Pi is 0002934300004125

The Evolution of Free Radicals andOxidative Stress

Joe M. McCord, PhD

The superoxide free radical has come to occupy an amazinglycentral role in a wide variety of diseases. Our metabolic focus onaerobic energy metabolism in all cell types, coupled with somechemical peculiarities of the oxygen molecule itself, contributeto the phenomenon. Superoxide is not, as we once thought, justa toxic but unavoidable byproduct of oxygen metabolism.

Rather it appears to be a carefully regulated metabolite capableof signaling and communicating important information to thecell’s genetic machinery. Redox regulation of gene expressionby superoxide and other related oxidants and antioxidants isbeginning to unfold as a vital mechanism in health and disease.Am J Med. 2000;108:652– 659. q2000 by Excerpta Medica, Inc.

Free radicals, oxidative stress, and antioxidants havebecome commonly used terms in modern discus-sions of disease mechanisms. The purpose of this

article is to provide some understanding as to why freeradical metabolism seems to occupy a central and re-markably common position in the mechanisms of somany seemingly unrelated types of human disease. In ar-ticles to follow in this series, experts in a broad spectrumof clinical specialties will examine more specifically thefree radical mechanisms known to be operative in theinflammatory process and in ischemia/reperfusion in-jury, as they affect a variety of organ systems. In addition,there are some free radical mechanisms that are unique tocertain tissue locations, especially those affecting the cen-tral nervous system.

In a general sense, substantial insight regarding freeradical production can come from the recognition of afew basic facts about the nature of the chemical reactionsthat support life as we know it, and from an understand-ing of the quirky structure of molecular oxygen, uponwhich our metabolism is based. We can gain further in-sight as we consider the course of our evolution in a redoxactive milieu. As evolutionary pressures tend to make thebest of any bad situation, we will see examples of evolu-tionary steps to contain, control, or avoid unpleasanttoxic species, and other examples of how these reactivespecies can be turned to constructive purposes contribut-ing to our defense, or even to our internal regulation andcommunication processes.

A REDOX PRIMER

At the risk of inducing unpleasant flashbacks to collegechemistry courses, let us consider the class of chemicalreactions upon which our bodies rely for energy produc-

tion. Reduction-oxidation (or redox) reactions are at thecore of our metabolic machinery. Redox reactions in-volve the transfer of electrons or hydrogen atoms fromone reactant to another. (The process of taking away elec-trons is called “oxidation,” because oxygen does it so well.The substance receiving electrons becomes “reduced.”)Life on our planet has evolved into two main categories oflifeforms: 1) photosynthesizing plants that capture solarenergy and use it to drive thermodynamically unfavor-able reactions producing reduced carbonaceous com-pounds, and 2) the rest of us, who eat these reduced car-bonaceous compounds and “burn” them in thermody-namically favorable reactions. The latter reactions allowthe electrons (or hydrogen atoms) to return to the mole-cule that wants them most, molecular oxygen, with therelease of large amounts of energy that we can cleverlytrap and use to power our lives. This process might bestraightforward but for the quirky electronic structure ofthe oxygen molecule. Oxygen itself is a diradical. (Chem-ically, a free radical is any molecule containing a single,unpaired electron. Usually, paramagnetic transitionmetal ions are not considered to be free radicals, althoughby technical definition they are.) Most molecules containonly pairs of electrons with opposite spins that reside indiscrete molecular orbitals and that may or may not par-ticipate in bond formation. Oxygen contains two elec-trons that are not spin paired, and each resides in an or-bital of its own. Thermodynamically, oxygen wants totake on additional electrons (two per atom, or four permolecule) to produce water molecules, which have muchlower free energy. The unconventional distribution ofelectrons in the oxygen molecule, however, makes it im-possible for oxygen to accept a spin-matched pair of elec-trons, as badly as it may want them, until one of its un-paired electrons undergoes a spontaneous spin reversal tomake pairing possible. At ordinary collisional frequency,the period of contact is too brief for this spin reversal tooccur, imposing a kinetic barrier (ie, a large energy ofactivation) to most oxidative reactions. It is this kineticbarrier that saves us from reacting explosively with anatmosphere of huge thermodynamic oxidizing potential.

From Webb-Waring Institute, University of Colorado Health SciencesCenter, Denver, Colorado.

Correspondence should be addressed to Joe M. McCord, PhD, BoxC-321, University of Colorado, 4200 East Ninth Avenue, Denver, Col-orado 80262.

652 q2000 by Excerpta Medica, Inc. 0002-9343/00/$–see front matterAll rights reserved. PII S0002-9343(00)00412-5

It is this same kinetic restriction that makes oxygen anideal terminal electron acceptor for biological systems.Enzymes have binding sites that can hold oxygen in con-tact with an oxidizable substrate for a much longer timethan would occur by simple collision, overcoming thekinetic barrier to reaction. At the same time, enzymesmay be designed to trap much of the energy released asthe oxidation occurs in a useful high-energy compound,such as ATP.

OXYGEN-DERIVED FREE RADICALS ANDOXIDANTS

Our relationship with oxygen is, at best, a difficult one tomanage. Occasionally, under normal biological condi-tions, oxygen does manage to steal away electrons fromother molecules by nonenzymatic autoxidations. Becauseit cannot accommodate a spin-matched pair, it must set-tle for stealing electrons one at a time. This breaking up ofelectron pairs results in free radical formation. The one-electron reduction product of oxygen is the superoxideradical, O2

•2. If two electrons are transferred, the productis hydrogen peroxide, H2O2, which is not a radical. It isnonetheless still eagerly receptive of two more electrons,causing hydrogen peroxide to be a cytotoxic oxidant.Certain chelates of ferrous iron and cuprous copper arecapable of transferring a third electron to hydrogen per-oxide, causing lysis of the O-O bond. One fragment isreduced to the state of water; the other fragment is thehydroxyl free radical, HO•, one of the most potent oxi-dants known. It can initiate lipid peroxidation, causeDNA strand breaks, and indiscriminately oxidize virtu-ally any organic molecule. The fact that it is so indiscrim-inate actually works in our favor, as most of its targets areexpendable. Reactivity and toxicity are not synonymous(1). Despite the much lesser reactivity of superoxide, itstoxicity appears to be substantial precisely because its tar-gets are focused. Cyanide, for example, is not particularlyreactive but is extremely toxic, because it effectivelystrikes one crucial target, cytochrome oxidase.

The family of reactive intermediates resulting from theincomplete reduction of oxygen therefore includes su-peroxide radical, hydrogen peroxide, and hydroxyl radi-cal. It is not correct to refer to this group as oxygen-derivedfree radicals, because one member, hydrogen peroxide, isnot a radical. Accordingly, several terms are now in use torefer to this family. Reactive oxygen metabolites (ROM)or active oxygen (AO) or variations thereon are the mostcommon. Occasionally, the terms are expanded to in-clude electronically excited oxygen (singlet oxygen), hy-pochlorous acid (produced from oxygen by the neutro-phil enzymes NADPH oxidase and myeloperoxidase),peroxynitrite, and even nitric oxide, as all of these oxi-dants are derived from molecular oxygen.

THE EVOLUTION OF THE AEROBICLIFESTYLE

Life on this planet first evolved in a reducing atmosphere.It was not until photosynthetic algae appeared that oxy-gen began to be introduced into the atmosphere in everincreasing quantities. This shift from a reducing environ-ment to an oxidizing one undoubtedly resulted in someserious evolutionary pressures. One might be surprisedwhen examining modern metabolic pathways to find thatvery few enzymes actually deal with molecular oxygen,despite the fact that our bioenergetics scheme is com-pletely dependent on the transfer of electrons to this ac-ceptor. In fact, approximately 98% of the oxygen we me-tabolize is handled by a single enzyme, the cytochromeoxidase in our mitochondria, which transfers four elec-trons to oxygen in a concerted reaction to produce twomolecules of water as the product. The enzyme is struc-turally quite complex, containing four redox centers (twohemes and two copper ions), each of which can store asingle electron. When all centers are reduced, the simul-taneous transfer of four electrons to an oxygen moleculeoccurs with no detectable intermediate steps. One prob-able reason for this dominance by cytochrome oxidase inthe reduction of oxygen is the chemical difficulty of car-rying out the reaction in a safe and controlled manner. Asdiscussed above, the reduction of oxygen by anything lessthan the full complement of four electrons results in theproduction of active oxygen species. Surely, as primitivemetabolic systems were struggling with the shift towardenergy-rich oxidative pathways, there must have beenmany contenders that lost out in the end, because unac-ceptable levels of active oxygen products were produced.Hence, one evolutionary strategy for survival in an oxi-dative environment is to restrict opportunities for poorlycontrolled transfer of electrons to oxygen. Althoughmany enzymes involved in redox pathways exist as re-duced intermediates with great thermodynamic potentialfor the transfer of their electrons to oxygen, the evolu-tionary pressure has led to kinetic barriers against suchreactivity. Instead, most of these high-energy electronsare transferred to NADP1 to produce NADPH, which isitself kinetically resistant to reaction with oxygen. Thus,our electron-conducting metabolic circuits are insulatedby evolutionary design to prevent the inadvertent devel-opment of short-circuits, much as the electron-conduct-ing circuits in a modern electronic circuit board are insu-lated to keep electrons flowing in the proper channels.Our insulation is not perfect. At least two sites have beenidentified in the electron-transport chain (Complex I andubisemiquinone) where electrons may leak out throughbreaks in the insulation to waiting oxygen molecules, re-sulting in the formation of superoxide (2). These sites arediagrammatically represented in Figure 1 as the primarysources of intracellular superoxide generation. It has been

The Evolution of Free Radicals and Oxidative Stress/McCord

June 1, 2000 THE AMERICAN JOURNAL OF MEDICINEt Volume 108 653

estimated that this leakage amounts to 1% to 2% of totalelectron flux through the mitochondria. Because workingmyocardium consumes oxygen at approximately 8 mMper minute, rates of superoxide production could exceed0.1 mM per minute. So, as well designed as we may be,mitochondria are still the major source of accidental freeradical production. During exposure to hyperoxia, therates of leakage from these sites in lung mitochondria arebelieved to increase in direct proportion to the increasedoxygen tension. Healthy adult rats will die within 72hours if placed in an atmosphere of 100% oxygen, onlyfive times the normal concentration at sea level (3).

THE EVOLUTION OF ANTIOXIDANTSAND ANTIOXIDANT ENZYMES

In addition to evolutionary attempts to avoid the produc-tion of reactive byproducts of oxidative metabolism, an-other very important direction was the ability to synthe-size or accumulate antioxidants—molecules that wouldavidly react with and annihilate active oxygen species be-fore they could inflict oxidative damage to vital compo-nents, such as DNA or cell membranes. The result washundreds of kinds of such antioxidant molecules, espe-cially in plants. Among the most successful of these mol-ecules are the water-soluble antioxidant ascorbic acid (vi-tamin C) and the lipid-soluble antioxidant a-tocopherol(vitamin E) (4).

The most efficient way to eliminate undesirable toxic

species, of course, is by means of catalysis. Families ofantioxidant enzymes have evolved for this purpose, in-cluding superoxide dismutases for the elimination of thesuperoxide radical, and catalases and glutathione peroxi-dases for the elimination of hydrogen peroxide and or-ganic peroxides. Humans have three genes encoding su-peroxide dismutases (SOD), which localize in the mito-chondria, the cytosol, or the extracellular spaces (5).These genes are derived from two ancestral genes. Onegene gave rise to the copper-and-zinc– containing en-zymes; the other gave rise to the manganese- or iron-containing enzymes. The genes can be traced back to themost primitive organisms with high degrees of homologyaround the active sites, indicating that the genes thatevolved early as life forms were figuring out how to sur-vive and thrive in the presence of oxygen. The SODs (6)catalyze the reaction:

O2•2 1 O2

•2 1 2H13H2O2 1 O2

This dismutation or disproportionation reaction makesuse of the fact that superoxide is both an oxidant and areductant, eager to get rid of its extra electron or to takeon another. The enzyme uses one superoxide radical tooxidize another. Catalases work in much the same way,because hydrogen peroxide can be a weak reductant aswell as a fairly strong oxidant:

H2O2 1 H2O23 2H2O 1 O2

In higher organisms, glutathione peroxidases appear tohave largely supplanted the need for catalase. These en-

Figure 1. A schematic representation of signal transduction pathways for superoxide radical. Mitochondrial respiration accounts formost of the superoxide generated in a cell with leakage sites at Complex I and at ubisemiquinone. The steady-state concentration ofsuperoxide is kept low in all compartments by SODs, not shown. The low levels of the radical remaining may modulate variouskinases, or may activate transcription factors directly to effect gene regulation in the nucleus. It is interesting to speculate on theexistence of a cell surface receptor for superoxide (R), which might transduce various responses within the cell by means of kinaseactivation, for example. There is presently no direct evidence for such a receptor.


654 June 1, 2000 THE AMERICAN JOURNAL OF MEDICINEt Volume 108

zymes use NADPH as the reducing species for hydrogenperoxide:

NADPH 1 H1 1 H2O23 2H2O 1 NADP1

They can reduce lipid peroxides as well as hydrogen per-oxide and are very important enzymes in the preventionof lipid peroxidation to maintain the structure and func-tion of biologic membranes.

THE REACTIVITY AND TOXICITY OFSUPEROXIDE RADICAL

Although the chemical reactivity of the superoxide radi-cal is modest, its toxicity is quite easily demonstrated.Escherichia coli contains three genes for SODs: one en-zyme uses manganese as its cofactor, one uses iron, oneuses copper and zinc. Disruption of the two major genesencoding the manganese and iron enzymes results in abacterium unable to grow aerobically in minimal me-dium but still able to grow anaerobically (7). Aerobically,it displays multiple auxotrophies and can grow if allamino acids are added to the minimal medium, indicat-ing that several biosynthetic pathways for amino acids aresensitive to inactivation by the radical. Indeed, certaindehydratases in E. coli have subsequently been shown tobe sensitive to inactivation by superoxide radical: the a,b-dihydroxyisovalerate dehydratase (8) and 6-phospho-gluconate dehydratase (9). More than a dozen other im-portant enzymes have similarly been shown to be inacti-vated by superoxide, including the following mammalianenzymes: catalase (10), glyceraldehyde-3-phosphate de-hydrogenase (11), ornithine decarboxylase (12), glutathi-one peroxidase (13), myofibrillar ATPase (14), adenylatecyclase (15), creatine phosphokinase (16), and glutaminesynthase (17).

The toxicity of superoxide is seen not only in its abilityto inhibit certain enzymes and thereby attenuate vitalmetabolic pathways, but also in its effects on other majorclasses of biological molecules. E. coli deficient in SODactivity show increased rates of mutagenesis (18), illus-trating the role of the radical, directly or indirectly, inDNA damage. In conditions of ischemia and reperfusion,the most acute problem resulting from the overproduc-tion of superoxide appears to be greatly increased rates oflipid peroxidation. Here, superoxide radical plays para-doxical roles, in that it can both initiate and terminatelipid peroxidation chains (19). This dichotomy of goodand bad will be explored further below. The practical re-sult, however, is that a proper balance between oxidantsand antioxidants is required. Superoxide radical is not allbad, as we shall see in other ways.

Knockout mice have now been produced for each ofthe three mammalian SOD genes separately, but not yetin combination. Surprisingly, SOD1 knockouts, missing

the cytosolic copper-zinc SOD, get along quite well untilthey are stressed. They do show increased neurologic andhistologic damage after focal cerebral ischema and reper-fusion (20) and increased motor neuron death after ax-onal injury (21). Similarly, SOD3 knockouts missing theextracellular SOD do well until stressed. They show in-creased pulmonary damage after exposure to hyperoxia(22). It is the homozygous SOD2 knockouts, however,that dramatically illustrate how toxic superoxide can be.SOD2 encodes the manganese-containing SOD that lo-calizes to the mitochondrion, the site that is responsibleby far for most of the cellular production of superoxide.SOD2 knockouts are born small, but alive, but they diewithin days of birth with a dilated cardiomyopathy (23).

IF LIFE GIVES YOU LEMONS, MAKELEMONADE

One of the most fascinating aspects of evolution is theability to make the best of a bad situation, to make a silkpurse from the proverbial sow’s ear. There are clear ex-amples of how active oxygen products, which we gener-ally try to avoid producing at all costs, can actually be putto constructive uses. The best example is the evolution ofour phagocytic NADPH oxidase. When first discoveredas a biologic metabolite, it appeared that the superoxideradical was simply a noxious cytotoxic byproduct thatserved no good purpose. That view changed when Babioret al (24) realized that the radical is an important player inour defense against invading microbes. It is now univer-sally accepted that the production of superoxide radicalby activated polymorphonuclear leukocytes and otherphagocytes is an essential component of their bactericidalarmamentarium (25). Precisely because superoxide is cy-totoxic, this NADPH oxidase has evolved to circumventthe kinetic stability of NADPH and specifically to allow itsoxidation by molecular oxygen with the production ofsuperoxide radical. It equips phagocytes (which haveevolved mechanisms to detect and engulf invading mi-croorganisms) with a way to destroy chemically the in-gested microbes. In effect, superoxide serves as an ex-tremely broad spectrum antibiotic. The neutrophil is alsodestroyed in the process, a Kamikaze mission, by its ownartillery. In addition, surrounding healthy host cells maybe injured or even killed in the crossfire (26 –28). In ef-fect, superoxide is a mediator of inflammation, and SODsdisplay anti-inflammatory activity. It should be notedthat our immune defense system tends to overreact to anychallenge, as too timid a response may be fatal. The dam-age associated with the inflammatory process is the pricewe pay for a vigilant defense system. From a medical ther-apeutic point of view, our own ingenuity may now haveevolved to a position of greater intelligence than our im-mune system, enabling us to treat ourselves with more



targeted synthetic antibiotics (that our bodies have notyet learned to produce) and allowing us to attenuate se-lectively our inflammatory response to spare ourselvesthe damage associated with it.

We now believe that the superoxide radical plays addi-tional constructive roles that may be more subtle in na-ture. When organisms evolved from single-celled crea-tures to complex, multicellular, multiorgan creatures, ahuge paradigm shift took place. For a single-celled organ-ism, the biological imperative is simply to grow and di-vide without restraint when times are good and food isplentiful. For higher organisms, only epithelial cells(which are continuously being sloughed) are in thisgrow-and-divide mode. Thus, nearly all the cells in ourbodies are under tight constraints that override the bio-logic imperative that tells our individual DNA moleculesto replicate themselves. Certain types of cells are able toescape from the restraints under certain circumstances.For example, fibroblasts are able to proliferate to formscar tissue that is necessary for wound closure and heal-ing. Lymphocytes capable of producing needed antibod-ies are able to proliferate to create a clone of such cellswhen appropriately stimulated. In both cases, it appearsthat superoxide may serve as the signal to override thepostmitotic constraints, and both cases may have evolvedas secondary responses to the oxidative nature of theprimitive immune system’s superoxide-generating ma-chinery.

The boundaries of an open wound become a battlefieldfor phagocytes versus microbes. The objectives are two-fold: to sterilize the wound, and to close the wound. TheNADPH oxidase of neutrophils becomes activated, andsuperoxide and other oxidants derived therefrom, in-cluding hydrogen peroxide and hypochlorous acid, ac-complish the first objective. It then appears that thephagocyte-generated superoxide serves to stimulate fi-broblasts to enter a proliferative mode (29), laying downcollagen fibrils and forming scar tissue to close and sealthe wound against further infection. The proliferative re-sponse of fibroblasts to exposure to superoxide is easilydemonstrated in the laboratory (30,31). Similarly, theclonal expansion of stimulated lymphocytes may bedriven largely by superoxide production. B lymphocytes,in particular, contain an NADPH oxidase closely relatedto the one found in neutrophils and macrophages. Whenthe oxidase is stimulated to produce superoxide by mito-gens, the result is cellular proliferation giving rise to aclone of antibody-producing cells.

REDOX REGULATION OF GENEEXPRESSION

If oxidative status can signal cells to respond in variousways, we must ask how these signals are transduced, car-

ried, and interpreted, especially by the cell’s genetic ma-chinery. The study of redox regulation of gene expressionhas exploded in recent years and clearly suggests that ox-idants are major determinants of gene expression. Reac-tive oxygen intermediates have been implicated in theactivation of a variety of kinases [such as the Src kinasefamily (32); protein kinase C (33); mitogen-activatedprotein kinase, MAPK (34); and receptor tyrosine kinases(35)] and transcriptional factors, such as AP-1 andNF-kB (34,36). An additional layer of complexity is of-fered by oxidant modification of redox-sensitive pro-teins, such as thioredoxin (37,38), which can regulate theactivity of certain stress kinases (39). Figure 1 depicts aschematic representation of how reactive oxygen speciesmay regulate gene expression. This extensive interface be-tween oxidants and reductants and the cell’s genetic ma-chinery results in responsiveness to exogenous oxidantexposure and to remarkably effective mechanisms gov-erning redox homeostasis under normal conditions. Theunfolding complexity of the system further suggests justhow badly things can go wrong when a cell’s redox statusis upset.

OXIDATIVE STRESS AND MALIGNANCY

Reining in a cell’s biological imperative to proliferate andplacing constraints on the natural inclination to replicateDNA and divide is no small feat. Indeed, it may requiremore sophisticated cellular engineering to squelch the de-sire to proliferate than to promote it. The connectionbetween mild oxidative stress and cellular growth maydate back to the primordial soup. When food is plentiful,metabolism is running at full speed, and there is sufficientenergy to support cell division, the rate of superoxideproduction will also be high (at least in aerobic organ-isms), producing a state of mild oxidative stress. Con-versely, when food supply nears exhaustion, the rate ofoxidant production within the cell would drop, possiblysignaling insufficient energy production to support thecell’s entry into a vulnerable period of replication.

For a normal postmitotic cell to become malignantlytransformed, several conditions may have to be met. Itmay be necessary to relieve certain evolutionary con-straints that tell the cell not to enter the cell cycle leadingto mitosis. It may be necessary to provide mild oxidativestress to serve as the driving force for proliferation. It mayeven be necessary to disable yet another set of evolution-ary constraints designed to prevent cells from runningamok by triggering apoptosis (40). This latter set of con-straints can, in fact, be triggered by oxidative stress per se(41). Although wild proliferation may be a mark of suc-cess for a bacterium, it is a very dangerous situation in acell that is part of a human being. The entire organism canbe brought down by what begins as a single errant cell that



has broken free of its evolutionary constraints. Thus, wehave evolved a failsafe system that can detect out-of-con-trol proliferation and can cause programmed self de-struction in any cell showing this behavior.

How might a “wannabe” cancer cell achieve and main-tain the condition of mild oxidative stress necessary todrive its proliferation? It has been shown that many typesof human cancer cells have reduced manganese superox-ide dismutase (MnSOD) (42). In most cases, the reducedactivity has been assumed to be the result of defectiveexpression of the gene (ie, changes in the promotor re-gion of the gene) (43). Oberley, St. Clair, and others haveobserved in numerous studies that transfection with thegene for human MnSOD can reverse the malignant phe-notype of tumor cells, suggesting that MnSOD functionsas a tumor suppressor (44 – 48). Very recently, Xu et al(49) reported finding a variant sequence containing acluster of three mutations in the promoter regions of theMnSOD genes from 5 of 14 human cancer cell lines ex-amined. All 5 cell lines were heterozygous for the variantsequence. The mutations change the binding pattern oftranscription factor AP-2 and cause a marked diminutionin the efficiency of the promoter using a luciferase re-porter assay system. Alternatively, mutations in the cod-ing region of MnSOD may adversely affect catalytic effi-ciency or the stability of the protein.

OXIDATIVE STRESS AND HUMANDISEASE

Perhaps the most noteworthy observation concerning therole of oxidative stress in human disease is the common-ality of it. Oxidative stress is now thought to make a sig-nificant contribution to all inflammatory diseases [ar-thritis (26,50), vasculitis (51), glomerulonephritis (52),lupus erythematosus (53), adult respiratory distress syn-drome (54)], ischemic diseases [heart disease (55), stroke(56), intestinal ischemia (57)], hemochromatosis (58),acquired immunodeficiency syndrome (AIDS) (59), em-physema (60), organ transplantation (61,62), gastric ul-cers (63), hypertension (64) and preeclampsia (65), neu-rologic diseases [multiple sclerosis (66), Alzheimer’s dis-ease (67), Parkinson disease (68), amyotrophic lateralsclerosis (69), muscular dystrophy (70)], alcoholism(71), smoking-related diseases (72), and many others.The reason that overproduction of free radicals is a fea-ture of such a broad spectrum of diseases derives from thefact that oxidative metabolism is a necessary part of everycell’s metabolism. If that cell is sick or injured in any waythat results in mitochondrial injury (calcium influx, leakymembranes, and so forth), then increased production ofsuperoxide is likely to result. In the series of articles tofollow, experts in many of these areas will delineate spe-

cific roles for free radicals and oxidative stress in a num-ber of the diseases mentioned above.

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