacco and the buccal epithelium; asbestos, benzo-pyrene, NO2 and the epithelium of the lung; nitrosamines and the gastric epithelium; polyomavirus and the epithelium of the cervix. The other cancer sites do not appear to benefit from the protective effect of antioxidants. The effect of each antioxidant varies with the cancer site: P-carotene and vitamin C share a risk-reducing effect for upper respiratory and digestive tract cancers and for cervical cancer. But there is stronger evidence of an association between lung cancer and carotenoids than between lung cancer and vitamin C, and of an association between gastric cancer and vitamin C rather than with carotenoids. This might reflect a specificity with regard to carcinogens. Vitamin E possibly decreases lung, stomach and cervical cancer risks, and selenium possibly lung and stomach cancers, but the findings are less consistent than for p-carotene and vitamin C. It is noteworthy that vitamin E and selenium seem to have a common effect and that this effect is mainly observed in men, as this might indicate that they react with carcinogens such as alcohol and tobacco. Prostatic cancer appears to be a special case, in that it has been inversely associated with lycopene intake [57] and vitamin E supplementation [4].

944

Pci^t Î- Disease processes

6.3 Public health implications Antioxidant nutrients are never as risk-reducing as the food they come from, essentially fruit and vegetables. However, it should be noted that fruit and vegetables are not the main contributors of vitamin E, which has been found to be protective in several studies. We, therefore, cannot be sure that these antioxidant nutrients, which are consistently inversely associated with some cancers, are effective by themselves. They might alternatively be markers for another risk-reducing component. This would explain the lack of protective effect of supplementation in intervention studies in the population not at risk (the Physician Health Study). Concomitant intake of synergic nutrients, such as provided by fruit and vegetables might be necessary: this would explain the moderate success of the Linxiang study, where a severely deficient population was supplemented with three antioxidants. One can therefore conclude that antioxidants are not the magic bullet capable of protecting against all cancers at all stages of carcinogenesis. 7

PERSPECTIVES

In the short term, we can await the findings of on-going intervention studies. Such studies are usually based on a combined treatment. A study into antioxidants, Helicobacter pylori and stomach cancer is being conducted in Venezuela to evaluate the progression rate of precancerous lesions of the stomach [118]. It combines vitamin C (250 mg), vitamin E (200 mg) and p-carotene (6 mg). In France, the SUVIMAX study [119] provides daily supplementation with vitamin C (120 mg), (3-carotene (6 mg), vitamin E (30 mg), selenium (100 mg) and zinc (20 mg), a combination equal to 1 to 3 times the recommended dietary allowances. If the combined supplementation provides no protection, this might instead indicate the role of other plant food components, such as glucosinolates, isothiocyanates, phenohc compounds, flavonoids, fiber etc.) The agenda for further research is marked by the uncertainties and the speculative hypothesis developed above. Is P-carotene the marker of increased risk through a lowered defense mechanism or through greater exposure to a specific carcinogen? Metabolism of carotenoids in situations relevant to human conditions must be investigated with special attention to doses, oxygen pressure and cell antioxidant environment. If P"carotene is the marker of risk reduction only for upper aerodigestive tract cancers, which is/are the component(s) responsible? Intense research into the various components of fruit and vegetables is already in progress to answer this question, but models relevant to human situations have still to be designed. In the meantime reanalysis of the many epidemiological observational studies should be carried out using recently acquired information on food composition. Should vitamin E always be studied in conjunction with vitamin C, keeping in mind that the main contributor of vitamin E is sunflower oil and mayonnaise.


945

which are generally used with raw vegetables. Such biochemical and epidemiological studies need to be supplemented by genetic and molecular studies to shed more light on the shift in the pro-oxidant/antioxidant balance occurring in the proliferating tumor cells. The main question is: should intervention studies be continued, mainly with (3carotene? Taking into consideration the risk level of the population sample, keeping doses at nutritional levels and combining several antioxidants may be not deleterious, but may also not provide much more helpful guidelines for prevention than the usual one of four to five helpings of fruit and vegetables per day Even if we think it impossible to influence human food habits and lifestyles, it is essential that research be continued, with the aim of defining which combination of antioxidants will contribute to better health, and for which population: the whole population or the population at risk. 8

ACKNOWLEDGMENTS

The author thanks Robert Goulevitch for his help in literature search and editing. 9

ABBREVIATIONS

ATBC: CARET: CDK: CI: DNA: GSHpx: LDL: MDA: NADH: OR: pRB:

oc-tocopherol P-carotene carotene retinol cyclin-dependent protein kinase confidence interval desoxyribonucleic acid glutathione peroxidase light density lipoprotein malondialdehyde nicotinic amine dehydrogenase odds ratios retinoblatoma protein

10 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Ames BN, Gold LS,Willett WC Proc Nat Acad Sci USA 1995;92:5258-5265. Simic MG. Mutat Res 1988;202:377-386. Pryor WA. Environ Health Perspect 1997;105:875-882. ATBC, The a-Tocopherol, P-Carotene Cancer Prevention Study GR. N Engl J Med 1994;330: 1029-1035. Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen MR, Glass A, Keogh JP, Meyskens FL,Valanis B, Williams JH, Barnhart S, Hammar S. N Engl J Med 1994;334:1150-1155. Hill MJ, Morson BC, Bussey HJR. Lancet 1978;1:245-247. Ponten J, Holmberg L, Trichopoulos D et al. Int J Cancer 1990;S5:5-21. Vogelstein B, Fearon EF, Hamilton SR et al. N Engl J Med 1988;319:525-532. Esterbauer H, Zollner H, Schaur RJ. ISI Atlas Sci Biochem 1988;1:311-317. Cavalieri E, Rogan E, Roth R. In: Floyd RA (ed) Free Radicals and Cancer. New York: Marcel

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Dekker, 1982:117-158. 11. Kensler TW, Egner PA, Moore KG, Taffe BG, Twerkok LE, Trush MA. Toxicol Appl Pharmacol 1987;90:337-346. 12. Cerutti PA. The Lancet 1994;344:862-863. 13. Harman D. In: Emerit I, Chance B (eds) Free Radicals and Aging. Basel, Switzerland: BirkhauserVerlag, 1992:1-10. 14. Wang XD, Russel RM, Liu C, Stickel F, Smith DE, Krinsky NY. J Biol Chem 1996;271:2649026498. 15. Krinsky NL In: Preventive Med 1989;18:592-602. 16. Krinsky NL Free Rad Biol Med 1989;7:617-635. 17. Burton GW, Ingold KU. Science 1984;224:569-573. 18. Palozza P, Krinsky NL Methods in Enzymology 1992;213:403-420. 19. Astorg P, Gradelet S, Berges R, Suschetet M. Nutr Cancer 1996;25:27-34. 20. Halliwell B, Gutteridge JMC. In: Halliwell B, Gutteridge JMC (eds) Free Radicals in Biology and Medecine. Oxford, England: Clarendon Press, 1985. 21. Herbert V. Am J Clin Nutr 1994;60:157-158. 22. Neve X Experientia 1991 ;47. 23. Cerutti PA. Science 1985;227:375-381. 24. Gerber M, Dornand J. In: C Pasquier, Olivier Ry Auclair C, Packer L (eds) Oxidative Stress, Cell Activation and Viral Infection. Basel, Switzerland: Birkhauser Verlag, 1994. 25. Packer L, Suzuki YJ. In: Pasquier C et al. (eds) Oxidative Stress, Cell activation and Viral Infection. Basel/Switzerland: Birkhauser Verlag, 1994:113-130. 26. Clurman BE, Roberts JM. J Nat Cancer Inst 1995;87:1499-1501. 27. Cerutti PA. Env Health Persp 1989;81:39-43. 28. Mitchel REJ, McCann R. Carcinogenesis 1993;14:659-662. 29. Everett SA, Dennis MF, Patel KB, Maddix S, Kundu SC,Willson R. J Biol Chem 1996;271: 3988-3994. 30. Szent-Gyorgyi A, Egyud LG, McLaughlin JA. Science 1967;155:539-541. 31. Burton GW,Traber MG. Ann Rev Nutr 1990;10:357-382. 32. Slater T, Benedetto C, Burton GW, Cheeseman KH, Ingold KU, Nodes JT In: Thaler-Dao H, Paoletti R, Crastes de Paulet A (eds) Icosanoids and Cancer. New York: Raven Press, 1984: 21-29. 33. Cheeseman KH, Collins M, Proudfoot K et al. Biochem J 1986;235:507-514. 34. Cheeseman KH, Emery S, Maddix SP et al. Biochem J 1988;250:247-252. 35. Masotti L, CasaU E, Galeotti T Free Radical Biol Med 1988;4:377-386. 36. Szabados GXTretter L, Horvath I. Free Rad Res Comm 1989;7:161-170. 37. Begin ME, Ells G, Horrobin DF J Nad Cancer Inst 1988;80:188-194. 38. Najid A, Beneytout J-L,Tixier M. Cancer Lett 1989;46:137-141. 39. Cogrel P, Morel I, Lescoat G, Chevanne M, Brissot P, Cillard P, Cillard J. Lipids 1993;28: 115-119. 40. Southgate J, Pitt E, Trejdosiewicz LK. Br J Cancer 1996;1996:728-734. 41. YoshikawaT, Kokura S,Tainaka K, NaitoY, Kondo M. Cancer Res 1995;55:1617-1620. 42. Schwartz JL, Antionades DZ, Zhao S. Ann NY Acad Sci 1993;680:262-278. 43. Anti M, Marra G, Franco A, Bartoli GM, Ficonelli R. Gastroenterology 1992; 103. 44. Ripple MO, Henry WF, Rago RP,Wilding G J Nad Cancer Inst 1997;89:40-48. 45. Gonzales MJ, Schemmel RA, Gray JI, Dugan L, Sheffield LG,Welsh CW Carcinogenesis 1991; 12:1231-1235. 46. Gerber M, Richardson S, Favier F, Crastes de Paulet A. In: Emerit I et al. (eds) Antioxidants in Therapy and Preventive Medicine. New York: Plenum Press, 1990. 47. Lhuillery C, Cognault S, Germain E, Jourdan ML, Bougnoux P. Cancer Lett 1997; 114: 233-234. 48. ButtkeTM, Sandstrom PA. Immunol Today 1994;15:7-10.


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115. Knekt P, Jarvinen R, Seppanen R, Heliovaara M,Teppo L, Pukkala E, Aromaa A. Am J Epidemiol 1997;146:223-230. 116. Matsukawa Y, Marui N, Sakai T, Satomi Y, Yoshida M, Matsumoto K, Nishino H, Aoike A. Cancer Res 1993;53:1328-1331. llT.Fotsis T, Pepper MS, Aktas E, Breit S, Rasku S, Adlercreutz H, Wahala K, Montesano R, Schweigerer L. Cancer Res 1997;57:2916-2921. 118. De Sanjose S, Munoz N, Sobala G, Vivas J, Peraza S, Cano E, Castro D, Sanchez V, Andrade O, Tompkins D, Schorah CJ, Axon AT, Benz M, Oliver W. Eur J Cancer Prev 1996;5:57-62. 119. Hercberg S, Galan P, Preziosi P, Roussel A-M, Arnaud J, Richard MJ, Malvy D, Paul-Dauphin A, Briancon S, Favier A. Intern J Vitam Nutr Res 1998;68:3-20. 120. lARC. Handbooks of cancer prevention vol 2. Carotenoids publ Int Ag Cen Res Lyon: 1998.


IT;2000 Elsevier Science B.V. All rights reserved. Handbook of Oxidants and Antioxidants in Exercise. C.K. Sen, L. Packer and O. Hanninen, editors.

Q^l yJ I

Part XI • Chapter 32

Alcohol and oxidative stress Charles S. Lieber Mount Sinai School of Medicine and Alcohol Research and Treatment Center, Section of Liver Disease and Nutrition, Bronx Veterans Affairs Medical Center (151—2), 130 West Kingsbridge Rd, Bronx, NY 10468, USA. Tel: +1-718-579-1646. Fax: +1-718-733-6257. E-mail: [email protected]

1 2

3

4 5 6 7 8

1

INTRODUCTION METABOLISM OF ETHANOL 2.1 Alcohol dehydrogenase pathway 2.2 Microsomal ethanol-oxidizing system (MEOS) 2.2.1 Role of MEOS in ethanol metabolism 2.2.2 Oxidative stress, xenobiotic and retinoid toxicity and carcinogenicity 2.3 Role of catalase TOXICITY OF ACETALDEHYDE, INCLUDING ITS ADVERSE EFFECTS ON ANTIOXIDANT SYSTEMS 3.1 Elevated acetaldehyde and mechanisms of toxicity 3.2 Alterations of antioxidant systems and phospholipids 3.2.1 Depletion of glutathione and S-adenosylmethionine 3.2.2 Vitamin E deficiency 3.2.3 Phosphatidylcholine depletion SUMMARY PERSPECTIVES ACKNOWLEDGMENTS ABBREVIATIONS REFERENCES

INTRODUCTION

Alcohol-induced oxidative stress predominates in the liver because of its link to the oxidation of ethanol which occurs predominantly in that organ. Each of the three pathways of ethanol metabolism contributes in a unique manner. In addition, they all produce acetaldehyde which can cause peroxidation. The link between oxidative stress and the specific steps in the metabohsm of alcohol will be reviewed for each pathway separately, followed by a more general discussion of the action of acetaldehyde and the alterations in defense mechanisms against oxidative stress. Finally, associative organ damage and possible therapeutic approaches will be reviewed. Specific pathologic alterations linked to a given aspect of the oxidative stress are discussed in connection with the corresponding disorders, while some more general pathologic consequences (e.g., fibrosis) are addressed in a separate chapter.

Part XI: Disease processes

952 2

METABOLISM OF ETHANOL

The hepatocyte's three main pathways for ethanol metaboHsm are each located in different subcellular compartments: 1) the alcohol dehydrogenase (ADH) pathway is in the cytosol (or soluble fraction of the cell; 2) the microsomal ethanol-oxidizing system (MEGS) in the endoplasmic reticulum; and 3) catalase in the peroxisomes (Fig. 1). 2.1 Alcohol dehydrogenase pathway The main pathway of ethanol metabolism proceeds via ADH, with transfer of hydrogen from the substrate to the co-factor nicotinamide adenine dinucleotide (NAD), converting it to its reduced form (NADH), and allowing for the produc-

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PROCOLLAGEN-^ COLLAGEN PROPEPTIDES

^-^-HYDROXYPROLINE

Fig. 1. Oxidation of ethanol in the hepatocyte. Many disturbances in intermediary metaboUsm and toxic effects can be hnked to (i) ADH-mediated generation of NADH; (ii) the induction of the activity of microsomal enzymes, especially the MEOS containing P4502E1 (CYP2E1); and (iii) acetaldehyde, the product of ethanol oxidation. GSH: reduced glutathione; GSSG: oxidized glutathione; — , pathways that are depressed by ethanol; -^-^^-^ stimulation or activation; -[, interference or binding [1].

Chapter 32: Alcohol and oxidative stress

953

tion of acetaldehyde (Fig. 1). This first step generates an excess of reducing equivalents in the cytosol, NADH, with a shift in the redox potential, as measured by changes in the lactate/pyruvate ratio [2]. The reducing equivalents can also be transferred to NADP, the increased nicotinamide adenine dinucleotide phosphate-reduced form (NADPH) being utilized for synthetic pathways in the cytosol and microsomes (vide infra). Some of the hydrogen equivalents formed in this reaction are transferred from the cytosol into the mitochondria. Since the mitochondrial membrane is impermeable to NADH, the reducing equivalents enter the mitochondria via shuttle mechanisms, such as the malate cycle, fatty acid elongation, and a-glycerophosphate cycles. Normally, fatty acids are reduced via (3-oxidation in the citric acid cycle of the mitochondria, which serves as a "hydrogen donor" for the mitochondrial electron transport chain. When ethanol is reduced, the generated hydrogen equivalents, which are shuttled into the mitochondria, supplant the citric acid cycle as a source and the mitochondria are shifted to a more reduced redox state, as measured by changes in the ratio of (3-hydroxybutyrate to acetoacetate [2]. The altered redox state is responsible for a variety of metabolic abnormalities: the increased lactate/pyruvate ratio results in hyperlactacidemia because of both decreased utilization [3] and enhanced production of lactate by the liver. The hyperlactacidemia contributes to the acidosis and also reduces the capacity of the kidney to excrete uric acid, leading to secondary hyperuricemia [4]. Another possible consequence of enhanced purine degradation increases production of activated oxygen species by xanthine oxidase (XO) (vide infra), as indicated by the protective effect of allopurinol against the alcohol-induced lipid peroxidation [5]. Other metabohc consequences include enhanced lipogenesis, depressed lipid oxidation, and hypoglycemia [6]. The increased NADH/NAD ratio raises the concentration of a-glycerophosphate which favors hepatic triglyceride accumulation by trapping fatty acids. In addition, excess NADH may promote fatty acid synthesis [7]. Theoretically, enhanced lipogenesis can be considered a means for disposing of the excess hydrogen. The activity of the citric acid cycle is depressed, partly because of a slowing of the reactions of the cycle that require NAD; the mitochondria will use the hydrogen equivalents originating from ethanol, rather than those derived from the oxidation of fatty acids that normally serve as the main energy source of the liver. The decreased fatty acid oxidation, whether it is a function of reduced citric acid cycle activity is secondary to the altered redox potential (vide supra), or as a consequence of permanent changes in mitochondrial structure and functions [6], results in the hepatic accumulation (as triglycerides) of fatty acids of different sources, lead to the development of a fatty liver. A characteristic feature of liver injury in the alcohoHc is the predominance of steatosis and other lesions in the perivenular zone, also called the centrilobular or zone three of the hepatic acinus. Multiple mechanisms for this zonal selectivity of toxic effects have been proposed. The hypoxia hypothesis originated from the

954


observation that liver slices from rats fed alcohol, chronically consume more oxygen than those of the controls. It was then postulated that the enhanced consumption of oxygen would increase the gradient of oxygen tension along the sinusoids to the extent of producing anoxic injury of perivenular hepatocytes [8]. Indeed both in human alcoholics [9] and in animals fed extensive amounts of alcohol [10,11], there was a decrease in hepatic venous oxygen saturation (PO2) [9,10], and in the tissue oxygen tension [11]; these have been found during the withdrawal state. However, these changes in hepatic oxygenation disappeared [10,12] or decreased [11] when alcohol was present in the blood. Acute ethanol administration increased splanchnic oxygen consumption in naive baboons, but the consequences of this effect on oxygenation in the perivenular zone were offset by an increased blood flow resulting in unchanged hepatic venous oxygen tension [10]. Ethanol actually induced an increase in portal hepatic blood flow [10,12—15]. In cats [3] and baboons [15] that were fed alcohol chronically, defective oxygen utilization rather than lack of blood oxygen supply characterized liver injury, that was produced by high concentrations of ethanol. The low oxygen tension normally prevailing in perivenular zones exaggerates the redox shift produced by ethanol [10]. By increasing NADH, hypoxia may occur and in turn inhibit the activity of NAD"^-dependent xanthine dehydrogenase (XD), thereby favoring that of oxygen-dependent XO [5]. Purine metabolism via XO leads to the production of oxygen radicals which can mediate toxic effects towards liver cells, including peroxidation. Physiological substrates for XO, hypoxanthine xanthine and AMP, significantly increased in the liver after the presents of ethanol, together with an enhanced urinary output of allantoin (a final product of xanthine metabolism). Allopurinol pretreatment resulted in 90% inhibition of XO activity, and also significantly decreased ethanol-induced lipid peroxidation [5]. In addition to the redox change, a main mechanism whereby ADH-mediated ethanol metaboHsm affects oxidative stress is through the production of acetaldehyde (vide infra). ADH is also present in extra hepatic tissues such as the kidney and the stomach but their isozyme composition differs from that of the liver and the impact of ethanol metabolism on the redox level and the oxidative stress in these tissues has not been well chartered as yet. 2.2 Microsomal ethanol-oxidizing system (MEGS) 2.2.1 Role ofMEOS in ethanol metabolism That a cytochrome P450 enzyme system can also metaboHze ethanol was demonstrated in liver microsomes in vitro and the system was found to be inducible by chronic alcohol feeding in vivo [16]; it was named the microsomal ethanol oxidizing system (MEOS) [16,17]. Its distinct nature was shown by: a) isolation of a P-450 containing fractions from liver microsomes which, although devoid of any ADH or catalase activity, could still oxidize ethanol,


955

as well as higher aliphatic alcohols (e.g., butanol, which is not a substrate for catalase) [18,19]; and b) reconstitution of ethanol-oxidizing activity using NADPH-cytochrome P-450 reductase, phospholipids, and either partially purified or highly-purified microsomal P-450 from untreated [20] or phenobarbital-treated [21] rats. That chronic ethanol consumption results in the induction of a unique P-450, was also shown by Ohnishi and Lieber [20] using a liver microsomal P-450 fraction isolated from ethanol-treated rats. An ethanol-inducible form of P-450, purified from rabbit liver microsomes [22], catalyzed ethanol oxidation at rates much higher than other P-450 isozymes, and also had an enhanced capacity to oxidize 1-butanol, 1-pentanol and anihne [23], acetaminophen [24], CCI4 [23], acetone [25,26], and A^-nitrosodimethylamine (NDMA) [27]. The purified human protein (now called CYP2E1 or 2E1) was obtained in a catalytically active form, with a high turnover rate for ethanol and other specific substrates [28]. MEOS has a relatively high K^ for ethanol (8—10 mM compared to 0.2—2 mM for hepatic ADH) but, contrasting with hepatic ADH, which is not inducible in primates, as well as most other animal species. A 4-fold induction of 2E1 was found in biopsies of recently drinking subjects, using the Western blot technique with specific antibodies against this 2E1 [29]. The presence of 2E1 was also shown in extrahepatic tissues [30] and in nonparenchymal cells of the liver, including Kupffer cells [31]. In rats, ethanol treatment caused a 7-fold increase in CYP2E1 content of Kupffer cells. It also increased the hydroxylation of/?-nitrophenol, a relatively specific substrate for CYP2E1, demonstrating that the induced CYP2E1 was catalytically active. 2.3 Oxidative stress, xenobiotic and retinoid toxicity and carcinogenicity There is increased evidence that ethanol toxicity may be associated with an enhanced production of reactive oxygen intermediates. Numerous experimental data indicate that free radical mechanisms contribute to ethanol-induced liver injury. Increased generation of oxygen- and ethanol-derived free radicals occurs at the microsomal level, especially through the intervention of the ethanol-inducible 2E1 [32]. This induction is associated with prohferation of the endoplasmic reticulum (vide supra), which is accompanied by an increased oxidation of NADPH, with resulting H2O2 generation [33]. There is also increased superoxide radical production (Fig. 1) [34]. In addition, the 2E1 induction contributes to the well-known lipid peroxidation associated with alcoholic liver injury. DiLuzio [35,36] was one of the first to report that ethanol produces increased lipid peroxidation in the liver and that the ethanol-induced fatty liver could be prevented by antioxidants. Lipid peroxidation correlated with the amount of CYP 2E1 in liver microsomal preparations, and it was inhibited by antibodies against 2E1 in the control and ethanol-fed rats [37,38]. Indeed, 2E1 is rather "leaky" and its operation results in a significant release of free radicals, including 1-hydroxyethyl-free radical intermediates [39,40], confirmed by detecting the hydro-

956

P(i^t XI: Disease processes

xyethyl radicals in vivo [41]. Ethanol can also be converted to acetaldehyde by liver microsomes through a nonenzymatic pathway involving the presence of hydroxyl radicals originating from the iron-catalyzed degradation of H2O2 [42]. The production of ethanolfree radicals may be due to an oxidizing species bound to cytochrome P450 and abstracting a proton from the alcohol a-carbon [43]. Since hydroxyethyl radicals appear to be involved in the all^lation of hepatic proteins, they may contribute to the damaging effects of ethanol. Furthermore, an in vitro-produced hydroxyethyl radical forms stable adducts with albumin or fibrinogen [44], and patients with alcoholic cirrhosis have increased serum levels of both immunoglobulin G (IgG) and immunoglobulin A (IgA) reacting with proteins of liver microsomes incubated with ethanol and NADPH [44], which do not cross react with the epitopes derived from acetaldehyde-modified proteins. Much of the medical significance of MEOS, and its ethanol-inducible 2E1, results not only from the oxidation of ethanol and from the unusual and unique capacity of 2E1 to generate reactive oxygen intermediates, but also from the activation of many xenobiotic compounds to their toxic metabolites, often free radicals. This pertains to carbon tetrachloride and other industrial solvents such as bromobenzene [45] and vinylidene chloride [46], as well as anesthetics such as enflurane [47] and halothane [48]. Ethanol also markedly increases the activity of the microsomal low K^ benzene-metabolizing enzymes [49] and this aggravates the hemopoietic toxicity of benzene. Enhanced metabolism (and toxicity) also pertains to a variety of prescribed drugs, including isoniazid and phenylbutazone [50], and some over-the-counter medications such as acetaminophen (paracetamol, A^-acetyl-/^-aminophenol) all of which are substrates for, or inducers of, cytochrome P4502E1 (CYP2E1). Therapeutic amounts of acetaminophen (2.5 to 4 g/day) can cause hepatic injury in alcoholics. In animals given ethanol for long periods, hepatotoxic effects peak after withdrawal [51] when ethanol is no longer competing for the microsomal pathway and when levels of the toxic metabolites are at their highest. Thus, heavy drinkers are most vulnerable to the toxic effects of acetaminophen shortly after the cessation of chronic drinking. There is an association between alcohol misuse and an increased incidence of upper alimentary and respiratory tract cancers [52]. Many factors have been incriminated, one of which is the effect of ethanol on enzyme systems involved in the cytochrome P-450-dependent activation of carcinogens. This effect has been demonstrated with the use of microsomes derived from a variety of tissues, including the liver (the principal site of xenobiotic metabolism [53,54], the lungs [53,54], and intestines [30,55], the chief portals of entry for tobacco smoke and dietary carcinogens, respectively, and the esophagus [56], where ethanol consumption is a major risk factor in the development of cancer. Alcoholics are commonly heavy smokers, and a synergistic effect of alcohol consumption and smoking on cancer development has been described, as reviewed elsewhere [52]. Indeed, long-term ethanol consumption was found to enhance the mutagenicity of tobacco-derived products [52].


957

Alcohol may also influence carcinogenesis in many other ways [57], one of which involves severe vitamin A depletion which is associated with alcoholic liver disease already at early stages [58,59]. Depressed hepatic levels of vitamin A were observed even when alcohol was given with diets containing large amounts of vitamin A [60]. New hepatic enzyme pathways of retinol metabolism, inducible by either ethanol or drug administration, have been discovered [61,62]. Depletion in hepatic vitamin is associated with lysosomal lesions [63] and decreased detoxification of NDMA [64]. Although vitamin A deficiency might adversely affect the liver [63], an excess of vitamin A is also known to be hepatotoxic [65]. Long-term ethanol consumption enhances this effect, resulting in striking morphologic and functional alterations of the mitochondria [66], along with hepatic necrosis and fibrosis [59]. Hypervitaminosis A itself can induce fibrosis and even cirrhosis, as reviewed elsewhere [65]. Thus, alcohol abuse narrows the therapeutic window for vitamin A, which hinders it therapeutic use. Retinol is an antioxidant but it is a weak one and, as noted earlier, its use is complicated by its intrinsic hepatotoxicity, exacerbated by ethanol. Unlike retinol, its precursor P-carotene is considered to lack toxicity Furthermore, in addition to acting as a retinol precursor, P-carotene is also an efficient quencher of singlet oxygen and can function as a radical-trapping antioxidant. Moreover, P-carotene has been shown to have the potential of acting as a more efficient antioxidant than retinol. Carotenoids were found to inhibit free radical-induced lipid peroxidation [67—69]. However, in a study in rats, Alam and Alam [70] reported no change in either blood or tissue lipid peroxides following ingestion of P-carotene (180 mg/kg/day), for a period of 11 weeks and carotenoids did not protect against peroxidation in chohne deficient rats [71]. By contrast, a study in guinea pigs noted a protective effect against in vivo lipid peroxidation when animals were pretreated with P-carotene [72]. Furthermore, Palozza and Krinsky [73] reported that P-carotene inhibited malondialdehyde production in a concentration-dependent manner and delayed the radical-initiated destruction of endogenous a- and y-tocopherol in the rat, and Kim-Jun [74] reported inhibitory effects of P-carotene on lipid peroxidation in mouse epidermis. Mobarhan et al. [75] also showed that subjects replenished with P-carotene had a decreased level of circulating lipid peroxides, but it was not reported whether this could be achieved in individuals who continue to drink, and at a dose of p-carotene that has no toxicity in the presence of alcohol. One may wonder whether the combination of alcohol and P-carotene, at the dose used for replenishment, can be useful in terms of preventing lipid peroxidation, without producing some signs of toxicity Indeed, enhanced hepatic toxicity of P-carotene in the presence of ethanol has been observed with the possible existence of a defect in utilization and/or excretion associated with liver injury and/ or alcohol abuse [76,77]. In addition, in men, heavy drinking was associated with a relative increase in serum p-carotene [78], and relatively moderate drinking in women also has a similar effect [79]. It is noteworthy that P-carotene increased pulmonary cancer

958

P(^^t XI: Disease processes

and cardiovascular complications in smokers [80]. This eflfect was confirmed in a study by Omenn et al. [81], and was found to be related to the amount of alcohol consumed [82], confirming the suspicion [83] that the toxicity resulted from an alcohol P-carotene interaction. Thus, vitamin A or P-carotene supplementation must be used cautiously in alcoholics, especially if P-carotene is given in beadlets, which potentiate the toxic alcohol-p-carotene interaction [84]. 2.4 Role of catalase Catalase can oxidize alcohol in vitro in the presence of an H202-generating system [85] (Fig. 1), and its interaction with H2O2 in the intact liver has been demonstrated [86]. However, its role is limited by the small amount of H2O2 generated [87]. Under physiological conditions, catalase thus appears to play no major role in ethanol oxidation. The catalase contribution might be enhanced if significant amounts of H2O2 become available through p-oxidation of fatty acids in peroxisomes [88]. However, the peroxisomal enzymes do not oxidize short chain fatty acids such as octanoate; peroxisomal p-oxidation was observed only in the absence of ADH activity. In its presence, the rate of ethanol metabolism is reduced by adding fatty acids [89], and conversely, the B-oxidation of fatty acids is inhibited by NADH produced from alcohol metabolism via ADH [89]. Similarly, the generation of reducing equivalents from ethanol by ADH in the cytosol inhibits H2O2 generation leading to significantly diminished rates of peroxidation of alcohols via catalase [90]. Furthermore, aminotriazole (a catalase inhibitor) has little, if any, effect on alcohol oxidation in vivo, as reviewed by Takagi et al. [91] and Kato et al. [92,93]. Thus, despite the considerable controversy that originally surrounded this issue, it was agreed by the principal contenders involved that catalase cannot account for microsomal ethanol oxidation [94,95]. However, catalase could contribute to fatty acid oxidation. Indeed, long-term ethanol consumption is associated with increases in the content of a specific cytochrome (P4504A1), this promotes microsomal co-hydroxylation of fatty acids which may compensate, at least in part, for the deficit in fatty acid oxidation due to the ethanol-induced injury of the mitochondria [6]. Products of o-oxidation increase liver cytosolic fatty acid-binding protein (L-FABPC) content and peroxisomal p-oxidation [96], an alternate but modest pathway for fatty acid disposition. 3.

TOXICITY OF ACETALDEHYDE, INCLUDING ITS ADVERSE EFFECTS ON ANTIOXIDANT SYSTEMS

Due to the inducibility of 2E1, chronic excessive alcohol consumption results in enhanced acetaldehyde production that, in turn, aggravates the oxidative stress directly, as well as indirectly, by impairing defense systems against it.


959

3.1 Elevated acetaldehyde and mechanism of toxicity Acetaldehyde, the product of ethanol oxidation, is highly toxic but is rapidly metabolized to acetate, mainly by a mitochondrial low K^ aldehyde dehydrogenase (ALDH). However, the activity of ALDH is significantly reduced by chronic ethanol consumption [97]. The decreased capacity of mitochondria of alcoholfed subjects to oxidize acetaldehyde, associated with unaltered or even enhanced rates of ethanol oxidation (and therefore acetaldehyde generation because of MEOS induction; vide supra), results in an imbalance between production and disposition of acetaldehyde. The latter causes the elevated acetaldehyde levels observed after chronic ethanol consumption in humans [98] and in baboons, with a tremendous increase of acetaldehyde in hepatic venous blood [15], reflected high tissue levels. The toxicity of acetaldehyde is due, in part, to its capacity to form protein adducts, resulting in antibody production, enzyme inactivation, [99] and decreased DNA repair [15,100]. It is also associated with a striking impairment of the capacity of the liver to utilize oxygen. 3.2 Alterations of antioxidant systems and phospholipids 3.2.1 Depletion of glutathione and S-adenosylmethionine Acetaldehyde promotes glutathione (GSH) depletion, resulting in free radicalmediated toxicity, and lipid peroxidation. Indeed, in isolated perfused livers acetaldehyde was shown to cause lipid peroxidation [101,102]. In vitro, metabolism of acetaldehyde via XO or aldehyde oxidase may generate free radicals, but the concentration of acetaldehyde required is much too high for this mechanism to be of significance in vivo. However, another mechanism to promote lipid peroxidation is via GSH depletion. One of the three amino acids of this tripeptide is cysteine. Binding of acetaldehyde with cysteine and/or glutathione (GSH) may contribute to a depression of liver GSH [103]. Rats fed ethanol chronically have significantly increased rates of GSH turnover [104]. Acute ethanol administration inhibits GSH synthesis and produces an increased loss from the liver [105]. GSH is selectively depleted in the mitochondria [106] and may contribute to the striking alcohol-induced alterations of that organelle. GSH offers one of the mechanisms for the scavenging of toxic-free radicals, as shown in Fig. 2, which also illustrates how the ensuing enhanced GSH utilization (and thus, turnover) results in a significant increase in a-amino-/t-butyric acid [108] (Fig. 3). The latter can serve to assess semiquantitative^ the increased GSH turnover in vivo. Although GSH depletion per se may not be sufficient to cause lipid peroxidation, it is generally agreed upon that it may favor the peroxidation produced by other factors. It is important in the protection of cells against electrophilic drug injury in general, and against reactive oxygen species in particular, especially in primates, which are more vulnerable to GSH depletion than rodents [110]. Iron

960

Part XI: Disease processes METABOLITES

LIPID PEROXIDATIONX f ADH

ETHANOL I

- A M I N O - n - B U T Y R I C ACID ^,^^^^. ^ RADICALS

MICROSOMES

I

-GSH

^

(x-KETOBUTYRICACID

(

K

^ - ^ ACETALDEHYDE METHIONINE

CYSTEINE -

SERINE—^/-

T^ S-ADENOSYL-METHIONINE—^-HOMOCYSTEINE ^ /

CYSTATHIONINE

^7=—• PHOSPHATIDYLCHOLINE

PHOSPHATIDYLETHANOLAMINE

Fig. 2. Link between accelerated acetaldehyde production and increased free radical generation by "induced" microsomes, resulting in enhanced lipid peroxidation. Metabolic blocks due to alcohol, folate deficiency, and/or alcoholic liver disease are illustrated with possible beneficial effects of GSH, its precursors (including S-adenosylmethionine), and phosphatidylchoHne [107].

overload may play a contributory role, as chronic alcohol consumption results in an increased iron uptake by hepatocytes [111]; iron exposure accentuates the changes of lipid peroxidation and of the glutathione status in the liver cell induced by acute ethanol intoxication [112]. Therapeutic use of GSH itself is complicated by the fact that its replenishment ^

50i

9 40 o < _

CD S 20 H

6 < I

>5

(NS) LiJ

10

t

4000 i5_ 2000^ S

llEQMi ADEQUATE DIET

5

n—

7 9 6 8 10 WEEKS ON METABOLIC UNIT

Q:

12

13

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Fig. 3. Effect of alcohol consumption on plasma a-amino-w-butyric acid. Consumption of 4 g of ethanol/kg/day in addition to an adequate diet in three human volunteers resulted in a doubling of plasma a-amino-«-butyric acid after 2—4 weeks which was reversed after cessation of drinking. A similar trend was observed for the branched-chain amino acids. This increase was not observed when fat was substituted isocalorically by ethanol [109].


961

through supplementation is hampered by its lack of penetration into the hepatocytes, except for its ethyl derivative, which is not suitable for the treatment of an alcoholic liver injury. Cysteine is one of the three amino acids of GSH, and the ultimate precursor of cysteine is methionine (Fig. 2). Methionine deficiency has been incriminated in alcoholics and its supplementation has been considered for the treatment of alcoholic liver injury, but some difficulties have been encountered. Indeed, excess methionine was shown to have some adverse effects [113], including a decrease in hepatic adenosine triphosphate (ATP). Horowitz et al. [114] reported that the blood clearance of methionine after an oral load of this amino acid was slowed in patients with cirrhosis. Because about half of the methionine is metabolized by the liver, the previously mentioned observations suggest impaired hepatic metabolism of this amino acid in patients with alcoholic liver disease. For methionine to be utilized it has to be activated to S-adenosylmethionine (AdoMet) (Fig. 2). However, Duce et al. [115] found a decrease in AdoMet synthetase activity in cirrhotic hvers, and AdoMet depletion ensues after chronic ethanol consumption [116]. Potentially, such AdoMet depletion may have a number of adverse effects. AdoMet is the principal methylating agent in various transmethylation reactions important for nucleic acid and protein synthesis, as well as membrane fluidity and functions. It includes the transport of metabolites and the transmission of signals across membranes and maintenance of membranes. Thus, depletion of AdoMet may promote membrane injury documented in alcohol-induced liver damage [117]. AdoMet is not only the methyl donor in almost all transmethylation reactions, but it also plays a key role in the synthesis of polyamines and, as already alluded to, it provides a source of cysteine for GSH production. Orally administered AdoMet is a precursor for intracellular AdoMet, both as unchanged AdoMet and by the methionine it provides. Compared to methionine, the administration of AdoMet has the advantage of bypassing the deficit in AdoMet synthesis (from methionine) referred to earlier (Fig. 2). The usefulness of AdoMet administration has been shown in various clinical studies [118,119]. It was also demonstrated in the baboon [116,120], with improvement of GSH depletion, and in the leakage, from the liver into the circulation of the enzymes, glutathione dehydrogenase (GDH) (Fig. 4) and aspartate aminotransferase (AST) (Fig. 5). Recently, in a multicenter placebo-controlled, randomized the double-blind clinical trial. Mato et al. [121] showed that AdoMet improves survival in patients with alcoholic liver cirrhosis. 3.2.2 Vitamin E deficiency Bjoneboe et al. [122] reported a reduced hepatic oc-tocopherol content after chronic ethanol feeding in rats receiving adequate amounts of vitamin E, as well as in the blood of alcoholics. Furthermore, hepatic lipid peroxidation was significantly increased after chronic ethanol feeding in rats receiving a low vitamin E diet [123], indicating that dietary vitamin E is an important determinant of hepa-


962 IZZI ^ ^ •ra

100

CONTROL BABOONS ETHANOL-FED CONTROL + AdoMet ETHANOL + AdoMet

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^ l- 95% of circulating the polymorphonuclear leukocytes [82]. Circulating monocytes represent a relatively small percentage of leukocytes (usually less than 10%). When these monocytes leave the circulation in response to signals indicating wounding or infection they differentiate to become macrophages. Their life cycle is complex and varied. The monocytes, like the neutrophils, have a broad arsenal of antimicrobial agents that are useful in the debridement and sterilization of infected areas [33]. Approximately 30% of circulating white cells are lymphocytes [25]. The lym-

Chapter 34: Regulation of inflammation and wound healing

1005

phocytes are the key cells that develop and process antibody development. There are two major subdivisions of lymphocytes and these are the bone marrowderived cells (B cells) and the thymus-derived lymphocytes (T cells). The understanding of the development of lymphocyte functioning has seen a rapid expansion in the last few years and describing these cells in wound healing has recently been reviewed [83—85]. The inflammatory response of clot formation, cessation of fluid loss and the initiation of neutrophil and macrophage infiltration into a wound site to initiate the repair process are the initial stages of wound heahng. In Section 3.1, the role of neutrophils and macrophages in the wound healing process in terms of their ability to generate abundant free radicals and oxidants will be discussed. 3.1 The role of neutrophils and macrophages in wounds The process of phagocytosis, using microscopic examination of tissue, was first noted by MetchnikoflF about 100 years ago [86]. Investigators have been able to document the movement of neutrophils based on microscopic examination of tissue samples, but the understanding of the molecular events that signal and stimulate the activation of neutrophils is now a topic of intense current investigation. Neutrophils contain a multilobed nucleus and granules that contain an assortment of enzymes such as myelperoxidase, lysozyme, collagenase, elastase, gelatinase and Cathepsin G [87]. Neutrophils are present in blood at a concentration of about 2,500—10,000/)il and they have a rather short life span in the range of 24—48 h [88]. Certainly, when isolated from human blood, neutrophils undergo rapid degeneration such that their respiratory burst activation is rapidly diminished within a few hours of separation from the blood. The fate of healthy neutrophils is also somewhat elusive. Many of the neutrophils eventually are located in the gut in the absence of an acute injury [25]. The physiology and cell biology of the life cycle of neutrophils is at best poorly understood. When neutrophils reach the site of an injury they have a finite time in which to aid in wound healing and then the cell degrades in a poorly understood process becoming among other fates, a major constituent of pus [25]. With the pus comes the removal of debris and microorganisms that impede the wound healing process. Probably the most studied and most intriguing aspect of neutrophil functioning at a wound site is its ability to phagocytose. In eukaryotes the cells that are able to phagocytose are know collectively as "professional phagocytes". Professional phagocytes are derived from bone marrow and includes neutrophils, macrophages and monocytes [89]. From an evolutionary perspective, the process of phagocytosis in higher organisms has likely derived from phagocytosis in more primitive unicellular cells such as amoebae. Amoebae phagocytose mainly for nutrition while the mammalian professional phagocytes are phagocytose in order to remove invading microorganisms. The mechanisms of the absorption of extracellular material in both cell types is, however, remarkably similar. Many mammalian cell types can pha-

1006

P^^t XI: Disease processes

gocytose and both endothelial and epithelial cells have been shown to phagocytose microorganisms. However, their efficiency at phagocytosis is far lower than that of the professional phagocytes [89]. As neutrophils enter an environment where they may need to phagocytose an invading microorganism they move through the endothelial capillary bed by changing their cell membrane topology to develop pseudopodia which facilitate the "squeezing" of the neutrophils between the endotheUal cells [90,91]. The fluid in a wound area contains many soluble antibodies which can bind to bacteria and result in a coating of antibody on an invading microorganism. Some antibodies coat bacteria in a wound site and are called opsonins. Opsonins allow for a more efficient recognition of the invading bacteria by the phagocytic cells [92—94]. When bacteria and other microorganisms are taken up by neutrophils, and the contents of the neutrophil granules fuse with the vacuole containing the microorganism, then the granule contents are "poured" into the vacuole containing the microorganism. It is believed that the primary role of neutrophils is to get to the wound site quickly and destroy bacteria rapidly [95]. Macrophages enter the wound environment later than the neutrophils, and early studies on guinea pigs in which the neutrophils were depleted, demonstrated that in the absence of a gross infection, macrophages supported adequate healing [96,97]. In a healing wound, as the neutrophil numbers decline, there is a buildup of macrophages. Histological examination of acute compared to chronic wound sites reveals an inverse relationship of the number of neutrophils and macrophages at the respective wound sites. Studies in which the macrophages are depleted show serious consequences for the animal as the wounds do not heal well, even in the absence of infection. It is now known that macrophages secrete numerous growth factors that stimulate adjacent cells to grow. This growth must occur at the correct time! Failure to provide these signals results in dramatically reduced wound healing [15,98—101]. Macrophages can originate from circulating monocytes or can be derived from dividing resident tissue macrophages. Lung alveolar macrophages, for example, are a combination of dividing and infiltrative cells. Similarly, liver Kupflfer cells are macrophages and are believed to be substantially derived from circulating monocytes, but they can divide and proliferate within the liver [33]. However, in an inflammatory condition, (and these can be pathological inflammatory conditions such as arthritis) the monocytes are recruited from the circulating blood and differentiate into macrophages as they move from the blood to the site of inflammation [33]. There is a notable differential migration of neutrophils and macrophages. It is not surprising that there are chemotactic signals that are specific for the different types of professional phagocytes. For instance type I collagen can stimulate macrophages but not neutrophils [102]. Fibronectin is deposited by macrophages (as well as other cells), and it is believed to be a stimulating protein for the formation of fibroblast orientation [103]. Fibronectin is an adhesive extracellular matrix protein and it plays important roles in embryogenesis, as well as wound healing. The cellular effects of fibronectin are on the integrin


\ 007

family of receptor/binding proteins on the surface of cells. The expression of integrins directs when and how the cells become attached. There is evidence that fibronectin may alter blood vessel formation, alveolar epithelial cell differentiation wound area growth and maturation [104,105]. Macrophages have other functions that are distinct from those of the neutrophil. As they come into a wound or inflammatory site, they are believed to be more likely involved in the breakdown of the extracellular degradation of connective tissue matrix than neutrophils. The macrophages can be stimulated to produce and secrete proteases [106,107]. Cathepsin S one class of proteases is present mainly in cells from a monocyte lineage including macrophages. Macrophage cathepsins have been noted to be involved in the destruction of basement membranes and extracellular matrix and cathepsin has recently been shown to degrade various extracellular matrix molecules [108]. Macrophages remove debris from a wound and are involved in the preparation of the wound area for angiogenesis. Angiogenesis occurs in the presence of and to a large extent is dependent upon macrophages. In addition to secreting macrophage-derived growth factors, macrophages must balance their activity between bactericidal and phagocytic activity, and the preparation of the area for new cell growth. There are at least 17 identified major cytokines and growth factors that are secreted by the macrophage. Growth of hematopoietic progenitor cells is coordinated by a number of both stimulatory and inhibitory cytokines [103]. 3.2 The respiratory burst The NADPH oxidase of leukocytes is an enzyme complex that is present in neutrophils, macrophages and certain other leukocytes [109]. It is a remarkable enzyme complex in that is reduces molecular oxygen (O2) to superoxide (O2—). The overall reaction is as follows: 2 O2 + NADPH -> 2 ©2^ • + NADP+ + H+ This reaction only occurs when the leukocyte is appropriately stimulated with a stimulant such as a phorbol ester, the LPS endotoxin from a gram negative bacteria, and certain peptide cell stimulants such as formyl-methione-leucine-phenylalanine (fMLP) [82]. The respiratory burst oxidase is not usually active in a circulating neutrophil or monocyte. In order to have activity, the enzyme requires activation, phosphorylation and protein assembly. This usually occurs when the cell has left the circulation and is at a site of injury or infection. The respiratory burst oxidase has at least four components required for activation. Two of these components are membrane bound and two are soluble and located in the inactive state in the cytoplasm [110—113]. In addition, there are other peptides that are involved in the activation process. The regulatory enzymes protein kinase C (PKC) has a role [114], and dephosphorylases are important in maintaining the activity of the phosphorylated and activated enzyme.

1008

P^^t Î- Disease processes

In spite of over 20 years of research on this important enzyme complex, there is not much known about how it is maintained in an active state, how it functions in vivo in microaerobic environments that are encountered in wound sites, and whether the enzyme once activated can inactivate and reactivate at a later time. The mechanism of oxygen reduction is also quite elusive [109]. NADPH is the electron donor and molecular O2 is the acceptor. The respiratory burst oxidase does contain a cytochrome b558 which is a likely candidate for electron transport and potentially a reduction site for oxygen. The binding site of NADPH is still elusive. The cytochrome bssg seems to complex between two of the membrane peptides and is present as two hemes per pair of membrane anchor peptides. Flavin adenine dinucleotide (FAD) is also present in this enzyme; however, the exact amount is also not clear [115,116]. There are some similarities to mitochondrial redox enzymes in that electrons reduce molecular O2 in the mitochondria in two separate ways. One is the cytochrome oxidase, which is a highly efficient four electron reductant of water. This enzyme has never been shown to produce superoxide as a by-product. The NADH-ubiquinone reductase, as well as the succinate-ubiquinone reductase have both been shown to leak electrons and result in a small but certainly significant rate of formation of superoxide that is derived from mitochondrial electron transport [117]. Cells contain two distinct forms of superoxide dismutases (SOD), a copper-zinc and a manganese enzyme. SOD is believed to be present in order to trap superoxide from reacting with such substrates as reduced thiols on proteins, or with other hemes, flavins or quinones. The mitochondrial respiratory chain enzymes contain Fe-S clusters which are electron carriers and potentially reductants for oxygen [118,119]. The NADPH linked respiratory burst oxidase, however, it has no Fe-S clusters, and there are more similarities to cytochrome P-450, which is an enzyme family that is important in drug and toxin metabolism [120]. The evolution of the respiratory burst oxidase is not clear. It has likely evolved from more than one type of enzyme when one considers the transient nature of its activation and deactivation, via PKC. The rapid burst of oxygen reduction and the fact that it has at least four subunits that are mixed between membrane bound and cytosolic are special characteristics [109]. What is the function of the respiratory burst? The respiratory burst makes oxidants that are essential for microbicidal killing [109]. Exactly how leukocytes kill invading pathogens is not at all clear. The respiratory burst enzyme's main product is the precursor for a number of oxidants and radicals. Some of these are produced by enzymes such as SOD, which makes H2O2 and myeloperoxidase, which makes HOCl and other oxidants such as chloramines, which are produced by nonenzymatic reaction between HOCl and amines. There is compelling evidence that the respiratory burst is essential for microbicidal activity as there is a human disease called Chronic Granulomatous Disease in which the patients are very susceptible to infections, and their neutrophils do not have an adequate respiratory burst [109]. Does the respiratory burst generate molecules that are signals? There is a diverse literature that demonstrates that oxidants are very


\ 009

important in the modulation of cell diflferentiation and growth. As superoxide is formed and subsequently reacts forming H2O2 principally, and a secondary diverse range of oxidants and radicals, it is quite likely that signals derived from the respiratory burst enzyme cause modulation of function in other cells [41,121-125]. 3.3 Cytokines and growth factors involved in oxidative stress, and healing Growth factors and cytokines are involved in almost every step of every change in the diflferentiation and movement of cells during wound healing, it is apparent that what we know today is merely a small portion of what is a complex, redundant and reliable mechanism to regulate tissue regeneration. There are different mechanisms as to how growth factors operate. For example, transforming growth factor-oc (TGF-a) is synthesized as a membrane-bound peptide that can act as a growth factor for an immediately adjacent cell (a juxtacrine) [126,127], or if the active portion of the peptide is cleaved by proteolysis then TGF-oc becomes a potent soluble growth factor that can be active at remote sites and it becomes a paracrine and autocrine growth factor [128]. The synthesis and the release of the cytokine is one of the factors in establishing the activation and activity of these growth factors in wound healing and cell regeneration. However, each cytokine must have a receptor in order for it to be functional so there is a duality in understanding the role of growth factors in that both the factor and its receptor must be active and necessarily regulated in order for the growth factor to function. This does complicate the analysis of growth factor analysis in vivo. The transforming growth factor (TGF-P) signals cells through serine/threonine receptors. A number of these receptors have been recently identified and partially characterized. It is possible to use genetically altered truncated fragments of TGF-P or its receptor to block the signal from the cytokine [129]. Inactivation of the "type 11" receptor reveals two receptor pathways for the diverse TGF-activities. There is a TGF-P that belongs to the epidermal growth factor (EGF) family [130]. Like other cytokines this peptide is also synthesized as a precursor that is transmembranous. The mature cytokine is released as a 50-amino acid peptide, and both the free and released forms have biological activity This cytokine has a role in oncogenesis (as do many growth factors) and is associated with numerous tumor types including carcinomas, melanomas and hepatomas [128,131,132]. TGF-p is a potent inducer of angiogenesis and as such, has distinct and critical importance in wound healing and regeneration [133]. Recent studies have shown that activator protein-2 (AP-2) (a gene expression product involved in oxidative sensitivity of cells) regulates the TGF-P promoter may be involved in both tumorigenesis and growth [134]. Vascular endothelial growth factor (VEGF) is one of the more recently discovered growth factors, and it is a dimeric glycoprotein that is structurally similar to platelet-derived growth factor [135,136]. There are several isoforms of VEGF each of which has varying activity and presumably differing affinity to VEGF

1010


receptors [137]. VEGF is very specific for endothelial cell growth at least in vitro. As endothelial cells must proliferate to regenerate the capillary bed that is essential for reoxygenation VEGF is surely a critical cytokine for wound [135,136,138]. It has recently been shown that VEGF mRNA in keratinocytes is enhanced during wound healing and VEGF seems to act in a paracrine manner [139]. Tumor necrosis factor-a (TNF-a) is a macrophage-derived cytokine that has a multitude of signaling effects on different cell types. These effects include cell killing, induction of apoptosis (programmed cell death), DNA fragmentation (that indicates DNA damage), lipid peroxidation induction (indicating damage to membranes), and inhibition of mitochondrial electron transport (indicating a decrease in the oxidative ability of the cell to survive) [140]. It also induces activation of nuclear factor K-B ( N F - K B ) [141]. N F - K B is a recognized cellular protein factor that is sensitive to oxidant stress. It activates a number of gene expression following its activation by oxidants [142,143]. Insulin-like growth factors Type I and II (IGF) are pleotropic polypeptides which have structural homologies to insulin. The IGF receptor binds IGF-I better than IGF-II or insulin. The receptor is a heterotetramer, consisting of subunits joined by disulfide. Insulin-like growth factors-I and -II differentially regulate endogenous acetylcholine released from the rat hippocampal formation [144]. Growth hormone stimulates peripheral tissue growth by involving the endocrine action of IGF-I from liver [145,146]. The IGF family of growth factors have a role in the wound healing process. The IGF-II circulates bound to an IGF receptor known as IGF-binding proteins. The IGF-II binds to the cation independent mannose-6-phosphate receptor. It reacts immunologically with the insulin, as well as the IGF-I receptor [147,148]. Conversely the IGF-I and insulin do not cross react with the IGF-II receptors [149]. The IGF-II receptor binds mannose-6-phosphate at different sites and there is no phosphorylation after binding that would suggest activation of a PKC However, there is a redistribution of this receptor upon binding, and it involves an okadaic acid sensitive serine-threonine phosphatase [150]. When EGF binds to its receptor it can elicit a number of cell specific responses that result in differentiation and cell proliferation. The binding of EGF causes dimerization and phosphorylation of the receptor [151]. When epithelial cells are confluent (i.e., the cells are all in direct contact with one another), they are far more stable than if growing as independent "bundles" of cells. Single cells growing in culture initiate apoptosis and die. If cells are restricted in their ability to contact an adjacent cell they too initiate apoptosis [152]. The EGF-like growth factors, known as heparin-binding EGF-like growth factor (HB-EGF), increases in the kidney in vivo in response to acute renal tubular injury [153]. HB-EGF is synthesized as a membrane bound precursor and is believed to act as a juxtracrine, when it is in direct contact with an adjacent cell. In cultures, addition of EGF prevents apoptosis initiation and is good reason to suspect that EGF is modulating cell growth [154]. IL-1 has been long recognized as a cytokine that


1011

has a role in cell proliferation and wound healing. The expression of the IL-1 receptor is dependent on PKC PKC and has a pivotal role in the activation of T cells. T cells are stimulated by the interaction of a specific antigen with T cell receptor-CD3 complex. This interaction activates the intracellular kinase cascade [155], one of which is mediated by PKC. Activated kinase cascade activates transcription factors which initiate transcription and expression of a variety of molecules including IL-2 and the high affinity IL-2 receptor chain (IL-2R). Due to IL-2 being a major T cell growth factor, the coordination of production IL-2 and IL-2 receptor is crucial for T cell proUferation and the immune response [156]. T cell stimulation with phorbal myristic acetate (PMA) alone is enough for IL-2R expression [157], and PKC is responsible for this effect [155]. Expression of IL-2R is transcriptionally regulated [158], and involves N F - K B activation [159]. N F - K B activation comprises of the phosphorylation and release of its inhibitory protein IKB [160]. The activated N F - K B translocates from the cytosol to the nucleus, where it binds to the (B consensus sequence of IL-2R gene promoter [159]. This binding is essential for NF-KB-regulated gene expression [114,161]. Interferons are a class of polypeptides that play a crucial role in the defense against viral and parasitic infections. They can stimulate the body's own immune system to acts against viral infections [162—164]. These cytokines can alter cell growth rates but have not yet proven to be useful in the treatment of cancers. There are several types of interferons which have a multitude of biological effects. The Y-interferon has been known for several years to stimulate the production of inducible nitric oxide synthase, iNOS in macrophages. P-Interferon is helpful in the treatment of multiple sclerosis and a-interferons have been partially successful in the treatment of chronic viral hepatitis B and C [165,166]. 3.4 Clinical implications Several well-characterized diseases inhibit wound healing directly due to "cellular" impairment. In diabetics there is a significantly increased problem in healing of surgical and traumatic wounds [25]. There are several possible causes for the wound healing problems associated with diabetes and these include the wellcharacterized degradation of capillary circulation in diabetics. The elevated serum glucose may result in an increased glycosylation of peripheral tissues, and the insulin deficiency or insulin resistance of the two types of diabetics are all factors that would likely disrupt normal wound healing. Diabetics have a propensity to develop infections from Staphylococcus aureus, Escherichia coli and Candida [167]. It is likely that a combination of diminished circulation, as well as the characteristic hyperglycemia are major contributing factors to diabetic wound healing retardation. Large vessel diseases such as atherosclerosis, due to sclerotic plaque buildup is exacerbated in diabetics. Whether this is due directly to the diabetes or its secondary effects of a tendency towards obesity and hypertension it is not immediately clear. While diabetes is a very common disease there are many less common diseases

1012


that can provide insight into the mechanisms of leukocyte physiology by understanding what defects exist in the pathology. Chronic granulomatous disease [109] is a fairly well-characterized disorder in which the leukocytes of the patients cannot generate superoxides due to a defect in the respiratory burst enzyme. Such patients do not usually live very long as they are very susceptible to infections. It is possible to have neutropenia (a low neutrophils count), and have almost normal wound healing. Patients with chronic granulomatous disease who have a wound with no infection or debris, have no difference in healing compared to a healthy subject [25]. With antibiotic therapy often these patients can live for several years, however, few patients with severe chronic granulomatous disease live into adulthood. There are numerous sub types of chronic granulomatous disease. A recent study [168] demonstrates that there are possibilities to repair a genetic defect such as chronic granulomatous disease. In this recent study patients with a defective p47^^°^ peptide, received intravenous infusions of autologous CD34^"^^ peripheral blood stem cells that had been transduced ex vivo with a recombinant retrovirus encoding normal p47^^°^. These patients showed signs of development of neutrophils with a normal p47^^^^. Corrected cells were detectable for as long as 6 months after infusion in some individuals although these cells represented a tiny fraction of total neutrophils. As described in this brief survey of wounding and inflammation each of the steps involved in inflammation and wound repair involves a multitude of signals that must be transmitted to and from adjacent cells. There are many redundancies built into the inflammatory repair mechanism. Patients with a defective and impaired myeloperoxidase often don't have any major problems [169]. Primary inherited defects in neutrophil function: etiology and treatment [170,171]. Deficiency of myelperoxidase is not a lethal defect and this indicates that there are compensatory mechanisms for loss of a single enzyme even if myelperoxidase comprises a large portion of the protein in a leukocyte. What we can learn from known pathologies? The known and characterized pathologies provide a clue that the balance of the inflammatory system can be disrupted without, necessarily, destroying the organism. Many subtle pathologies involved in wound healing are likely manifestations of defects in the inflammatory response. While some of these have now been identified, there is much to be learned about the pathophysiology of the immune system as it relates to wound healing. 4

LINKS BETWEEN WOUND HEALING AND INFLAMMATION

4.1 The balance of radical production and control by antioxidants Leukocytes that generate copious quantities of reactive radicals that are important in the killing of bacteria, viruses and fungi have to have control systems for their own protection. Neutrophils contain considerably more ascorbate (vitamin C) than do other cells [172]. Ascorbate is an important reductive antioxidant


1013

that has been shown to reduce vitamin E radical directly [173]. The earliest wound healing research determined that ascorbate was essential for the prevention of scurvy. Subsequently the molecular role of ascorbate being involved in the formation of hydroxyproline from proUne defined the role of ascorbate in the wound environment [174]. Vitamin E is strongly linked to the prevention of lipid peroxidation and it is believed to act as a chain breaking antioxidant which stops the progression of the nonenzymatic radical reactions of lipid peroxidation. Ascorbate is also believed to be important in the reduction of other radicals preventing radical damage to important cellular sites such as DNA, membranes and regulatory enzymes. Vitamin E is located in the membranes as it is very lipophilic. It can be recycled by enzyme linked reduction, such as in the mitochondrial respiratory chain where it is unlikely ascorbate in fact would remain oxidized [175]. When neutrophils have their respiratory burst stimulated by PMA they undergo a rapid respiratory burst. In an in vitro model system in which there is far more oxygen than in a "physiological" condition, there is no measurable decrease in the cell's ascorbate, vitamin E or glutathione [176]. This indicates that the neutrophils have a significantly enhanced cellular system to prevent their own oxidants from destroying the cell that made them. It is important to recognize that the production and protection of radicals in a cell such as a neutrophil is a balance. A diminution of one antioxidant such as ascorbic acid may not result in a measurable increase in oxidative damage as there are other antioxidant systems that can compensate for a decrease of one antioxidant. It is now common practice in the developed world to take vitamin supplements especially antioxidant vitamins such as vitamin C and E [177]. While the evidence that these vitamin supplements can actually result in any measurable health benefit is not very clear, there is a wealth of good scientific evidence that indicates that these two vitamins in particular are most influential in the prevention of cellular oxidative damage at all levels in the cell from the DNA to the cell membrane [178—183]. 4.2 The role of oxygen in wound healing and inflammatory resolution Oxygen in tissues is usually found in the range of 10—20% of ambient air saturated water (15—30 mmHg or 24—48 |iM O2). In the "center" of a wound the oxygen concentration can be decreased by an order of magnitude again [25]. When there is a stressful event for an organism such as a wound, the oxygen consumption will rise both locally and systemically. Accordingly the concentration of oxygen available for healing become a limiting and important aspect of a healing wound. In contrast, almost all cell cultures are performed in air saturated liquids that the cell would never be exposed to in a physiological condition. It is becoming increasingly popular to utilize hyperbaric oxygen therapy clinically to stimulate wound healing. The logic is that increasing oxygen tension into an "anaerobic" wound environment can stimulate leukocyte antimicrobial activity in turn leading to wound resolution. A recent re-evaluation of several sur-

1014

Pciyt î Disease processes

gical cases has shown the effectiveness of hyperbaric oxygen and other procedures that can increase tissue oxygen tension and enhance chnical wound healing [71]. Sometimes a combination of rather simple, well-defined regimens can result in the resolution of wounds that might otherwise not heal well. For example, inexpensive techniques such as preoperative evaluation of nutrition, hyperglycemia, and steroid use can be controlled. Postsurgical considerations of maintenance of body warmth, adequate oxygen supply and the use of hyperbaric oxygen are all factors that aid wound resolution [25]. 4.3 Wound healing and multiple cell signals The complexities of wound healing and the interactions between intercellular messages and the eventual survival from an event such as a traumatic wound depend on a vast number of interacting events. A recent study [184] has begun to define a new way to evaluate leukocytes and their ability to encounter and respond to multiple chemoattractant signals in complex spatial and temporal patterns. As leukocytes are exposed to multiple chemoattractants (and potentially chemorepellants) they must process the signals that they receive so that they can move up a gradient of one type of attractant towards another. They must process signaling in a combinatorial manner in order not to be rendered immobile and unable to function properly The "symphony" of cellular control of inflammation becomes more complex the more we have been able to understand it. 5

SUMMARY

The wound healing process is a complex cellular process that involves a number of cell types which must all interact harmoniously for the wound to resolve and heal. It differs in complexity from embryogenesis in that the wound is frequently infected or exposed to debris such as necrotic tissue that must be removed before the wound is completely healed. While the main cell types discussed in this chapter have been the neutrophils and macrophage, the interaction between platelets, fibroblasts, epithelial, endothelial cells, as well as specialized cells in selective tissues such as neural, bone, muscle are all tissues that are frequently damaged in injuries. Each cell type has specific requirements and limitations as to when and how it can replicate. Cytokines are a source of messages between cells and their production and subsequent interaction with "remote" cells can signal information about the wound environment that enables the wound peripheral cells to begin replicating and allow for the regrowth of the wounded area. The necessity of having cells "stick" together is certainly critical in getting wound resolution. The expression and timing of the expression of adhesion molecules is a research area of considerable interest as it precedes tissue growth. The one item that is often overlooked in wound healing and in cell biology in general is the role of oxygen tension. At the very best most tissue concentrations of oxygen are far lower than might be found in a cell culture growing in ambient


1015

air. In wounds the oxygen supply substantially dictates the rate of neutrophil and macrophage production of O2" which precedes the production of numerous other oxidants. Oxygen is also required for generation NO-. NO- is certainly formed in human macrophages and has a role in the bactericidal arsenal of the immune system. 6

PERSPECTIVES

From a cellular and biochemical perspective the future for investigations into inflammation and wound repair are likely to unravel many new and currently ill-defined pathways of regulation. Understanding a concept such as how a cell can sense two separate chemoattractants, or how a cell can know when it must turn on a set of genes to reconstruct a wounded area in an area that is separated by microns from an environment that would destroy the cell, this represents an example of the balance that exists between the inflammatory "cleanup" cells and the reconstruction cells that follow into the wound site. Clinically, as new pathways of cellular regulation and control are defined, there exist possibilities to understand the nature of pathological wound healing. As has been the case for centuries, observations made by clinicians about their patients can aid basic cellular sciences and clinicians can learn much by understanding the cellular events that result in wound healing. Keeping up with all the new information represents a major challenge. This brief review attempts to bridge this gap. 7

ACKNOWLEDGMENTS

The author is supported in part by a grant from The National Institutes of Health, Grant GM27345. I thank Prof Thomas K. Hunt and Dr John Feng for many helpful comments. The artwork of Fig. 1. was drawn by Fred Kim and Ronald Moy who have assisted in the manuscript preparation. 8

ABBREVIATIONS

AP: EGF: FAD: fMLP: HB-EGF: IGF: ICAM: IL: IL-2R: iNOS: LPS:

activator protein epidermal growth factor flavin adenine dinucleotide formyl-methione-leucine phenylalanine heparin-binding EGF-like growth factor insulin-dependent growth factor intercellular adhesion molecule interleukin interleukin 2-receptor inducible NOS lipopolysaccharide

1016


NOS: PKC: PMA: ROS: SOD: TGF: TNF: VCAM: VEGF: 9 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

nitric oxide synthase protein kinase C phorbal myristic acetate reactive oxygen species superoxide dismutase transforming growth factor tumor necrosis factor vascular cell adhesion molecule vascular endothelial growth factor

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i( 2000 Elsevier Science B.V. All rights reserved. Handbook of Oxidants and Antioxidants in Exercise. C.K. Sen, L. Packer and O. Hanninen, editors.

1091 i.\J^


Oxidative stress induced by the metabolism of medical and nonmedical drugs Moreno Paolini^ and Giorgio Cantelli-Forti^ 'Department of Pharmacology, Biochemical Toxicology Unit, The University of Bologna, Via Irnerio 48, 40126 Bologna, Italy Tel: +39-051-20917-83. Fax: +39-051-2488-62. '^Division of Environmental Toxicology and Community Health, University of Texas Medical Branch, Galveston, TX, USA.

1 2 3

INTRODUCTION DRUGS AND FREE RADICALS DRUGS YIELDING FREE RADICAL METABOLITES 3.1 Carbon tetrachloride 3.2 Clozapine 3.3 Aminopyrine 3.4 Phenylbutazone 3.5 Nitrofurantoin 3.6 Doxorubicin 3.6.1 Free radicals generation by redox-cycling 3.6.2 Free radicals generation by drug iron complex 4 OXIDATIVE DAMAGE BY DRUGS DEPLETING GLUTATHIONE 5 INDUCERS OF CYTOCHROME P450 ISOZYMES 6 INDUCERS OF PEROXISOME PROLIFERATION 7 SUMMARY 8 PERSPECTIVES 9 ACKNOWLEDGEMENTS 10 ABBREVIATIONS 11 REFERENCES

1

INTRODUCTION

Reactive oxygen species (ROS) such as oxygen-derived free radicals, singlet oxygen, hydrogen peroxide, hypochlorite and peroxynitrite, are now known to be implicated in numerous pathologies, including the physical and mental decline associated with the aging process. However, it has been shown that certain ROS evidently form part of our immune system defenses against invading microorganisms and may also act as essentially intracellular second messengers for several cytokines and growth factors [1—3]. Indeed, oxy-radicals are able to mimic, in many ways, the actions of polypeptide factors. For example, calcium-ion mobilization from mitochondria and endoplasmic reticulum, and influx from the extracellular spaces are some of the earliest events occurring after ROS exposure [4]. The consecutive changes in the activities of phosphatases and kinases that transduce the initial signal to a family of transcription factors are only beginning to

1022

P(î^t XI: Disease processes

be explored [5,6]. These changes are a part of a highly complex network of activities that allows an immediate response to various forms of cellular stress. In some instances, however, oxidative stress may result more directly in the alteration of the activity of trascription factors such as nuclear factor K-B, tumor necrosis factor a, interleukin-1 and the oncoproteins Fos and Jun [7,8], followed by their enhanced synthesis and the transactivation of subfamilies of effector genes [9]. It is becoming increasingly recognised that activation of these gene expression circuits by oxy-radicals can contribute to various forms of cellular injury, including cancer [10] and the involvement of free radicals in many degenerative diseases. Nevertheless, with the exception of some conditions caused by environmental toxins and ionizing radiation, it is not currently known whether ROS generation is the causal factor of illnesses or the result of their progression. Formation of ROS, like the superoxide anion O2 " and its secondary radicals, is continuously induced by such factors as cellular respiration, oxidation during the metabolism of xenobiotics and arachidonic acid, and respiratory burst in phagocytic cells [11]. Under normal physiological conditions, about 1—3% of the oxygen we breathe is used to make superoxide. Since human beings consume a lot of oxygen, it is possible to produce over 2 kg of superoxide in the body every year. Some examples of ROS generation during endogenous metabolism are shown in Table 1. The key role of constitutive levels of superoxide dismutase (SOD), catalase (CTS) and glutathione peroxidase (GPX) activities in protection against oxygen-mediated biological damage is extensively documented [12]. However, several unrelated phenomena that pose a risk to health, such as the metabohsm of specific drugs, induction of the cytochrome P450 superfamily by drugs and environmental pollutants, inflammation and reoxygenation of ischemic tissues (Fig. 1), and alcoholism or tabagism can also lead to increased production of radicals. Tissues can often respond to mild oxidative stress by creating extra antioxidant defences, but severe increases of steady-state concentration of oxygen radicals can cause cell injury or even death by necrosis or apoptosis [13]. Here, we briefly summarize some of the scenarios where certain medical and nonmedical drugs can impose an oxidative stress either by the production of free radicals, (the toxin may itself be a free radical, be metabolized to a radical. Table 1. Endogenous generation of ROS. Enzyme or system

ROS produced

Mixed ftmction oxidase Monoammine oxidase (brain) Xantine oxidase NADPH oxidase in neutrophils Nitric oxide synthase Superoxide dismutase Oxidative phosphorilation in mithocondria Fenton reaction (on surface of proteins)

O2 ~ H2O2 O2 " 02^ NO H2O2 O2 ~ HO

Chapter 35: Oxidative stress induced by the metabolism of drugs

Injury Exercise to excess Radiation Infection Cold Ischaemia/reperfusion Traumatic Toxins Thermal

1023

Drug Metabolism Cytochrome P450 induction Arachidonic acid release, enzymic peroxide formation Haem protein release Metal ion release from storage sites Phagocyte recruitment and activation Interference with antioxidant defence systems

sU Induction of antioxidant defences

1

Oxidative Stress

Tissue injury

Fig. 1. ROS overproduction by tissue injury.

or generate oxygen free radicals), by depleting antioxidant defences, or also indirectly by the induction of cytochrome P450 isoenzymes and peroxisome proliferation. 2

DRUGS AS FREE RADICALS

Nitrogen dioxide (NO2 ) is a free radical contaminant member of an extended family of nitrogen oxides and oxy-acids interrelated by a number of reactions. Respiratory exposure to NO2, which is produced primarily by atmospheric oxidation of nitric oxide, produces a well-documented sequela of epithelial injury in both the conducting airways and the proximal alveoli. Since NO2 per se is eliminated by absorption, pulmonary-surface-lining-layer-derived products are probably critical to the genesis of NO2 -induced lung disease. N02 exists in equilibrium with its dimer [14], NO2O4: 2NO*

^

N2O4

ich in aqueous solution forms nitrite and nitrate [15]: water N2O4 —> NO,- -

+

NO^

Nitrogen dioxide is formed in the clean troposphere primarily through the rapid reaction of nitric oxide with ozone [16]:

1024


NO

+ 0 3 ^

NO2 + O3

and it is removed by photolysis: NO2 + /?!/ leading to the generation of O3. 0('P)

-^

+

NO

+

0(^P)

0 2 ^ 0 3

The net result is a photo-stationary state, where the relative concentrations are set by light intensity NO2 can react with ozone [16] NO2

+

0 3 ^

NO3

+

O2

and the nitrate radical so formed is also photolyzed [17]. NO3 + /iz/ ^ NO3 + /iz/ ^

NO NO2

+ +

O2 or 0(^P)

Further nitric oxide reactions yield: NO3 + NO -^ 2N02or[16] NO3 + NO2 ^ N2O5 which is the major source of the highly reactive NO3 at night [18]. NO2 is also generated in the troposphere by the reaction of NO with the hydroperoxyl radical [17]: NO

+

HO2

^

NO2

+

OH

or with other peroxyl radicals, and finally by the reaction: 2NO

+ 0 2 ^

2NO2

which, however, is too slow to be an important source of NO2. In polluted atmosphere, the situation is far more complex, with the formation of many possible nitrogen-containing products [18]. Nitrogen dioxide can enter the body by inhalation and also can be generated in vivo [16—20] in many ways. Indeed, nitric oxide has been sound to constitute an intermediate key in a number of physiological processes, and this strongly supports the concept that NO2 might also be frequently reduced in biological systems. A fundamental reaction which may result in nitrogen dioxide generation in the body is:

Chapter 35: Oxidative stress induced by the metabolism of drugs NO

+

O2OONO

OONO

^ ^

H+ —^

1025

" OONO to give NO^ or OH

+

NO2

Nitric oxide also appears to react rapidly with organic peroxyl radicals: NO

+

RO2

-^

ROONO

forming peroxynitrites that have been proposed as intermediates in the nitrozation of organic hydroperoxides by N2O4. NO2 might also be formed by the oxidation of nitrite in solution. For example, it has been suggested that the reaction of oxyhemoglobin with nitrite could yield NO2 and hydrogen peroxide [21]: Hb02 NO2

+ +

NO2

HbOz

^ ^

Hb+3 + Hb3+ +

NO2 + H2O2 NO2- + O2

or, as occurs in gaseous phase, nitrogen dioxide might be formed in physiological systems through the reaction of NO with O2. NO2 may undergo various different types of reactions [16]. These include hydrogen atom abstraction from C-H bonds [22,23] (e.g., n-alkanes, phenols), additions to unsaturated bonds [24,25] (dialkenes, alkenes, including polyunsaturated fatty acids), electron transfer reactions [25,26] (morpholine, ascorbate, atocopherol) and additions to many inorganic free radicals in atmospheric chemistry [27,28] and in hquid phase [29,30] (methanol, 2-propanol, benzene). These reactions, yielding oxygen- and carbon-centered radical formations, can lead to oxidative lesions (i.e., inflammation of the respiratory tract, lipid peroxidation) when the oxidant-antioxidant balance is altered [31]. It is well known, for example, that the interaction of nitrogen dioxide with plasma, can lead to depletion of antioxidants such as ascorbic acid, uric acid, protein thiol groups, a-tocopherol [32], thus damaging biological macromolecules [33—38]. 3

DRUGS YIELDING FREE RADICAL METABOLITES

Free radicals are produced during the metabolism of many medical and nonmedical drugs by enzymes such as cytochrome P450-dependent monooxygenases or peroxidases. In some cases, chemicals can redox cycle using reductases (e.g., cytochrome P450-reductase) by catalysing an electron reduction; some redox cycling drugs reduce molecular oxygen by one electron to generate superoxide which, in the presence of transition metals such as iron, can produce the very reactive hydroxyl radical (HO) by the well known iron-catalized Haber-Weiss reaction. Therefore, chemicals able to release iron from storage proteins can be very toxic, causing protein, lipid and nucleic acid oxidation. A wide variety of chemical toxins, including some medical drugs, have been


1026

Table 2. Free radicals by the metabolism of medical and nonmedical drugs. Chemical

Radical metabolite

References

CCI4 Daunomycin (daunorubicin) Adriamycin (doxorubicin) Mitoxantrone Nitrofurantoin Nitrofurazone Metronidazole Acetaminophen 6-Hydroxydopamine 4-Hydroxyanisole Etoposide(VP-16) Benzidine Aminopyrine Clorazil Phenylhydrazine 3-Methylindole Probucol FeS04 Methimazole Chlopromazine Salicylanilides

CCI3 Q, O2 Q , O2 N' RNO2 , O2 RNO2 , O2 RNO2 , O2 ArO Q , O2 Q , ArO Q , OH ArNHs'"" RN(CH3)2" R Ar, O2 N" ArO OH RS CPZ^ Ar

[39] [40] [40] [40] [41] [41] [41] [42] [42] [42] [42] [42] [43] [44] [45] [46] [46] [46] [46] [47,48] [49]

found to metabolize to free radicals in biological systems (Table 2). In some cases, the free radical metabolite is responsible for the toxic effect of a given agent, while in others it may react further to give both radical and nonradical species. Below are some examples. 3.1 Carbon tetrachloride One of the earliest free radical metabolites known to be implicated in the toxicity of a xenobiotic is the trichloromethyl radical (CCI3). In 1961, Butler [39] proposed that this radical was responsible for the hepatotoxicity of carbon tetrachloride (CCI4). It is generally accepted that the reductive dehalogenation product of CCI4 results from metaboHc activation by cytochrome P450-dependent monooxygenases. The immediate consequences of the metabolic activation of CCI4 are the lipid peroxidation and covalent binding to proteins, but the subsequent events leading to centrolobular necrosis of the liver remain unclear. The mechanism probably involves reductive cleavage of the C-Cl bond [46,50,51], yielding the trichloromethyl radical: CCI4

cytochromeP450

+

CCI3

+

Cl-

O2

CI3COO


1027

A second carbon-centered radical derived from CCI4 metabolism was discovered more recently by Connor et al. [52]. This is the carbon dioxide anion radical (CO2 ), which was identified in the effluent perfusate of the isolated perfused liver following infusion of halocarbon. It was initially suggested that the formation of this second radical involved molecular oxygen [52]. In the proposed pathway, the trichloromethylperoxyl radical formed from the reaction of CCI3 with O2 was seen as a secondary product of the reductive CCI4 metabohsm. The decomposition of the trichloromethylperoxyl radical to OC -CI could be followed by hydrolysis to CO2 . The reaction sequence most likely responsible for CO2 radical formation involves the trichloromethyl peroxyl radical (CCI3OO), which is converted to the trichloromethoxy radical (CCI3O) by a two-electron reduction followed by the promotion and elimination of a water molecule: CI3COO

2e- + 2H+ > CI3CO-H2O

OH-^ CICO

+

HOCl

+

CI-

The carbon dioxide anion radical C02^ could be generated directly from hydrolysis of the chloro-carbonyl radical: H2O > CO2 -HCl -H+ The carboin dioxide anion radical is known to reduce oxygen to superoxide: O2 C02~ > CO2 + O2 CICO

forming also carbon dioxide, the final product of CCI4 metabolism. However, recent investigations have cast doubt on this pathway and the most likely explanation foresees the cytosolic formation of CO2" radical from water via an intermediate containing glutathione (GSH), which might play an important role in the overall process [53,54]. 3.2 Clozapine The use of clozapine, a unique antipsychotic drug, has been restricted due to a 1—2% incidence of drug-induced agranulocytosis [55]. Hypersensitivity reactions in general appear to be linked to its metabolic activation by human myeloperoxidase (MEP) and horseradish peroxidase (HRP) in monocytes and neutrophils [56]. During phagocytosis, these cells are activated and release significant quantities of myeloperoxidase and hydrogen peroxide [57]. The peroxides are generally able to bioactivate aromatic amines via one-electron transfer reactions, leading to the formation of free radicals metabolites. MEP activity may thus, convert hydrogen peroxide and chloride ions to hypochlorous acid, which is a powerful oxidant catalyzing 2-electron oxidations [57]. The evidences currently available seem to indicate that clozapine is metabolized to a free radical by both HRP


1028 GSSGO'/

O2

GSSG"

CZP+GS'

Czp

HRP/MEP H2O2

CZP

ACTH *-

CZP+NADPH'

CLZ + ACT'

J^5™I?

NADPH

NADP+ + O2

Fig. 2. Scheme for the clozapine (CZP)-metabohsm by HRP/MEP system involving subsequent reactions with glutathione (GSH), NADPH or ascorbate (ACT). HRP: horseradish peroxidase; MEP: myeloperoxidase; ACTH: monodehydro-ascorbate.

and MEP systems. A clozapine cation radical would be a very electrophilic species, generated as reported in Fig. 2. The adduct formation could proceed through different mechanisms such as radical-radical coupling or addition of the nucleophilic glutathione anion to the clozapine radical cation, as depicted in Fig. 3. The resulting neutral radical adduct could then be oxidized by a second CH3

'} CkJOi ACTH

I H2O2

NADPH GSH

-^°XX GS

IH

X^ Fig. 3. Proposed clozapine (CZP) bioactivation. HRP: horseradish peroxidase; MEP: myeloperoxidase; ACTH: monodehydro-ascorbate.


1029

clozapine cation radical or by another oxidant to produce the final stable adduct. Another possibility foresees a second-order radical disproportionation forming the two-electron oxidation product, di-imine [58]. Although glutathione conjugation is usually considered to be a detoxification process, conjugation with semiquinones or aminophenoxyl free radicals does not necessarily lead to less redox-active species [59—61]. The clozapine cation radical itself also behaves as a strong oxidant and, if thiyl radicals are formed in excessive amounts, oxidative stress may result, due to the depletion of the glutathione pool and the formation of the ROS that are probably responsible for clozapineinduced disorders. On the other hand, ascorbate at physiological concentration is able to reduce the clozapine cation free radical, thereby inhibiting not only oxidative stress but also protein and glutathione adduct formation [62—66]. The coadministration of clozapine with antioxidants such as ascorbic acid to patients should be beneficial in reducing the risk of agranulocytosis [57—67]. 3.3 Aminopyrine The clinical use of aminopyrine was sharply curtailed when it was found to be responsible for potentially fatal bonemarrow toxicity and agranulocytosis [68]. However, this substrate is still widely employed to assess either in vivo or in vitro the activity of the cytochrome P450-dependent monooxygenase system. It has been shown that the hypochlorous acid formed by the neutrophil myeloperoxidase system, is capable of producing free radical electrophilic metabolites as reported in the proposed scheme (Fig. 4), which are probably coresponsible for the drug toxicity [68]. 3.4 Phenylbutazone During the prostaglandin H synthase-(PHS)-catalyzed reduction of the hydroperoxy endoperoxide PGG2, a number of xenobiotics including phenylbutazone are CI2 + H2O2 ' ^ ^ ' ^ ^ H O C I + OH (CH3)2NR + HOCI

*^ (CH3)2NMCI) + OH -

(CH3)2NR + c r

O^ R

CeHs ^ N ^ /CH3 L==-1_QH3

(CH3)2NR (CH3)2N + o r

Fig. 4. Proposed scheme for the formation of aminopyrine (AMP) cation radical AMP"^ by the myeloperoxidase (MEP)-H202-C1 - system. MEP: myeloperoxidase.


1030

cooxidized by PHS-hydroperoxidase [69,70]. This nonsteroidal anti-inflammatory drug, firstly introduced in the 1950s to relieve the symptoms of rheumatoid arthritis, can produce serious side-effects [71]. The metabolic pathway of phenylbutazone to 4-hydroxyphenylbutazone occurs via the formation of a carbon-centered radical in the diketone moiety (position C-4). This radical can react with molecular oxygen to form a peroxyl radical [72]. The abstraction of a hydrogen atom results in the formation of 4-hydroperoxyphenylbutazone, which is thought to be reduced to 4hydroxyphenylbutazone by PHS hydroperoxidase [72]. The proposed PHSdependent metabolism is shown in Fig. 5. A potential mechanism for the formation of 4-hydroxyphenylbutazone could involve the so-called "self-reaction", a chain termination reaction between two phenylbutazone peroxyl radicals forming a tetroxide [73]. The last intermediate rearranges to two alkoxy radicals and molecular oxygen in the triplet state [74], leading to production of 4-hydroxyphenylbutazone, after the abstraction of hydrogen atoms by the reactive alkoxy radical [75]. Therefore, it seems that the phenoxybutazone peroxyl radical can either abstract a hydrogen atom to yield 4hydroxyperoxyphenylbutazone, or react with another peroxyl radical and eventually rearrange to 4-hydroxyphenylbutazone [76]. Considering the potency of phenylbutazone to inhibit PHS and prostacyclin synthase, the proposed inhibited species was the phenylbutazone peroxyl radical. Alternatively, if the peroxyl radicals self-react to form the tetroxide, the decomposition product of this intermediate, the alkoxy radical could be the inhibitor [76,77]. It was recently reported that, hem protein systems such as myoglobin- and hemoglobin-H202-dependent systems oxidize phenylbutazone into a free radical metabolite that is capable of initiating lipid peroxidation and inactivating a i-anti-

^^^"^

PHS^

>.. C4H9

C4H9

02 .0^'^C4H9 PBN

HO—0

C4H9

A^ô

0-0-'

o nC4H9.

^ HO

-1/2 02

C4H9

•0'"^C4H9

Fig. 5. Prostaglandine H syntase (PHS) hydroperoxidase-mediated metabolism of phenilbutazone (PNB).


1031

proteinase [71]. This is particularly deleterious at the sites of chronic inflammation and tissue injury, where haem proteins can become available to catalyze such reactions [77—79]. 3.5 Nitrofurantoin Nitrofurantoin (NTF) is widely used as an antibacterial agent known to be particularly effective in the treatment of acute urinary tract infections [80,81]. Although it does not seem to have carcinogenic properties [82], prolonged administration of the drug has frequently been found to result in pulmonary fibrosis [83]. NTF can be reduced by NADPH-cytochrome c-reductase and xanthine oxidase [84]. In both enzymatic reactions, the drug is able to catalyze or stimulate ethylene formation from methionine via the production of a strong oxidant (cripto-HO radical) [85]. The production of this reactive species does not need to involve O2 directly, but the presence of high amounts of SOD in the xanthine oxidation reaction resultes in a total inhibition of ethylene formation [86]. It has been proposed that at or very close to the xanthine oxidase system, the following reactions can take place: NTF + e - - -^ NTF0, O2 + e-- -. NTF+ 0 2 ^ N T F + O2" 2O2- + 2H+ ^ H2O2 + O2 NTF+ H2O2 ^ (NTF" H2O2) crypto - H O

Reactions occurring some distance away from xanthine oxidase may be: N T F + O2 ^ 20," + 2H+ ^ F + H2O2 ^

NTF+ O2 H2O2 + O2 (NTF" H2O2) crypto — H O

depending on the capability of O2 to diffuse away from the immediate vicinity of the enzymatic system to the general reaction mileau, where the NTF/NTF ratio would be greater than in proximity to the enzyme. The reaction between superoxide and the drug is reversible, and near the enzyme the equilibrium would be expected to favor the generation of O2 . Away from xanthine oxidase, the equilibrium would be reversed and the NTF radical increased. This radical can subsequently react with H2O2, whether derived from the enzyme or O2 dismutation, leading to the formation of crypto-HO radicals which can further oxidize the methionine to ethylene [86]. Additional evidence showed that even in conditions of complete anaerobiosis, the reductase system was able to generate the highly reactive crypto-HO radical from H2O2 and NTF [87]. This radical.

1032


together with ROS, may also play a central role in the mechanism of pulmonary toxicity of the compound [86,88—90]. 3.6 Doxorubicin The anthracycline antibiotic doxorubicin (DXC) exerts a powerful action against a wide range of human malignancies such as acute leukemia, non-Hodgkin lymphomas, breast cancer, Hodgkin's disease and sarcomas [91]. Apart from several side-eflFects that are common to many cancer chemotherapeutics, it is currently thought that free radical metabolites are critically involved in the cytotoxic mechanism of doxorubicin against tumor cells. It is known that suitable flavoproteins catalyze the formation of reduced semiquinone radicals by accepting electrons from NADPH or NADH and donating them to the drug. By reducing oxygen to superoxide the parental chemical is then regenerated. This "redoxcycling" reaction is potentially harmful to cells since a relatively small quantity of the drug is able to generate numerous superoxide radicals [92,93]. The formation of an oxidized semiquinone in a different ring of doxorubicin is known to occur in the presence of iron when no reducing system is available. The reaction of the semiquinone iron complex reacting with molecular oxygen yields a fully oxidized form of doxorubicin and oxygen radicals [94,95]. 3,6A Free radicals generation by redox-cycling The reductive redox-cychng of doxorubicin was first described in 1977 [96], using the NADPH cytochrome P450-reductase as a motor enzyme, and later NADH dehydrogenase and xanthine oxidase were also shown to catalyze the oneelectron reduction of the drug [97,98]. For this reason, the metaboHsm can occur in mithocondria, endoplasmic reticulum, cytoplasm and nucleus. The nucleuscatalyzed redox-cycling of doxorubicin led to the knowledge of how site-specific activation of the drug after DNA binding lead to the formation of free radicals. Redox cycling, however, is rapid in the presence of oxygen and slow under hypoxic conditions. The oxygen-dependent mechanism of doxorubicin metabolism involves the generation of hydroxyl radicals as DNA damaging species. The drug can be reduced by cellular flavoproteins to yield a 1-electron reduced semiquinone form. In the presence of oxygen, the semiquinone free radical is oxidized back to the parental quinone with the formation of superoxide radicals, as depicted in Fig. 6:

2O2- + 2H+ ^ H2O2 + O2 O2- + Fe^+ ^ 0 2 + Fe+ H2O2 + Fe+ ^ HO* + O H - + Fe


Interaction with macromoleciies (DNA, RNA)

1033

(C7-deoxyaglicone) MUTAGENESIS

NADP\

/FH2 DNA

(H202,HO •, etc) NADPH Lipids

Lipid peroxidation OCH3 O"

OH

O Sugar

(SEMIQUINONE) O

vv; o

o

Interaction with macromolecuies

C7-Free Radical

Fig. 6. Free radical generation by doxorubicin (DXC) metabolism.

These oxygen radicals are responsible for DNA damage, since the presence of SOD, CTS and iron chelator inhibit their cytotoxicity in several experimental models [99]. In the absence of molecular oxygen (Fig. 6), the unstable semiquinone loses its sugar moiety, yielding an intermediate C7 free radical which can bind covalently to cellular macromolecuies or become again reduced forming a relatively stable product, the C7-deoxyaghcone [100,101]. A tautomer of C7-deoxyaghcone is the C7-quinone-methide, which is a strong DNA alkylating agent and is potentially toxic for tumor cells [102,103]. In the presence of submicromolar concentrations of iron and at low partial pressure of oxygen, the semiquinone of doxorubicin can react with H2O2 to cause the breakdown of deoxyribose [104]. The very low iron requirements for the reaction and the optimal oxygen tension required (approximatively 4 mmHg) may mimic the situation occurring in tumor cells [105]. Again, both in the presence or in the absence of oxygen, lipid peroxidation can be induced by the doxorubicin semiquinone radical, probably mediated by the HO species [106].


1034 3,6,2Free radicals generation by drug iron complex

Doxorubicin-iron complex can support free radical formation either by reducing system or by itself in the absence of reducing systems. The doxorubicin-iron III complex (DXC-Fe^"^), in the presence of a reducing system such as NADH cytochrome P450-reductase or of thiols like glutathione (GSH) and cysteine, may be reduced to DXC-Fe^"^ (Fig. 7). The latter complex can react with oxygen or hydrogen peroxide leading to the generation of superoxide or hydroxyl radical while the complex is oxidized to yield DXC-Fe^"" again [95,107,108]. This GSH driven to HO production is a typically cycHc process, which in the presence of sufficient amounts of GSH can proceed indefinitely, in a very similar manner to redox cycling [109]. Without the presence of a suitable reducing apparatus, DXC-Fe^"^ can reduce its chelate irons by means of an intramolecular redox reaction either by oxidation on the C9 side chain or the hydrochinone moiety at ring B, producing a doxorubicin free radical chelated with Fe^"^ [94,110,111]. This intramolecular reaction is probably catalyzed by DNA, which in the presence of oxygen yields superoxide radical and with H2O2 can also produce hydroxyl radical (Fig. 7) [112,113]. Oxidation of the side chain will ultimately lead to the gen-

NADP+

DXC-Fe^Xt

NADPH

,rGSSG

GSH

DXC-Fe2+ H2O2

HO' DXC°'-Fe2+ H2O2

HO' DXC^^Fe^""

9-COOH-DXC Fig. 7. Free radical generation by doxorubicin (DXC)-iron complex. GSH: glutathione.


1035

eration of 9-dehydroxyacetyl-9-carboxyl doxorubicin (9-COOH-DXC) [111]. Contrary to intercalated doxorubicin, the drug-iron complex preserves its ability to catalyze oxygen radicals in the presence of double strand DNA [107]. Therefore, doxorubicin-iron complex-driver hydroxyl radical generation can proceed near DNA and has a high potential to induce DNA insults, especially in the light of its own probable ability to catalyze HO production by this complex [112,113]. Free radical generation in cardiac cells is due to one-electron reduction of the drug occurring in several subcellular compartments, and seems to be responsible for the cardiotoxicity which is an important dose-limiting factor of its use in cancer treatment [106,114]. 4

OXIDATIVE DAMAGE BY DRUGS DEPLETING GLUTATHIONE

Acetaminophen is an acetanilide widely used as an antipyretic and analgesic drug, which can cause hepatocellular necrosis in man and experimental animals [115,116]. Evidence from a variety of studies has indicated a model where the microsomal cytochrome P450-dependent system plays an important role in the oxidation of the drug to a chemically reactive and potentially toxic metabolite, highly reactive both as an electrophile and as reactive oxidant [117]. At low doses, the nonenzymatic conjugation of the reactive metabolite of acetaminophen with glutathione is believed to serve as a detoxification mechanism [117]. However, at high doses, intracellular glutathione levels are significantly depleted allowing the reactive metabolite to bind covalently to hepatic protein and to stimulate peroxidative events [118]. In other words, the cellular toxicity occurs only when glutathione stores fall below critical concentrations. In addition to the glutathione conjugate of acetoaminophen, other thioether metabolites have been identified (e.g., 3-mercapturic acid), and these are considered to represent biotransformation products of the glutathionyl precursor [119,120]. The 3thioether structures of these conjugates support the contention that the reactive metabolite of acetaminophen is the quinone derivative, the A^-acetyl-p-benzoquinone imine, which is prone to nucleophilic attack (e.g., by sulphydryl agents NCOCH-

NOCH3

S-Protein

\

N-Protein

GSH depletion

Fig. 8. Proposed pathway of acetaminophen metaboHsm. NAPQI: A^-acetyl p-quinoneimine. GSH: glutathione.

1036


such as glutathione) at the 3-position as shown in Fig. 8. The drug is initially metabolized by cytochrome P450 isoforms to give A^-hydroxyacetaminophen which has been shown to be more toxic than the drug itself [121,122]. However, the formation of this intermediate has been questioned [123]. The metabolite is relatively instable at physiological pH and temperature and presumably undergoes water loss to form the A^-acetyl-p-quinoneimine (NAPQI) [124]. This reactive metabolite can either react with GSH to form GSH-conjugates or can oxidize GSH to its disulfide GSSH with the concomitant production of acetaminophen. In cells with a functioning GSH-reductase, the rapid increase in GSSG is transient and is rapidly reduced back to GSH [118]. NAPQI may then undergo enzyme mediated conjugation or reduction by GSH. A number of investigations have shown that NAPQI is able to bind covalently to cellular macromolecules which, along with the concomitant GSH depletion, can have a pivotal role in acetaminophen cytotoxicity [125,126]. Because of these properties, NAPQI may stimulate lipid peroxidation, probably, with the aid of the released proinflammatory cytokines which are capable of mediating cytolytic effects by means of the induced overgeneration of ROS [123,127,128]. 5

INDUCERS OF CYP ISOZYMES

The superfamily of cytochrome P450 genes encodes a variety of metal-isoenzymes with a wider but overlapping substrate specificity that catalizes the metabolism of endo- and xenobiotics either into biologically inactive metabolites, or in some cases, into reactive ones. Reactions by these structurally different catalysts, involving the use of molecular oxygen and NADPH or NADH as a source of reducing power are considered to exhibit various types of activities such as monooxygenases, peroxidases, oxidases, reductases and desaturases. The utilization of molecular oxygen for the combined oxidation of NADPH and target xenobiotics makes the cytochrome P450 superfamily of isoenzymes a potentially significant source of ROS. In protecting living organisms by facilitating the metabohsm of various toxins, the cytochrome P450-dependent system potentially endangers an organism by causing activation of oxygen. During the monooxygenase catalytic cycle, this hemoprotein forms the pivot of the oxygen indispensable for its monooxygenase function: monooxygenase CYP RH + NADPH + H+ > ROH + NADP+ + H2 As observed by Gillette et al. [129] as long ago as 1957, the oxidation of NADPH also involves the generation of hydrogen peroxide: oxidase CYP NADPH + H+ > NADP+ + H2O2 Although several possibilities have been hypothesized, the production of H2O2 either in the presence or in the absence of substrate, seems to originate via


1037

autoxidation of the oxy-cytochrome P450 complex: Fe3+02

> Fe3+

+

O2

liberating O2 , which in turn spontaneously dismutates to give H2O2 [130]: 2H+

+

O2

+

O2

> H2O2

+

O2

In the presence of transition metals hydrogen peroxide may be important for the microsomal generation of the very reactive hydroxyl radicals according to the Haber-Weiss reaction: O2-

+

H2O2

> HO

+

O2

+

OH-

Alternatively hydrogen peroxide can also be reused by cytochrome P450 when operating in its peroxidase mode of activity, depicted as: peroxidase CYP RH + H2O2 > ROH + H2O It should be noted that the overall significance of ROS generation by the cytochrome P450 apparatus would consider not only the toxicological consequences of the phenomenon, but also the role that O2 /HO radicals may have in xenobiotic metabolism. Many chemicals such as adrenaline, benzoate, ethanol, aniline, safrole, dimethylsulphoxide and benzene are actually biotransformed by these active oxygen species that are derived from the cytochrome P450-system [130]. In some cases, the metabolic pathway involves the production of free radical metabolites (i.e., a-hydroxyethyl radical from ethanol or hydroxycyclohexadienyl radical and phenol from benzene) which can impose an oxidative stress [131]. The active oxygen production by the microsomal monooxygenase system is then increased by cytochrome P450 inducers, leading to a magnification of the processes described above [129,130]. It has recently been reported that the overproduction of O2 by cytochrome P450 overexpression, is not a prerogative of the induction of certain isozymes, as was initially discovered for cytochrome P450 2B1 and cytochrome P450 2E1 [130,132,133], but instead parallels the "unspecific" cytochrome P450 induction [134]. More generally, a strict correlation exists between the induction of each cytochrome P450 gene family and the O2 yield [134]. The importance of this phenomenon is underlined by the fact that an impressive number of chemical compounds, some of which are reported in Table 3, are to various extent capable of inducing cytochrome P450 enzymes. Many of these inducers are widely used medical drugs (Table 4). Cytochrome P450 induction occurs by various mechanisms currently poorly understood in organisms that have diverged extensively during evolution. It is likely that the induction is advantageous from an evolutionary point of view, possibly as a mechanism by which terrestrial organisms confront potentially toxic


1038 Table 3. Cytochrome P450 inducers. Xenobiotics

References

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

[135] [136] [137] [138] [139] [135] [140] [140] [140] [140] [140] [135] [141] [142] [140] [140] [140] [143] [140] [140] [140] [140] [140] [140] [140] [144] [140] [140] [135] [135] [135] [139] [135] [135] [135] [145] [140] [140] [146] [140] [140] [140] [135] [136] [136] [146] [147]

1,1,1,2-Tetrachloroethane 1,1 -Dichloroethylene 1,2-Benzanthracene 1,2-Dimethylhydrazine 1,4-Bis[2-(3,5-chloropyridyloxy)]benzene 1,4-Dioxane 1,9-Cichlorotetrachloro-p-dibenzodioxin 1 -Aminoanthracene 1 -Aminomethyltetrachloro-p-dibenzodioxin 1 -Cyanotetrachlorodibenzodioxin 1 -Methylchrysene 11 -Aminoundecanoid 16a-Cyanopregnenolone 16a-Hydroxyestrone 2,3,4,4',5,5'-Hexachlorobiphenyl 2,2',4,4',5,5'-Hexachlorobiphenyl 2,2',4,4'-Tetrachlorobiphenyl 2,3 -Terbutylhydroxyanisole 2,3,3',4,4',5-Hexachlorobiphenyl 2,3,3',4,4'-Pentachlorobiphenyl 2,3,4,4',5,6-Hexachlorobiphenyl 2,3,4,4',5-Pentachlorobiphenyl 2,3,4,4'-Tetrachlorobiphenyl 2,3,4,5-Tetrachlorobiphenyl 2,3,7,8-Tetrachloro-p-dibenzodioxin 2,3,7,8 -TetrachlorodibenzofUran 2,3,7,9-Tetrachloro-p-dibenzodioxin 2,3,7-Trichloro-p-dibenzodioxin 2,4,5-Trichlorophenol 2,4,5-Trimethylaniline 2,4,6-Trichlorophenol 2,4-Dichlorophenoxyacetic acid 2,4,5-Trichlorophenoxyacetic acid 2,4-Dimethoxyaniline 2,6 Bis(L -methylethyl)phenoxy]sulfonylcarbamic acid 2,6 Bis(L-methylethyl)phenylester]carbamic acid 2-Acetylaminfluorene 2-Acetylfluorene 2-Allylisopropylacetamide 2-Aminoanthracene 2-Aminobiphenyl 2-Aminofluorene 2-Chloro-p-phenylenediamine 2-Chloroethanol 2-Chloromethylpyridine 2-Chloropromazine 2-Hexanone

(Continued.)


1039

Table 3. Continued. Xenobiotics

References

48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88.

140] 144] 144] 144] .140] 140] 140] 139] 135] 140] 136] 142] 140] 144] 148] 148] 140] 140] 140] 141] 141] 140] 140] 140] 140] 140] 140] 140] 140] 140] 140] 140] 135] 147] 140] 139] 139] 140] 135] 139] 149]

2-Methylcrysene 3,3',4,4'-Tetrachlorobiphenyl 3,3',4,4'-Tetrachloroazobenzene 3,3',4,4'-Tetrachloroazoxybenzene 3,3',4,4',5-Pentachlorobiphenyl 3-Aminobiphenyl 3-Methylchrysene 3 -Methylcholanthrene 4,4'-Oxydianiline 4-Aminobiphenyl 4-Chloroacetylacetanilide 4-Hydroxyestrone 4-Methylchrysene 5,5-Diphenylhydantoin 5-Ethyl-5-phenylhydantoin 5-Ethyl-5-phenyloxazolidinedione 5-Methylchrysene 6-Methylchrysene 6-Aminochrysene 6a-Methylprednisolone 7-Br-Dichloro-p-dibenzodioxin 7-CF3-dichloro-p-dibenzodioxin 7-Cl-dichloro-p-dibenzodioxin 7-CN-dichloro-p-dibenzodioxin 7-C02-methyldichloro-p-dibenzodioxin 7-H-dichloro-p-dibenzodioxin 7-1-Dichloro-p-dibenzodioxin 7-Methyldichiloro-p-dibenzodioxin 7-NH2-dichloro-p-dibenzodioxin 7-N02-dichloro-p-dibenzodioxin 7-OH-dichloro-p-dibenzodioxin 7-Phenyldichloro-p-dibenzodioxin 8-Hydroxyquinoline a-Hexachlorocyclohexane a-Naphthylamine Acetone Acetylsalicylic acid Acridine orange Aflatoxin Bl 5,5'-Diallylbarbituric acid 3-(10,ll-Dihydro-5H-dibenzo[a,d]cyclohepten-5-ylidene)-A^,7V-dimethyl-lpropanamine 89. 5-Ethyl-5-isopentylbarbituric acid 90. Anisole,2,3,5,6-tetrachloro-4-nitro 91. Anthracene 92. 2,3-Diniethyl-l-phenyl-3-pyrazolin-5-one 93. Aroclor 1254 94. Asbestos (Continued.)

[139] 136] 140] 150] 151] 152]


1040 Table 3. Continued. Xenobiotics

References

95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119.

150] 139] 140] 139] :i40] 140] 139] 140] 140] 140] 153] 139] 139] 139] :i35] 135] 154] 150] 155] 146] 139] 156] 139] 140] 140]

120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137. 138. 139. 140.

Ascorbic acid P -Naphthoflavone P -Naphthylamine 5,5-Diethylbarbituric acid 2-(4-Chlorophenyl)-a-methyl-5-benzoxazolacetic acid Benzo[a]anthracene Benzene Benzo[a]chrysene Benzo[a]pyrene Benzo[b]chrysene 1 -([ 1,1 '-Biphenyl]-4-ylphenylmethyl)-1 H-imidazole Bromobenzene 5-sec-Butyl-5-ethylbarbituric acid Butylated hydroxyanisole Butylated hydroxytoluene C.I. solvent yellow 14 Cannabidiol 5H-dibenz[b,f|azepine-5-carboxamide A^-(aminocarbonyl)-2-bromo-2-ethyl-carbanamide Chlordane Chlorendic acid Chlorometazole 2-Chloro-A^,7V-dimethyl-1 OH-phenotiazine-10-propanamine Chrysene A^-Câno-A^'-methyl-A^"-[2-[[(5-methyl-lH-imidazol-4-yl)methyl]thio]ethyl] guanidine 2-[4(2,2-Dichlorocyclopropyl)phenoxy]-2-methylpropanoic acid 2-(4-Chlorophenoxy)-2-methylpropanoic acid 5-(2-Chlorophenyl)-l,3-dihydro-7-nitro-2H-l,4-benzodiazepin-2-one 1 -[(2-Chlorophenyl)diphenylmethyl]-1 H-imidazole 5-(l -Cyclohexen-1 -yl)-5-ethylbarbituric acid Decabromodiphenyl oxide Dehydroepiandrosterone (11P, 16a)-9-Fluoro-11,17,21 trihydroxy-16-methylpregna-1,4-diene-3,20-dione Diallyl sulfide Dibenzo[a,h]antracene Dichlorobenzene Dichlorodiphenyltrichlorohetane Dichlorvos Dieldrin Diphenylhydantoin Disperse orange 11 2-[a-(2-Dimethylaminoethoxy)-a-methylbenzyl]pyridine Enldrin Erythromycin Ethanol 1,2-Dichloroethylene

(Continued.)

[157] 153] :i39] 147] 139] 136] 139] 158] 147] 140] 136] 140] 136] 146] 150] 135] 139] :i35] 139] 139] 139]


1041

Table 3. Continued. Xenobiotics 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 161. 162. 163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183. 184.

Eucalyptol Fluorine «-Methyl-[4-(trifluoromethyl)phenoxy]-benzenepropanamine 5-Methoxy-1 -[4-(trifluoromethyl)-phenyl]-1 -pentanone-o-(2aminoethyl)oxime Geniconazole 2-Ethyl-2-phenylglutarimide 7-Chloro-2,4',6-trimethoxy-6'-methylspiro-[benzoftiran-2(3H),r-[2]cycloexene] -3,4'-dione 5-(l-Cyclohepten-l-yl)-5-ethylbarbituric acid 5-(l-Cyclohexen-l-yl)-l,5-dimethylbarbituric acid a-Methyl-4-(2-methylpropyl)benzene acetic acid 5-(3-Dimethylaminopropyl)-10,11 -dihydro-5H-dibenzo[b,flazepine Indole-3-carbinol Interferon Interleukin-2 Imidazole Isonicotinic acid hydrazide Isopropanol 5-(l-Propenyl)-l,3-benzodioxole kepone CI-1 -actyl-4-[[2-(2,4-dichlorophenyl)-2-( 1 H-imidazol-1 -ylmethyl)-1,3-dioxolan2- yl]methoxy]phenyl]piperazine Lansoprazole Lindane 4-(8-Chloro-5,6-dihydro-llH-benzo-[5,6]cyclohepta[l,2-b]pyridin-l 1-ylidene)-1 -piperidinecarboxylic acid ethyl ester Mandrax (6a)-17-Hydroxy-6-methylpregn-4-ene-3,20-dione 5-Ethyl-5-methyl-5-phenylbarbituric acid 2-Methyl-2-propyl-1,3-propanediol dicarbamate Methylpyrazole Methyltrichlorometane 2-Methyl-1,2-di-3-pyridyl-1 -propanone 1 -[2,4-Dichloro-j?-[(2,4-dichlorobenzyl)oxy]-phenethyl]imidazole MK-0571 MK-7512 Monur Musk ambrette Musk xilene A^-esane A^-nitrosodimethylamine A^-phenyl-p-phenylenediamine Nafenopin Naphthalene Nicotinic acid A^,7V-diethylnicotinamide Octachlorostyrene

(Continued.)

References 150] 140] 159] 160] 153] 150] 150] 139] 139] 161] 149] 144] 162] 163] 144] 139] 144] 139] 135] 139] 164] 150] 139] 150] 150] 139] 150] 144] 139] 165] 139] 139] 144] 136] 143] 143] 139] 166] 135] 135] 140] 139] 150] 144]

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Table 3. Continued. Xenobiotics 185. 5-Methoxy-2-[[(4-methoxy-3,5-dimethyl-2-pyridinil)methyl]sulfinyl]-lH. benzimidazole 186. 1,1 -Dichloro-2,2-bis-(p-chlorophenyl)ethane 187. Pantoprazole 188. rra«^(-)-3-[l,3-Benzodioxol-5-yloxy)methyl]-4-(4-fluorophenyl)piperidine 189. Pentamethylbenzene 190. 5-Ethyl-5-(l-methylbutyl)barbituric acid 191. Perchloride ethylene 192. Perfluoroacetic acid 193. Perfluorobutyric acid 194. Perfluorodecanoic acid 195. Perfluorooctanoic acid 196. A^-(4-ethoxyphenyl)acetamide 197. Phenantrene 198. 5-Ethyl-5-phenylbarbituric acid 199. Phenothiazine 200. 4-Butyl-1,2-diphenyl-3,5-pyrazolidinedione 201. 5,5-Diphenyl-2,4-imidazolinedione 202. Pentamethylbenzene 203. Propanol 204. 3-Hydroxypregn-5-en-20-one 205. Pyrazinecarboxamide 206. Pyrazole 207. Pyrene 208. Pyridine 209. RG-7512 210. Rifampicin 211. 5-(2-Propenyl)-1,3-benzodioxole 212. 5-Allyl-5-(l-methylbutyl)barbituric acid 213. SKF-525A 214. 7-(Acetylthio)-17-hydroxy-3-oxo-pregn-4-ene-21 -carboxylic acid y-lactone 215. 1,2-Diphenyl-4-[2-(phenylsulfinyl)ethyl]-3,5-pyrazolidinedione 216. 9-Amino-l,2,3,4-tetrahydroacridine 217. 5-Ethyl-5-(l-methylbutyl)-2-thiobarbituric acid 218. Toluidine 219. Toxaphene 220. trans-Reiinoic acid 221. Tridiphane 222. Trichloroacetic acid 223. Trichloro ethylene 224. Oleandomycin triacetate ester 225. 5-[(3,4-Dimethoxyphenethyl)methylamino]-2-(3,4dimethoxyphyl)-2isopropylvaleronitrile 226. 5-Ethyl-5-{1-methyl-l-buthenyl)barbituric acid 227. Vinyl chloride

]References 167] 135] 168] 159] .144] 139] .139] '169] .169] ;i69] .169] :i70] 140] 171] :i39] .150] ;i39] .144] [144] 139] [172] 139] [140] 139] .173] 175] 140] [139] 174] [150] 150] 175] 139] 140] 135] 176] 177] 139] 144] 139] 178] [139] 139]


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Table 4. Cytochrome P450 inducers. Medical drugs 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. (Continued.)

Acetylsalicylic acid Allobarbital Amitryptilyn Amobarbital Antipyrine Ascorbic acid Barbital Benoxaprofen Butobarbital Butabarbital Carbamazepine Carbromal Chlorimipramina Chlormetazole Chlorpromazine Cimetidine Ciprofibrate Clofibrate Clonazepam Clotrimazole Cyclobarbital Dehydroepiandrosterone Dexamethasone Doxylamine Eldrin Ethanol Erythromicin Fluoxetine Fluvoxamine Glutethimide Griseofulvin Heptabarbital Hexobarbital Ibuprofen Imidazole Imipramine Interferon Isoniazid Ketoconazole Lansoprazole Medroxyprogesterone Mephobarbital Meprobamate Miconazole Nikethamide Omeprazole Pantoprazole

References [139] [139] [149] [139] [150] [150] [139] [140] [139] [139] [150] [136] [150] [156] [139] [140] [157] [153] [147] [147] [139] [139] [158] [177] [135] [139] [139] [159] [160] [150] [150] [139] [139] [161] [148] [149] [162] [139] [139] [164] [150] [139] [150] [139] [150] [167] [168]

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Table 4. Continued. Medical drugs 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65.

References Paroxetina Pentobarbital Phenacetina Phenobarbital Phenothiazine Phenylbutazone Phenytoin Pregnenolone 16a-carbonitrile Pyrazinamide Rifampicin Secobarbital Spironolactone Sulfinpyrazone Tacrine Thiopental Troleandomycin Verapamil Vinbarbital

[159] [139] [170] [139] [139] [150] [139] [139] [172] [139] [139] [150] [137] [175] [139] [139] [178] [139]

compounds in the diet and the environment [179]. From a more basic standpoint, however, along with the alteration of endogenous metabolism (where these catalysts are physiologically involved) coadministered drugs, and the metabohsm of xenobiotics, cytochrome P450 induction determines a strong increase of O2 ~ yield. This is particularly evident with drugs such as barbiturates (CYP2B1), ethanol (CYP2E1) and pregnenolone 16-a carbonitrile (cytochrome P450 3A) which are able to generate more severe oxidative stress [130—134]. This unremitting free radical production, imposed upon the cell by medical and nonmedical drugs, may perturb cellular defence mechanisms and thus, trigger cytotoxicity. The induction phenomenon is even more complex since the overexpression of cytochrome P450 lAl, for example, which metabolizes polycycHc aromatic hydrocarbons, exibits a genetic polymorphism. Not surprisingly, in the presence of 100% oxygen, mice that are genetically unresponsive to cytochrome P450 induction survive significantly longer than responsive mice [180]. Again, the increase of certain cytochrome P450 isoforms such as cytochrome P450 lAl and cytochrome P450 2B1 is associated to a pleiotropic response which involves the induction of metabolizing enzymes belonging to both phase-I and phase-II systems together with the induction of noncorrelated enzymes [134]. Under these conditions, the production of O2 ~ from different cytochrome P450s can leads to a greater level of oxidative stress as compared to the one produced by the induction of single isoforms.

Chapter 35: Oxidative stress induced by the metabolism of drugs 6

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INDUCERS OF P E R O X I S O M E PROLIFERATION

Peroxisomes are organelles widely distributed in most animal and plant cells with many vital cellular functions, such as the synthesis and the metabolism of complex biomolecules [180—185]. They contain over 40 enzymes (e.g., D-amino acid oxidase, a-hydroxyacid oxidase, polyamine oxydase and fatty acyl-CoA oxidase) including some that produce H2O2 (e.g., FAD-containing monooxygenases) and others that degrade H2O2 (e.g., CTS, GPX and SOD). A variety of structurally dissimilar xenobiotics including hypohpidemic drugs (such as dofibrate, ciprofibrate and nafenopin), phthalate ester plasticizers, trichloroethyl erbicides, and dietary factors and hormones have been reported to cause prohferation of peroxisomes [185—189]. The phenomenon of peroxisome proliferation has attracted considerable attention during the past decade due to the discovery that peroxisome proliferators have a role in the multistep process of carcinogenesis [188,189]. During an adaptive phase these compounds produce hepatomegaly and induction of peroxisomal and endoplasmic reticulum enzymes [190—194]. Chronic administration of proliferators results in the development of hepatocellular carcinomas by nonmutagenic mechanisms [194—196]. Peroxisome proliferators, which are also typical inducers of cytochrome P450 isoforms, in particular the one of the cytochrome P450 4A family, are included in Tables 3 and 4. Under physiological conditions, peroxisomes are estimated to consume between 10 and 35% of the total oxygen utilized by liver, but since they are 10 times less abundant than mitochondria, individual peroxisomes must consume a significant amount of oxygen as compared to mitochondria [197,198]. The oxygen consumed is then converted into H2O2 and possibly to O2 , as suggested by the presence of O2 - and H202-producing and -degrading enzymes in peroxisomes [199—201]. Hydrogen peroxide is then degraded by catalases, but it is estimated that 2% of H2O2 may diffuse out into cytosol [202]. However, because of the fact that ciprofibrate-treated liver, for example, has a 25- to 30-fold increase in H2O2 producing oxidases, a high proportion of H2O2 is expected to escape from peroxisomes into cytoplasm [202]. This excessive release of H2O2 from peroxisomes due to a disproportionate induction of the H2O2producing, FAD-containing oxidase may be a crucial factor in peroxisomalproliferator-induced oxidative stress along with the simultaneous reduction in CTS activity in peroxisomes and an increased production of O2 , derived by the simultaneous cytochrome P450 induction [194]. This scenario is particularly important since it may lead to a situation of excessive H2O2 and O2 escaping from organelles into the cytoplasm, which in turn could gain access to other intracellular compartments (e.g., nucleus), because of the parallel loss of cytosolic GPX and CuZn SOD activities [203], which could be responsible for the production of highly reactive HO derivatives and alteration in cellular proteins, enzymes and DNA [186—188]. Administration of clofibrate and other peroxisome prohferators also results in the induction of cell proliferation and in variable increases

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of a number of enzymes (pleiotropic response), and this provides support for the hypothesis that by generating active forms of oxygen the induction of the peroxisomal fatty acid |3-oxidation system may play a key role in liver tumorigenesis [204]. 7

SUMMARY

Although reactive oxygen species (ROS) have a prominent role in immune system defenses against invading microorganisms and in activating cytoplasmic signal transduction pathways that are related to differentiation, proliferation, cytoskeletal organization and cell death, they have been conventionally regarded as having pathological potential in a number of processes including aging, inflammation, cataract formation, arthritis, heart desease and cancer. ROS are continuously produced in living cells as by-products of endogenous metabolism during metabolism of xenobiotics, and by radiation. However, because antioxidant defences are not completely efficient, increased ROS formation in the body (oxidative stress) is likely to induce damage. One way of imposing oxidative stress is by the action of certain drugs, namely those that produce free radicals or deplete antioxidant defences. Thus, there is mounting interest in the possibility that the side-effects of several drugs involve increased oxidative damage. 8

PERSPECTIVES

A great deal of medical and nonmedical drugs are able to impose an oxidative stress and many pathologic states are known to involve the overgeneration of ROS. To date, however, it is not known to what extent these phenomena are due to ROS formation, or if their production is a result of the disease itself. In addition, the impressive amount of literature on this argument neither proves that ROS are involved in the induction of various illnesses, nor that drugs with antiROS activity are involved in their beneficial effects to these properties. However, there are models in which ROS generation is able to provoke illnesses, whose pathologic outcome can be attenuated by the use of anti-ROS drugs. Typical examples are the alloxan model of diabetes, the MTP-induced Parkinson's disease, experimental ischemia and inflammation. ROS formations also form part of our immune system defenses and may be involved in some signaling processes. So, it would not be a medically sound approach to block their formation indiscriminately. Of course, our requirement for antioxidant vitamines evidences that ROS production must be carefully controlled, but abnormally high intakes of these vitamines appear to be of little benefit. Enzymes of antioxidant machinery have a fundamental role for the prevention of ROS-related diseases, but the overexpression of one or more of these catalysts may have a deleterious rather than a beneficial effect. Considering that many people live in misery due to debilitating diseases such as polyarthritis, Parkinson's and Alzheimer's syndrome in which ROS generation


1047

is believed to play a leading role, appropriate site-specific anti-ROS drugs for the control of such diseases would be most welcome. 9

ACKNOWLEDGEMENTS

We thank Prof G.F. Pedulli of the Department of Organic Chemistry "A. Mangini" of the University of Bologna for critical comment and helpful advice. We also thank Drs L. Pozzetti, S. Trespidi, M.A. Antonelli for the assistance. The Authors are greatful to Drs B. Esposito, A. Camerino and Mr A. Caporali for their artwork. This work was supported by the National Research Council of Italy (CNR) and MURST (Ministery of the University and of the Scientific and Technological Research) 40 and 60% grants. 10 ABBREVIATIONS 9-COOH-DXC: 9-dehydroxyacetyl-9-carboxyl doxorubicin ACT: ascorbate ACTH: monodehydro - ascorbate aminopyrine AMP: catalase CTS: cytochrome P450 CYP: clozapine CZP: doxorubicin DXC: DXC-Fe^^: doxorubicin-iron III complex glutathione peroxidase GPX: glutathione GSH: horseradish peroxidase HRT: myeloperoxidase MEP: A^-acetyl p-quinoneimine NAPQI: nitrofurantoin NTF: PHS: prostaglandin H synthase phenylbutazone PNB: reactive oxygen species ROS: superoxide dismutase SOD: 11 REFERENCES 1. 2. 3. 4. 5. 6. 7.

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107. Eliot H, Gianni L, Meyers C Biochemistry 1984;23:928-936. 108. Meyers CE, Gianni L, Simone C et al. Biochemistry 1982;21:1707-1713. 109. Zweyer JL. Biochem Biophys Acta 1985;83:209-213. 110. Zweyer JL, Gianni L, Muindi J, Meyers CE. Biochem Biophys Acta 1986;884:324-336. 111. Gianni L,Vigano L, Lanzi et al. J Natl Cancer Inst 1988;80:1104-1111. 112. Muindi JRF, Sinha BK, Gianni L, Meyers CE FEBS Lett 1984;172:226-230. 113. Muindi JRF, Sinha BK, Gianni L, Meyers CE Molec Pharmacol 1985;27:356-365. 114. Green MD, Speyer JL, Muggia FM. Eur J Cancer Clin Oncol 1984;20:293-296. 115. Boyd EM, Bereczky GM. Br J Pharmacol 1996;26:606-614. 116. BoyerTD, Rouff SL. J Am Med Assoc 1971;218:440-441. 117. Jollow DJ et al. J Pharmacol ExpTher 1973;187:195-202. 118. Albano E, Poh G, Chiarpotto et al. Chem Biol Interact 1983;47:249-263. 119. Nelson SD,VaishnavX Kambara H, BaillieTA. Biomed Mass Spectrometry 1981;8:244-248. 120. Jollow DJ,Thorgeirsson SS, Potter WZ et al. Pharmacology 1974;12:251-256. 121. Gemborys MW, Gribble GW, Mudge GH. J Med Chem 1978;21:649-655. 122. Healey K, Calder IQYong AC et al. Xenobiotica 1978;8:40-44. 123. Holtzman JL. Drug Metab Rev 1995;27:277-297. 124. Miner DJ, Kissinger PT. Biochem Pharmacol 1979;28:3285-3290. 125. Streeter AJ, Dahlin DC, Nelson SD, BaillieTA. Chem Biol Interact 1984;48:349-366. 126. Devalia GL, Ogilvie RC, Mc Lean AM. Biochem Pharmacol 1982;31:3745-3749. 127. Sumoski W, Baquerizo H, Rabinovitch A. Diabetologia 1989;32:792-796. 128. Ruddle NH. Immun Today 1987;8:129-130. 129. Gillette JR, Brodie BB, Ladu BR. J Pharmacol ExpTher 1957;119:532-540. 130. PerssonYQTereliusY, Ingelman-Sundemberg M. Xenobiotica 1990;20:887-900. 131. Knecht KT, Bradford BU, Mason RP,Thruman RG. Molec Pharmacol 1990;38:26-30. 132. Bast A. Trends Pharmacol Sci 1996;7:266-270. 133. Ekstrom MG, Ingelman-Sundemberg M. Biochem Pharmacol 1989;38:1313-131. 134. Paolini M, Pozzetti L, Pedulli GFet al. J Invest Med Clin Res 1996;44:470-473. 135. Kitchin K, Brown J, Kulkarni AP Mutat Res 1991;266:253-272. 136. Kitchin K, Brown J, Kulkarni AP Teratogen Carcinogen Mutagen 1993;13;167-184. 137. Li SJ, Rodgers EH, Grant MH. Chem Biol Interact 1995;97:101-118. 138. Rosenberg DW, Mankowski DC. Carcinogenesis 1994;15:73-78. 139. Amdur OM, Doull J, Klaassen CD. Toxicology — the basic science of poison, 4th edn. New York: Pergamon Press, 1991. 140. David F, Lewis V. Drug Metab Rev 1997;29:589-650. 141. Douglas MH, Erin JG, Joyce LB et al. Molec Pharmacol 1981;21:753-760. 142. Martucci CP, Fishman J. Pharmacol Ther 1993;57:237-257. 143. Iwata N, Suzuki K, Minegishi K et al. Eur J Pharmacol 1993;248:243-250. 144. Okey AB. Pharmacol Ther 1990;45:241-298. 145. Robertson DG, Krause BR,Welty DFet al. Biochem Pharmacol 1995;49:799-808. 146. Whitlock JP, Denison MS. Induction of cytochrome P450 enzymes that metabolize xenobiotics. In: Ortiz di Montellano PR (ed) Cytochrome P450 — Structure, Mechanism and Biochemistry, 2nd edn. New York: Plenum Press, 1995;367-390. 147. Lubet RA, Dragnev KH, Chauhan DP et al. Biochem Pharmacol 1992;43:1067-1078. 148. Nims RW, Sinclair PR, Sinclair JFet al. Chem Res Toxicol 1993;6:188-196. 149. Hodgson AV, White TB, White JW, Strobel HW Molec Cel Biochem 1993;120:171-179. 150. Gibson GG, Progress in Drug Metabohsm, vol 11. London: Taylor and Francis, 1988. 151. Roelandt L, Dubois M,Todaro A et al. Ecotoxicol Environ Saf 1995;31:158-163. 152. Qamar R, Sikandar GK, Shahid A. Chem Biol Interact 1990;75:305-314. 153. Sabzevari, Hatcher M, O'Sullivan M et al. Xenobiotica 1990;25(4):395-403. 154. Bornheim LM, Everhart ET, Li J, Correia MA. Biochem Pharmacol 1994;48:161-171. 155. KetterTA, Flockart DA, Post RM et al. J Clin Psycopharmacol 1995;15:387-398.


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( 2000 Elsevier Science B.V. All rights reserved. I ^ ^ ^ S ^ Handbook of Oxidants and Antioxidants in Exercise. ^ H ^ C.K. Sen, L. Packer and O. Hanninen, editors.

10^'^ lUJ J


The paradoxical relationship of aerobic exercise and the oxidative theory of atherosclerosis Robin Shern-Brewer^ Nalini Santanam^ CarlaWetzstein\ Jill E.WhiteWelkley^, Larry Price^ and Sampath Parthasarathy' Departments of ^Gynecology and Obstetrics, ^Physical Education, Emory University Medical School, Atlanta, Georgia 30322, USA. Tel: +01-404-727-8604. Fax: +01404-727-8615. E-mail: [email protected]

1

2 3

4 5 6 7 8 9

1

INTRODUCTION 1.1 Benefits of exercise 1.2 Oxidative stress during exercise 1.3 Oxidation theory of CAD 1.4 Antioxidants and LDL oxidation THE PARADOX EXERCISE AND OXIDATION OF LDL 3.1 Study 1 3.1.1 Conclusion and limitations of study 1 3.2 Study 2 COMPARISON OF STUDY 1 TO STUDY 2 4.1 Definition of chronic exercise IMPLICATIONS SUMMARY ACKNOWLEDGEMENTS ABBREVIATIONS REFERENCES

INTRODUCTION

The irony of the 20th century American health picture is that while many people strive for optimal health, the majority of Americans engage in very little physical activity as part of their daily life. Currently only about 24% of all adult Americans exercise regularly, a level that has remained basically unchanged since 1985 [1]. In 1990, the US Department of Health and Human Services published a report entitled Healthy People 2000: National Health Promotion and Disease Prevention Objectives [2]. The broad national goals proposed by this document are to increase the span of healthy life for all Americans, to reduce health disparities among Americans, and to secure access to preventive health services for all Americans. A major health promotion objective from this document is to increase the physical activity level of Americans. There is little doubt in the scientific community that regular aerobic exercise imparts important benefits. How-


1054

ever, a degree of caution may be warranted with regard to specific health claims made by individuals or groups of people that are publicized largely via lay magazines, health clubs, heaUh food stores, and/or health products concerning aerobic exercise, its benefits and its consequences. A case in point is the oxidative stress associated with aerobic exercise and the need for additional antioxidants. It is almost impossible to read through a medical journal, or even the newspaper, without encountering an article that deals with oxidative stress or antioxidant involvement in a disease process. It is imperative to develop a better understanding of the delicate balance between the pro-oxidant and antioxidant factors during aerobic exercise before nutrient supplementation recommendations can be made. 1.1 Benefits of exercise Scientific research suggests that programmed, regular exercise have several important health benefits. In addition to enhancing physical fitness, exercise appears to reduce the risk of dying from heart disease, reduce the risks of developing diabetes and developing high blood pressure, help in the control of body weight, and promote psychological wellbeing [3—7]. Table 1 lists several potential benefits of exercise.

Table 1. Benefits of regular exercise and physical fitness. Physical Benefits 1. Increased life expectancy 2. Decreased risk of developing and dying from cardiovascular disease and stroke 3. Decreased risk of developing and dying from colon and rectal cancers 4. Decreased risk of adult-onset diabetes 5. Decreased risk of bone fractures from osteoporosis improved cardiac function 6. Control of blood pressure levels 7. Improved blood fat concentrations 8. Improved regulation of blood clotting 9. Improved ability to deliver oxygen to tissues

Mental Health Benefits 1. Tension relief and resistance to mental fatigue 2. Reduced symptoms of stress 3. Improved sleeping habits 4. Increased energy level

10. Increased protection against physiological effects of stress 11. Quicker recovery from illness and injury 12. Increased resistance to fatigue 13. Improved posture and body mechanics 14. Strengthened tendons, bones, and muscles 15. Increased lean body mass 16. Decreased body fat 17. Decreased risk of injury 18. Reduced risk for low-back pain 19. Improve joint health 20. Improved performance in sport, work, and recreational activities

5. Increased positive interaction with others 6. Improved appearance 7. Improved self-image 8. Improved quality of life

Source: A report of the Surgeon General: physical activity and health. US Department of Health and Human Services, Centers for Disease Control and Prevention, 1996.

Chapter 36: Aerobic exercise and the oxidative theory of atherosclerosis

1055

1.2 Oxidative stress during exercise Conversely, strenuous exercise involves the consumption and utilization of oxygen, and cellular oxidative processes are enhanced. Free radicals are generated and if left unquenched can have deleterious effects on lipids, membranes, and other cellular macromolecules [8—10]. Damage can occur via oxidative reactions of polyunsaturated fatty acids in cellular membranes, nucleotides in DNA, and critical sulfhydryl bonds in proteins. Exercise has been shown to increase oxidative stress indices including lipid peroxidation, oxidative DNA damage, and intrinsic antioxidant defense systems as shown in Table 2. Antioxidants such as vitamin E and ascorbic acid are commonly thought to protect against oxidative damage by reacting with these free radicals [11]. As a result, they may be depleted in oxidative stress. 1.3 Oxidation theory of coronary artery disease (CAD) It has been suggested that the oxidation of low density lipoprotein (LDL) is a key step in the pathogenesis of Coronary artery disease (CAD) [12—14]. Evidence for the in vivo occurrence of lipoprotein oxidation includes: 1) immuno cytochemical demonstration of epitopes formed during the oxidation of lipoproteins (malondialdehyde-MDA-lysine) in atherosclerotic lesions [15], 2) LDL extracted from lesions has physical and biological properties of oxidized LDL [16], 3) circulating antibodies against oxidized LDL are present in plasma of cardiac patients [17], 4) immune complexes between these autoantibodies and oxidized LDL are present in lesions [18], and 5) animal and human studies demonstrating the beneficial effects of antioxidants on the development of atherosclerotic lesions [19,20]. This oxidized LDL exhibits a number of potent pro-atherogenic properties, shown in Table 3. 1.4 Antioxidants and LDL oxidation Lipophilic antioxidants, such as vitamin E, are carried in the lipoprotein and, it has been proposed that they may protect LDL against this oxidative modification [21—23]. When antioxidants are depleted from LDL, the LDL is more readily oxidized. Recent animal studies have shown that animals given antioxidants are protected against atherosclerosis despite very high levels of plasma cholesterol [24—26]. Human studies, although not as extensive as animal studies, are encouraging [27,28].


1056 Table 2. Selected studies of exercise and oxidative indices. Exercise

Sample

Assay

[78] [79] [80] [81] [82] [78] [80] [81] [83] [84] [85] [86] [86] [87] [88] [89] [90] [91] [92] [93] [80] [94] [83] [95]

Running Running Running Running Running Running Running Running Swimming Running Running Ergometer Ergometer Running Ergometer Running Running Running Running Walking Running Electrically stimulated Running Running

Rat muscle Rat muscle Rat muscle Rat muscle Rat muscle Rat liver Rat liver Rat liver Rat heart Rat urine Mouse muscle Human plasma Human plasma Human plasma Human serum Human serum Human serum Human serum Human urine Human synovial fluid Rat hind limb Rat muscle Rat heart Human urine

[87] [96]

Running Running

Human plasma Human plasma Rat plasma Rat heart Rat liver Rat liver Rat liver Rat skeletal muscle Rat skeletal muscle Rat skeletal muscle Rat skeletal muscle Rat skeletal muscle Rat skeletal muscle Rat heart Rat platelet Rat serum Rat heart Rat skeletal muscle Rat skeletal muscle Human plasma Rat plasma Human plasma

MDAMDAt MDAT MDAt MDAt MDAt MDAt MDAt MDAt MDAt MDAMDAt MDACDMDAMDAt MDAt MDAt MDAt MDAEPR signals t EPR signals t EPR signals t 8 -Hydroxydeoxy Guanosine t GSH i GSSGGSSGt GSSGt SOD activity t SOD activity t SOD activity t SOD activity t SOD activity t SOD activity t GPX activity GPX activity \ GPX activity \ GPX activity t GPX activity t GPX activity t Vitamin E \ Vitamin E \ Vitamin E J, Vitamin E \ Vitamin E t Vitamin E \ Vitamin E —

Reference

[97] [81] [98] [78] [98] [99] [100] [99] [82] [101] [102] [103] [83] [83] [104] [105] [106] [107] [87] (Continued.)

Running Running Running Running Swimming Running Swimming Running Running Running Running Running Running Running Running Running Running Running Running

Chapter 36: Aerobic exercise and the oxidative theory of atherosclerosis

1057

Table 2. Continued. Reference

Exercise

Sample

Assay

[108] [109] [110] [111]

Running Running Cycle ergometer Downhill Running

Human expired air Rat expired air Human plasma Human plasma

Pentane j Hydrocarbons | MPO protein ] MPO protein j

| : increase; j : decrease; —: no change; MDA: malondialdehyde; CD: conjugated diene; EPR: electron paramagnetic resonance spectroscopy; GSH: reduced glutathione; GSSH: oxidized glutathione; SOD: superoxide dismutase; GPX: glutathione peroxidase.

2

THE PARADOX

There is an apparent paradox between the beneficial effects of exercise on decreasing the risk for vascular disease and the potentially damaging consequences of exercise on vascular injury, i.e., promoting free radicals that may be generated in the course of physical exertion. The use of antioxidant supplements among exercisers to prevent free radical-induced injury appears to be widely promoted; yet our current understanding of the effect of exercise on oxidative processes provides little justification for their use. The oxidation of LDL is known to be involved in atherosclerosis. Current medical science considers sustained physical activity and exercise to be a deterrent to cardiovascular ailments. Yet, exercise represents a severe form of oxidative stress. Understanding this paradox will increase our understanding of when and how oxidation may have pro- and anti-atherogenic effects and how nutrition may be tailored to the oxidative demands of the exercising population.

Table 3. Atherogenic properties of oxidized LDL. Reference

Atherogenic properties of oxidized low density lipoprotein.

[112--114] [113,115] [116--118] [119--122] [123--125] [126--128]

Chemotactic for human monocytes, T-lymphocytes and smooth muscle cells Inhibits macrophage chemotaxis Avidly degraded by macrophages and generates foam cells Inhibits endothelium-dependent relaxation of aorta Cytotoxic to cells and causes endothehal injury Modified LDL increases adhesive properties of endothelial cells; Ox-LDL induces VCAM-1 gene expression in endothehal cells Causes DNA fragmentation and apoptosis of lymphoblastoid cells and smooth muscle cells Stimulates collagen production Enhances platelet aggregation Is immunogenic and can elicit autoantibody formation

[129--131] [132] [133,134] [135,136]


1058 3

EXERCISE AND OXIDATION OF LDL

Few have investigated the potential diflferences in susceptibility of LDL to oxidative modifications isolated from chronic exercisers and sedentary subjects. In order to address this paradox, we performed two studies. 3.1 Study 1 In the first study, we compared the oxidizability of LDL from short-term "chronic" exercisers (aerobically active more than 6 h/week) with the LDL from sedentary subjects [29]. The exercisers and sedentary subjects were significantly different with regard to fitness and lipid profile parameters. The sedentary subjects had lower aerobic capacity, higher body fat composition and a more atherogenic profile than the exercisers. These findings are in agreement with many studies reporting the benefits of aerobic activity [3—7]. The lag time of isolated LDL subjected to in vitro 2.5 jiM copper oxidation was significantly shortened in the exercisers as compared to the sedentary subjects (Fig. 1). This increased sensitivity was not due to the decreased presence of vitamin E since the amount of plasma and LDL vitamin E was not different between the two groups. Instead, these findings suggest that the LDL of exercisers contain increased amounts of preformed hpid peroxides, which could account for the increased susceptibility to oxidation. It is possible that plasma lipoproteins contain trace amounts of lipid peroxides [30—32]. Lipid peroxides could be formed by endogenous lipid peroxidation reactions and transferred to LDL. SanchezQuesada [33] reported an increase in minimally oxidized LDL following a prolonged exercise bout. The authors attributed this to acute oxidative stress concomitant with exercise.

p < 0.01

120-1 Study 1 100-

100

p < 0.01

97

93

•

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