reactive oxygen species and the central nervous system

8/3/2019 Reactive Oxygen Species and the Central Nervous System

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Journal of NeurocheniistryRaven Press, Ltd., Ne w York0 992 International Society for Neurochemistry

Short ReviewReactive Oxygen Species and the Central Nervous System

Barry HalliwellDivision of Pulmonary-Critical Care Medicine, UC-Davis Medical Center, Sacramento, California, U S A .

Abstract: Radicals are species containing one o r more un-paired electrons, such as nitric oxide (NO). The oxygenradical superoxide (Oz*-)an d the nonradical hydrogen per-oxide (H z 0 2 are produced du ring normal metabolism an dperform several useful functions. Excessive production of02*-nd H2OZ an result in tissue damage, which ofteninvolves generation of highly reactive hydroxyl radical(OH) an d other oxidants in the presence of catalytic ironor copper ions. An importa nt form of antioxidant defense isthe storage and transport of iron an d copp er ions in form sthat will not catalyze formation of reactive radicals. Tissueinjury, e.g., by ischemia or trauma, can cause increasedmetal io n availability and accelerate free radical reactions.This may be especially important in the b rain because areasof this organ are rich in iron and CS F canno t bind releasediron ions. Oxidative stress on nervous tissue can produce

damag e by several interacting mech anisms, inc ludin g in-creases in intracellular free Ca 2+ an d, possibly, release ofexcitatory am ino acids. Recent suggestions tha t free radicalreactions are involved in the neurotoxicity of aluminumand in dam age to the substantia nigra in patien ts with Par-kinsons disease are reviewed. Finally, th e na tur e of antioxi-dants is discussed, it being suggested that antioxidant en-zymes and chelators of transition metal ions may be m oregenerally useful protective agents than chain-breaking an-tioxidants. Careful precautions must be used in the designof antioxidants for therapeutic use. Key Words: Radical-Superoxide-Hydrogen peroxide-Oxidative stress-An-tioxidant enzyme-Chelator-Parkinsons disease. Halli-well B. Reactive oxygen species and the central nervoussystem. J. Neziroclzem. 59, 1609-1623 (1992).

Although aerobes need oxygen (0,) or survival,O2concentrations greater than those present in normalair have long been known to cause damage (reviewedby Balentine, 1982). The signs of O2 oxicity dependon the organism under study, its age, physiologicalstate, and diet. For example, pure 0, is less toxic toadult humans than to adult rats, and less toxic to new-born rats than to adult rats. High-pressure0, causesacute CNS toxicity in humans, leading to convul-sions; this has been a problem in diving and must beconsidered when using hyperbaric oxygen therapy,e.g., in the treatment of gas gangrene, multiple sclero-sis, and in combination with radiotherapy (0, aggra-vates the damage done to cells by ionizing radiation).The acute effects of hyperbaric 0, have often beenattributed to direct inactivation of enzymes by 0,(Balentine, 1982), although the evidence for this isnot convincing. However, the slower-acting toxic ef-fects of elevated0, have often been suggested to in-

volve oxygen radicals (Gerschmann et al., 1954;Fridovich, 1978; Balentine, 1982). Such radicals mayalso be involved in the acute effects produced by high-pressure O2because administration of antioxidant en-zymes offers some protection (Turrens et al., 1984).THE NATURE OF RADICALS

Electrons within atoms and molecules occupy re-gions of space known as orbitals. Each orbital canhold a maximum of two electrons. For example, thetwo electrons that form a covalent bond occupy thesame (molecular) orbital, but have opposite spins. Ifan orbital contains only one electron, that electron issaid to be unpaired. A free radical is defined as anyspecies capable of independent existence (hence theterm free) that contains one or more unpaired elec-trons. This broad definition encompasses a widerange of species, as summarized in Table 1. Nitric

Address correspondence and reprint requests to Dr. B. Halliwellat Division of Pulmonary-Critical Care Medicine, UC-Davis Medi-cal Center, 4301 X St., Sacramento, CA 95817, U.S.A.Abbreviations used: DOPA, 3,4-dihydroxyphenylalanine; SH,reduced glutathione; M PTP , I-methyl-4-phenyl- 1,2,3,6-tetrahy-dropyridine; PBN, N-tert-butyl-a-phenylnitrone; SOD, superoxidedismutase; U74006F, tirilazad mesylate.

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1610 B. HALLIWELLTABLE 1 . Some tvpes offiee radical

Type of radical ExampleHydrogen centered Hydrogen atom , HCarb on centered Tnch lorom ethyl, CCl;Sulfur centered Glutath ione thiyl, GSOxygen centered Superoxide. 0;-

Hydroxyl, OH (often written as OH)Lipid peroxyl, lipid-0;Phenoxyl, C6H,0 (electron delocalizedinto benzzne ring)Nitric oxide, NO (often written as NO)Th e superscript dot (*) denotes the presence of one or more un-paired electrons. Carbon-centered and sulfur-centered radicalsusually react rapidly with oxygen, e.g.:

CCI; + O2+ CCI3O; (trichloro niethyl peroxyl radical)RS + O 2-,SO; (thiyl peroxyl radical)

Electron delocalized

oxide, thought to be identical with or closely related tothe endothelium-derived relaxing factor (Moncada etal., 1991), is also a free radical. Most molecules foundin vivo are nonradicals, however.The diatomic oxygen molecule (0,) ualifies as aradical because it has two unpaired electrons, eachlocated in a different orbital, but both with the samespin quantum number. This parallel spin is one rea-son for the poor reactivity of 02 ,espite its powerfuloxidizing nature. According to thermodynamics, thecomplex organic compounds of the human bodyshould immediately combust in the0, ofthe air. How-ever, if 0, attempts to oxidize a molecule directly byaccepting a pair of electrons from it, both of theseelectrons must have spins opposite to that of the un-paired electrons in the 0, so as to fit into the vacantspaces in the 0, orbitals. A pair of electrons in thesame orbital (e.g., a covalent bond) would not meetthis criterion because they have spins opposite to eachother. This spin restriction is one factor that slowsdown the reaction of 0, with nonradicals (most bio-logical molecules). 0, much prefers to react with radi-cals (Table 1 ), accepting its electrons one at a time.For example, combination of carbon-centered orsulfur-centered radicals with 0 , is often very fast(Table 1 ).More than 90% of the 0, taken up by the humanbody is used by mitochondria1 cytochrome oxidase,which adds four electrons onto each 0, molecule togenerate two molecules of water:

O2+ 4H++ 4e- -+ 2H,O ( 1 )This enzyme, like most others that use O,?has transi-tion metal ions at its active sites. Transition metalssuch as iron, vanadium, copper, and titanium havevariable oxidation states; changing between thesestates allows them to transfer single electrons and soto facilitate oxidation-reduction reactions (reviewed

by Halliwell and Gutteridge, 1989). Indeed, reduc-tion of 0, to 2 H 2 0 by cytochrome oxidase proceedsin a stepwise fashion, with various partially reducedforms of oxygen held firmly bound to metal ionswithin the enzyme and not released into free solution.Thus, cytochrome oxidase does not release reactiveoxygen radicals into its surroundings.Several nonradical compounds autoxidize on ex-posure to air; examples are epinephrine, norepineph-rine, 3,4-dihydroxyphenylalanine DOP A), ascorbicacid, 6-hydroxydopamine, and thiols such as reducedglutathione (GS H) , cysteine, and homocysteine. Therates of these autoxidations depend on the amountof contaminating transition metal ions in the reactionmixture (Table 2 ) and it may be that the autoxidiz-able compounds would not be oxidized at all if metalion contamination could be removed completely (animpossible task! ) .For example, manganese ions accel-erate the oxidation of catecholamines to producequinones, semiquinones, and oxygen radicals; this

has been suggested to explain the degeneration of cate-cholamine neurons that has been reported in minersof manganese-containing ores (discussed by Halli-well, 1984; Archibald and Tyrce, 1987; Liccione andMaines, 1988), producing the so-called locura man-ganica syndrome. The nervous system is rich in oxidi-zable catecholamines (Cohen, 1988) and mixtures ofiron or copper salts with DOPA or dopamine havebeen reported to stimulate free radical damage to lip-ids (Sotomatsu et al., 1990).

SUPERO XIDE RADICAL: A US EFU L SPECIES?Acceptance of a single electron by an 0, moleculeforms the superoxide radical, 0,*-,which has oneunpaired electron. The discovery, by McCord andFridovich, of enzymes that appear to have evolvedspecifically to scavenge0,.- (superoxide dismutases;SODs) led to the proposal that O,*- is a major agentresponsible for 0, toxicity and that SODs are impor-tant antioxidant defenses (Fridovich, 1978, 1989).This antioxidant role of SODs is supported by a widerange of evidence, including results obtained usingthe techniques of modern molecular biology (e.g., seeFridovich, 1989; Touati, 1989; Chan et al., 1990).Superoxide is formed in vivo in a variety of ways. A

major source is the activity of electron transportchains in mitochondria and endoplasmic reticulum.Some of the electrons passing through these chainsleak directly from intermediate electron carriersonto 0,. Because 0, accepts electrons one at a time,0,- is formed. The rate of leakage at physiological0,concentrations is probably


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REACTIVE OXYGEN SPECIES AND CNS 1611TABLE 2. Role of metal ions in converting less reactive to more reactive species

Fe/Cu" * 'OHHzOzFelCub RO ' (alkoxyl), ROz' (peroxyl), cytotoxic aldehydes

02'-ILipid peroxides (ROOH)

FeICu plus 0;FeICu plus 0;

Cu/Fed

Thiols (RSH) + Oz'-, HzOz, hiyl (RS'), 'OHNAD(P)H NAD(P)', 0 2 ' - , HZOZ, 'OH

Ascorbic acid Semidehydroascorbate radical, 'OH, H2OZrdegradation products of ascorbateFelCu/Mn plus 0;Catecholamines, related autoxidizable molecules 02*- ,202, OH , semiquinones (or other radicalsderived from the oxidizing compounds)

' he iron- or copper-catalyzed Haber-Weiss reaction.Lipid peroxide decomposition is metal ion dependent, and eventually produces highly cytotoxic products such as 4-hydroxy-2,3-truns-Most so-called autoxidations are s timulated by traces of transition metal ions, and proceed by free radical mechanisms.Copper ions are especially effective in decomposing ascorbic acid, and ascorbatelcopperor ascorbateliron mixtures are cytotoxic.

nonenal, and less toxic ones such as malondialdehyde.

autoxidations of such molecules as catecholamines)beyond the ability of antioxidant defenses to cope. Inaddition, activated phagocytic cells produce 02'- ,which plays an important part in the mechanism bywhich engulfed bacteria are killed (Curnutte and Ba-bior, 1987). Phagocytes able to produce 02'- ncludemonocytes, neutrophils, eosinophils, and macro-phages of various types, including the microglial cellsof the brain (Colton and Gilbert, 1987). The latterinvestigators have suggested that microglial02'- en-eration helps to protect the CNS against infectiousorganisms. Again, excessive activation of phagocyticcells (as in chronic inflamation) can lead to free radi-cal damage.The evidence supporting the superoxide theory ofO2 oxicity is substantial, but the exact mechanism bywhich the putative excess 02'- generation at in-creased O2 evels could exert toxic effects is not com-pletely clear. Superoxide itself has limited reactivity.It is capable of inactivating a few enzymes directly,examples being mammalian creatine kinase (McCordand Russell, 1988) and some iron-sulfur proteins inbacteria, such as E. coli phosphogluconate dehy-dratase and aconitase (Gardner and Fridovich,199 a,b). Superoxide is also capable of inactivatingthe NADH dehydrogenase complex of the mitochon-drial electron transport chain in vitro (Zhang et al.,1990), although its ability to do this in vivo has notyet been demonstrated.Hence, the number of targets within mammaliancells that are known to be sensitive to 02'- s small.Indeed, under certain circumstances, controlled 02'-production is a useful process, e.g., in the bacterialkilling mechanisms of phagocytes referred to previ-

ously (Curnutte and Babior, 1987). Evidence is alsoaccumulating that several cell types, such as fibro-blasts, lymphocytes, and vascular endothelial cells,produce and release small amounts of02'- n physio-logical reactions (e.g., Maly, 1990; Meier et al., 1990;Murrell et al., 1990). This 02'-may be involved ingrowth regulation and intercellular signaling, as sug-gested by Halliwell and Gutteridge ( 1986).

NITRIC OXIDEAnother free radical released by several cell types,especially vascular endothelial cells and phagocytes, isnitric oxide, NO' (Moncada et al., 1991). NO' reactswith 02-- t physiological pH to yield a nonradicalproduct, peroxynitrite (Saran et al., 1990). There isconsiderable debate in the literature about whetherthis interaction of 02'- nd NO' might be damagingto cells. Thus, peroxynitrite may be directly cytotoxic,perhaps by oxidizing thiol groups, and it might alsodecompose to form OH' (Beckman, 1991;Radi et al.,1991). By contrast, interaction of 02'- and NO'

might represent a physiological regulatory process af-fecting vascular muscle tone. Thus, it has been sug-gested that vascular endothelial cells might produceboth 02'- nd NO' as "antagonistic agents," to givefine control of vascular tone (Halliwell, 1989a). Ineither case,02'- as a vasoconstrictor effect in biologi-cal systems, by removing NO' (e.g., Laurindo et al.,1991).Nitric oxide synthetase is widespread in brain tissue(Hope et al., 1991) .Nitric oxide has been suggestedto be involved in both the normal functioning of excit-

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1612 B. HALLIWELLatory amino acids such as glutamate and in the damag-ing effects produced by their generation in excess(Dawson et al., 1991; Forstermann et al., 1991). Itappears likely that the interaction of 0,'- with NO'(whether protective or dangerous) is highly relevantto normal brain metabolism and to neurodegenera-tive diseases (Beckman, 1991; Terada et al., 1991) .

HYDROGEN PEROXIDESODS remove 02'-by converting it into hydrogenperoxide.

20,'- + 2H++H,O, + 0 , (2)Several other enzymes that produce H,02 exist in hu-man tissues, such as L-amino acid oxidase, glycollateoxidase, and monoamine oxidase. Indeed, oxidativedeamination of dopamine by monoamine oxidase isthe main catabolic pathway for dopamine within do-pamine nerve terminals (Cohen, 1988). Cohen( 1988) suggested that an accelerated turnover of do-pamine in patients with Parkinson's disease, leadingto increased H ,02 formation, may provoke an oxida-tive stress (i.e., an increase in the generation of oxy-gen-derived species beyond the ability of antioxidantdefenses to cope with them) within surviving dopa-mine terminals, so accelerating their destruction. An-other possibility is that the side effects of prolongedtherapy with L-DOPA in patients with Parkinson'sdisease might be related to increased H202 ormation(Cohen, 1988;Olanow, 1990).Zhang and Piantadosi( 1991) reported that the monoamine oxidase inhibi-tor pargyline could partially protect rats against theCNS toxicity produced by hyperbaric 0 2 .H202 an act as an oxidizing agent, although it ispoorly reactive. Unlike 02'-,owever, H20 2 rossescell membranes easily. H,02 does not qualify as a radi-cal because it contains no unpaired electrons, and itcan be removed within human cells by the action oftwo types of enzyme, catalases and selenium-depen-dent glutathione peroxidases. The latter are probablymore important in the brain (Cohen, 1988;Jain et al.,1991).Despite the undoubted importance of SOD as aphysiological antioxidant (Fridovich, 1978, 1989;Touati, 1989), there is evidence that an excess ofSOD in relation to the activities of peroxide-metabo-lizing enzymes can be deleterious (Scott et al., 1987;Avraham et al., 1988;Groner et al., 1990;Remacle etal., 1991 ). For example, the gene for one SOD isoen-zyme (that containing copper and zinc) is located onchromosome 2 1. Evidence consistent with the viewthat the excess of copper /zinc-containing SOD activ-ity in trisomy 2 1 may contribute to the symptoms ofDown's syndrome has come from work with trans-

genic mice carrying the human gene encoding CuZn-SOD (Avraham et al., 1988;Groner et al., 1990;Ce-ballos-Picot et al., 1991 ).HYDROXYL RADICAL ANDRELATED SPECIES

H,O, is known to be toxic to many systems, includ-ing nervous tissue (Colton et al., 1989, 199 1;Pellmaret al., 1991). However, toxicity is not usually me-diated by a direct effect of the H,O,, except at high(and probably unphysiological concentrations. In-stead, the H, 0 2 is a precursor of highly oxidizing,tissue-damaging radicals. H,02 reacts with Fe2+ onsto form hydroxyl radical, 'OH, by the Fenton reac-tion, which may be represented by the overall reac-tion:Fe2++ H202 +Fe3++ OH + OH- ( 3 )

Hydroxyl radical is probably not the only damagingspecies formed when iron and H 2 0 2are mixed (e.g.,Bielski, 1991), but its formation is well established(Halliwell and Gutteridge, 1990a; Yamazaki andPiette, 1990, 1991). It may be that the initial productof reaction of Fe2+and H,02 is another highly oxidiz-ing species, an iron-oxygen complex called ferryl,that then decomposes to yield 'OH (reviewed by Hal-liwell and Gutteridge, 1990a). Copper ions also reactwith H,02 to form 'OH (discussed by Halliwell andGutteridge, 1990a; Aruoma et al., 1991).Hydroxyl radical reacts at great speed with almostevery molecule found in living cells, including DNA(causing strand breakage and chemical alterations ofthe deoxyribose and of the purine and pyrimidinebases), membrane lipids, and carbohydrates. Indeed,the cytotoxic action of H,O, on most, if not all, mam-malian cells seems to involve DNA damage(Cochrane, 1991 ) . Depending on the cell type stud-ied, some or all of this DNA damage may be mediatedby reaction of H,O, with iron and/or copper ions,bound at or close to the DNA, to form 'OH (reviewedby Halliwell and Aruoma, 1991).An additional mech-anism of DNA damage is the increasing of intracellu-lar free Ca2+concentrations by oxidative stress, lead-ing to the activation of nuclease enzymes within thenucleus (Orrenius et al., 1989).Another potentially devastating effect of 'OH is itsaction on membrane lipids. Various reactive speciesthat are generated on mixing02'-, 20 2, nd iron orcopper ions are capable of initiating the process oflipid peroxidation by abstracting a hydrogen atomfrom a polyunsaturated fatty acid side chain (writtenlipid-H below) in a membrane lipid. Polyunsaturatedfatty acid side chains (those with two or more carbon-carbon double bonds), such as arachidonic acid, aremuch more sensitive to free radical attack than satu-rated or monounsaturated side chains. Abstraction of

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REACTIVE OXYGEN SPECIES A N D CNS 1613

a hydrogen atom leaves behind a carbon-centered rad-ical (lipid) in the membrane:lipid-H + radical -+ lipid + radical-H ( 4 )

The most likely fate of carbon-centered radicals invivo is reaction with 0, to form peroxyl radicals (of-ten shortened to peroxy radicals).

lipid + 0, -+ lipid-0, ( 5 )Peroxyl radicals can attack membrane proteins (sodamaging receptors and enzymes) and can also ab-stract hydrogen atoms from adjacent fatty acid sidechains.

lipid-0, + lipid-H -+ ipid-02H + lipid ( 6 )Thus, abstraction of a single hydrogen can set off afree radical chain reaction (Eqs. 5 and 6 ) that leads toconversion of many membrane lipids into lipid hy-droperoxides ( ipid-0,H) .The existence of lipid per-oxides within a membrane severely disrupts its func-tioning, altering (usually decreasing) fluidity and al-lowing ions such as Ca2+ to leak across themembrane. This is in addition to the damage pro-duced by attack of peroxyl radicals on membrane pro-teins.Iron and copper ions can contribute to lipid peroxi-dation in two ways. First, they catalyze formation ofinitiating (H-abstracting ) species. Second, they stimu-late peroxidation by reacting with lipid hydroperox-ides and decomposing them to peroxyl radicals andalkoxyl radicals ( ipid-O), which can abstract H andlead to further peroxidation (Halliwell and Gutter-idge, 1989, 1990a). Products of these complex de-composition reactions include hydrocarbon gases anda wide range of toxic carbonyl compounds, includingaldehydes. Of these aldehydes, much attention in theliterature is usually devoted to malonaldehyde (some-times called malondialdehyde or MDA), but this ismuch less noxious than such highly cytotoxic unsatu-rated aldehydes as 4-hydroxy-2,3-trans-nonenalEs-terbauer, 1985). These aldehydes can damage adja-cent cells; membrane-bound enzymes and receptorscan also be inactivated. For example, the binding ofserotonin to its receptors in rat cortex membranes(Muakkassah-Kelly et al., 1982) and the binding ofspiroperidol to receptors in human caudate and puta-men (Andorn et al., 1988)are decreased by peroxida-tion of the membranes. Proteins essential for mem-brane function can thus be damaged not only by at-tack of peroxyl radicals, but also by covalentmodification by aldehyde end products of peroxida-tion.Most formation of OH and other reactive speciesoccurs by a reaction between reduced iron (Fe2+) rcopper (Cu) ions and H,O, (Eq. 3) . The Fe3+ or

Cu2+generated can be re-reduced by ascorbic acid orby 02-. hus, under certain circumstances, both0,- and ascorbic acid can accelerate the copper- oriron-dependent formation of OH from H202.Somecatecholamines can probably do the same (Halliwelland Gutteridge, 1989) . Reaction of Fe3+ with 02*-proceeds with intermediate formation ofperferryl,yetanother oxidizing species. Perferryl formed in biologi-cal systems appears to be a much weaker oxidant thanferryl or OH.Fe3++ 0,- P (Fe3+- 0,- - e2+- 0,) S

(Perfem1F e 2 + + 0 , ( 7 )

Combining this equation with Eq. 3, we arrive atthe overall reaction that has been called the iron-cata-lyzed Haber- We iss reaction:Fe catalyst0,-+H20, + O H + O H - + O , ( 8 )

Copper ions also catalyze this overall reaction (dis-cussed by Aruoma et al., 199 1 ) .Equation 8 implies that much of the toxicity of02-and H 202 n vivo is due to their reaction withiron or copper ions to form OH and other oxidizingspecies. Considerable evidence exists to support thissuggestion (Halliwell and Gutteridge, 1986, 1989,1990a; Imlay and Linn, 1988, Kyle et al., 1988; Me-neghini, 1988; Halliwell and Aruoma, 199 1 ;Pellmaret al., 199 1 ), although reaction 8 is certainly not theonly mechanism of cell injury by oxidative stress(Orrenius et al., 1989; Cochrane, 1991) . It followsthat the nature of the dam age done to cells by excessformation of H 2 0 2 a nd 02*- ill be afected by thelocation of me tal ion catalysts of reaction 8 within thecells. It also follows that, ijno catalytic me tal ions ayeQ VQ ~ ! Q ~ [ P ,h p n 02-nnd H 2 0 2 will have limited, ifany, damaging eflects (Halliwell and Gutteridge,1984b, 1986, 1990b). These fundamental principlesunderlie the importance of examining the availabilityand distribution of catalytic metal ions in explain-ing oxidative damage to cells. In general, iron andcopper ions act to convert poorly reactive species(0,-, H,02, thiols, lipid-0,H) into more reactive,cytotoxic ones (OH, ferryl, aldehydes, etc.) as sum-marized in Table 2. As discussed previously, they alsoaccelerate the oxidation of catecholamines.

IRON-DEPENDENT FORMATION OFREACTIVE RADICALS: WHERE DOESTHE IRON COME FROM?Iron is a remarkably useful metal in nature; we de-pend on it to transport (hemoglobin), store (myoglo-bin), and use (cytochromes, cytochrome oxidase,

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1614 B. HALLIWELL

non-heme-iron proteins) oxygen for respiration. It isalso an essential component in the active sites ofmany enzymes (e.g., aconitase, proline hydroxylase),including the antioxidant defense enzyme catalase. Inmammals, iron is absorbed from the gut and entersthe plasma attached to the protein transferrin, whichbinds 2 mol of ferric iron per mole of protein withvery high affinity at pH 7.4. In normal humanplasma, the average iron loading of transfenin is 20-30% of maximum. Hence, there is an excess of iron-binding capacity, which means that the content offree ionic iron in plasma should be effectively nil(Weinberg, 1992), a result confirmed by direct mea-surement using the bleomycin assay for free iron (re-viewed by Gutteridge and Halliwell, 1987).Transferrin enters cells by endocytosis, and the pHof the vacuole containing it is then lowered. This facili-tates release of iron from the protein. The unloadedtransferrin (apotransferrin) is ejected from the cell,and the iron ions released from it are used for thesynthesis of intracellular iron proteins. Excess iron isstored in the protein ferritin. At present, the chemicalnature and subcellular distribution of the non-pro-tein-bound iron pool within cells are completely un-known, except that the pool seems to be kept verysmall (Crichton and Charloteaux-Waters, 1987).Iron ions attached to the transport protein trans-ferrin, the neutrophil-derived protein lactofemn, theiron-storage proteins fenitin and hemosiderin, the0,-binding proteins hemoglobin, myoglobin, or leghe-moglobin, or to other iron proteins ( e g , ferredoxin)are thought to be incapable of reacting with H202 oform OH detectable outside the protein (discussed byHalliwell and Gutteridge, 1986, 1989, 1990a,b; Bo-lann and Ulvik, 1990). Lactoferrin and transferrinare resistant to damage by0,-,H,O,, and other oxi-dants, and iron bound to them will not catalyze freeradical reactions, which makes these proteins a safeform of transport for iron at pH 7.4 (Aruoma andHalliwell, 1987). By contrast, some (but only limitedamounts) of iron can be reductively mobilized fromferritin by 02* -nd by radicals generated during lipidperoxidation (Biemond et al., 1986; Bolann and U1-vik, 1990). Iron catalytic for free radical reactionsthus appears to be available within cells, both as amobile pool and as iron releasable from fenitin.Hence, an important function of intracellular antioxi-dant defense enzymes is to scavenge0,*- and H,02before they can come into contact with this availableiron (Halliwell and Gutteridge, 1986, 1989).In blood plasma from healthy humans, no iron isavailable to stimulate lipid peroxidation or OH radi-cal formation (Halliwell and Gutteridge, 1986,1990b). Human blood plasma contains very little, ifany, catalase or GSH ( 1-2 pM ) and only low activi-ties of SOD. Halliwell and Gutteridge ( 1986, 1990b)have argued that 02* -nd H2 0, generated extracellu-

larly may be employed for usefit1 purposes, such ascellular signaling, growth regulation, and inactivationof NO. They are not dangerous because, in the ab-sence of catalytic iron ions, they cannot lead to forma-tion of OH and other highly oxidizing species (al-though the reaction of NO* with 02*- ight be animportant exception; see Beckman, 1991). Thus, amajor orm ofantioxidant defense in human plasma isthe prevention of metal ions from participating in thegeneration of reactive radical species. Copper ions aresimilarly unavailable in human plasma (Gutteridge,1984; Evans et al., 1989); most or all of plasma cop-per is attached to the protein ceruloplasmin, whichhas antioxidant properties (Gutteridge and Stocks,1981).HEMOGLOBIN: A DANGEROUS PROTEINHemoglobin is transported in erythrocytes, whichare rich in the antioxidant defense enzymes catalase,

SOD, and glutathione peroxidase. However, isolatedhemoglobin (and myoglobin) are degraded on expo-sure to excess H,0,, with release of iron ions, capableof catalyzing free radical reactions, from the hemering (Gutteridge, 1986; Puppo and Halliwell, 1988).In addition, hemoglobin reacts with H202 o form aprotein-bound oxidizing species capable of stimulat-ing lipid peroxidation (reviewed by Kanner et al.,1987). The nature of this oxidizing species is unclearas yet. Reaction of H,O, with the protein probablygenerates a heme ferry1 species plus an amino acidradical. In the case of myoglobin, it has been sug-gested that a tyrosine peroxyl radical, capable of ab-stracting hydrogen and initiating lipid peroxidation,is formed on exposure of the myoglobin to H,O, (Da-vies 1990, 1991) .However, the identity of this radicalis still uncertain (Kelman and Mason, 1992).Thus, as Fig. 1 emphasizes, hemoglobin outside theerythrocyte is potentially a dangerous protein (Sadr-zadeh et al., 1987). Indeed, spasm ofcerebral arteriescan be a significant late complication of hemorrhagicstroke, and it has been proposed that release of hemo-globin from erythrocytes in the clot, and subsequentfree radical reactions, are involved (Steele et al.,1991) .Hemoglobin also binds NO avidly (reviewedby Macdonald and Weir, 1991).In general, however, the availability of catalyticiron or copper ions to stimulate free radical reactionsin most body tissues and extracellular fluids is ex-tremely limited, and can often control the extent oftissue damage being done ( e g , 0,- and H202. reunlikely to cause widespread damage unless such ionsare present). However, cell injury by almost anymechanism, including traumatic or ischemic injury,has the potential to accelerate ree radical reactions.This is partly because injured and lysed cells releasetheir intracellular iron into the surrounding environ-

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REACTIVE OXYGEN SPECIES A N D CNS 1615

Oxyhaemoglobin

rryl an d - - - -+ Haemino Acid Degradatior~ d i c a l s I

itiationIron IonRelease

Other Oxidative Damage

FIG. 1 . Hemoglobin: a dangerous protein. Hemoglobin is nor-mally transported inside erythrocytes, cells rich in antioxidant de-fense enzymes. Free hemoglobin is very sensitive to attack byH,02, resulting n an exacerbationof oxidative damage. Hemoglo-bin is also an avid binder of nitric oxide.ment, where iron is then available to accelerate for-mation of damaging oxygen-derived species (Halli-well and Gutteridge, 1984a, 1985; Halliwell et al.,1988 . Other reasons include recruitment of phago-cytes to the injured area with their subsequent activa-tion, and increased leakage onto 0 , of electronsfrom damaged electron transport chains. Bleeding asa result of injury, and subsequent hemoglobin libera-tion, may have a compounding effect (Gutteridge,1986; Sadrzadeh et al., 1987; Macdonald and Weir,1991). In severe muscle injury, as in crush syn-drome, the release of myoglobin into the circulationalso poses a risk of oxidative damage (Odeh, 1991) .OXIDANTS, IRON, COPPER, AND THE BRAIN

CNS damage is a major clinical problem. Indeed,ischemic or traumatic injuries to the brain or spinalcord are often said to result in more extensive tissuedamage than do equivalent insults to other tissues.Free radical reactions have often been implicated insuch damage (Demopoulos et al., 1982;Hailiwell andGutteridge, 1985; Traystman et al., 1991), particu-larly when ischemic tissues are reoxygenated (e.g.,Patt et al., 1988 .Ischemia appears to prime tissues(including brain) to respond on reoxygenation withincreased rates of generation of 02.- and H,O,(McCord, 1985). These increased rates of oxidantgeneration in the brain may sometimes involve theenzyme xanthine oxidase in rat, bovine, and gerbil

tissues (Betz, 1985; Patt et al., 1988; Terada et al,,1991).Xanthine oxidase oxidizes xanthine to hypo-xanthine and then on to uric acid, with simultaneousreduction of 0, to both 0,*-and H,O, (McCord,1985). However, the role of this enzyme in mamma-lian reoxygenation injury is increasingly questioned(Kehrer, 1989; Betz et al., 1991; Lindsay et al., 1991)and its significance in human brain is unclear at pres-ent. In addition, the importance of free radical genera-tion on reoxygenation as an injury mechanism in thebrain may be critically dependent on the extent andperiod of the oxygen deprivation (Agardh et al.,1991).The brain and nervous system may be especiallyprone to radical damage for a number of reasons(Halliwell and Gutteridge, 1985; LeBel and Bondy,1991). First, the membrane lipids are very rich inpolyunsaturatedfatty acid side chains, which are espe-cially sensitive to free radical attack. Ischemia leads tomembrane breakdown and a rapid increase in freefatty acids within the brain (reviewed by Traystmanet al., 1991) ; hese can provide substrates for free radi-cal attack and for the synthesis of excess prostaglan-dins and leukotrienes on reoxygenation. Second, thebrain is poor in catalase activity and has only moder-ate amounts of SODand glutathione peroxidase (Co-hen, 1988). Third, several areas of the human brain( e g , he globus pallidus and substantia nigra) are richin iron (Youdim, 1988a,b), yet CSF has no signifi-cant iron-binding capacity because its content oftransferrin is very low (Gruener et al., 1991). By spec-trophotometric and atomic absorption techniques,total iron values in CSF from normal subjects havebeen reported to be in the range 0.2-1.1 pM . Becausethe transferrin content of CSF is -0.24 KMand 1 molof transferrin binds 2 mol of iron ions, these data sug-gest that CSF transferrin is often at, or close to, ironsaturation (reviewed by Gutteridge, 1992).Most brain iron is protein bound, but little isknown of its molecular nature. Iron seems to playessential roles in the brain (Youdim, 1988a,b; Rose-bush and Mazurek, 1991), especially in learning andmemory. Thus, it has been suggested that iron ionsare required for the correct binding of certain neuro-transmitters to their receptors (Youdim, 1988a,b).Thus, a high content of brain iron may be essential,particularly during development, but its presencemeans that injury to brain cells may release iron ionsthat can stimulate free radical reactions. Homoge-nates of brain peroxidize very rapidly in vitro; indeed,peroxidation of ox brain homogenate has been usedfor many years as an assay to measure antioxidantactivity, and the peroxidation is inhibited by severaliron-chelating agents (Stocks et al., 1974). Ischemicor traumatic injury might be thought of as essentiallycausing a partial disruption ofbrain (partial homoge-nization). Oxidants can release free iron from hemo-

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1616 B. HALLIWELLglobin; heme might also be released and is anotherpowerful prooxidant (Gutteridge, 1986; Gutteridgeand Smith, 1988; Balla et al., 1991), as is hemoglobinitself in the presence of H 2 0 2 Sadrzadeh et al., 1987;Puppo and Halliwell, 1988).In addition, there is a high concentration of ascor-bic acid in the gray and white matter of the CNS. Thechoroid plexus has a specific active transport systemthat raises ascorbate concentrations in the CSF, reput-edly to - 0-fold the plasma level, and neural tissuecells have a second transport system that concentratesintracellular ascorbate even more (Spector and Eells,1984). Ascorbic acid in the absence of transitionmetal ions has well-established antioxidant properties(discussed by Frei et al., 1989; Halliwell, 1990). How-ever, ascorbate/ iron and ascorbate / copper mixturesgenerate free radicals (Halliwell and Gutteridge,1990~) . hus, if catalytic iron were generated in theCNS as a result of injury, ascorbic acid might thenstimulate OH generation within the brain and CSF.Indeed, injecting aqueous solutions of iron salts orhemoglobin into the cortex of rats has been shown tocause transient focal epileptiform discharges, lipidperoxidation, and persistent behavioral and electricalabnormalities (e.g., Rosen and Frumin, 1979; Will-more et al., 1980, 1986).The brain also contains copper, but nothing isknown of its molecular nature or whether it ever be-comes available to stimulate free radical reactions.

OXIDATIVE STRESSTHE MOLECULAR TARGETSIschemia/ reoxygenation injury and traumatic dam-age to the brain can produce liberation of catalyticmetal ions and an increase in the formation of reac-tive radicals. How then could further cellular damageoccur? Lipid peroxidation is one possibility, and sev-eral groups have found evidence consistent with in-creased CNS lipid peroxidation after ischemia (e.g.,Michel et al., 1987; Watson and Ginsberg, 1988),trauma (Hall and Braughler, 1988), iron salt injec-tion (Willmore et al., 1980), or damage by methylmercury (Sarafian and Verity, 1991). It must be em-phasized, however, that damage caused by oxidativestress need not necessarily involve lipid peroxidation(Halliwell, 1987; Orrenius et al., 1989; Cochrane,

I99 1). Hence, the failure of some groups to find lipidperoxides in injured nervous tissue does not rule outthe occurrence of oxidative damage; damage to DNAand proteins may be of equal, or even greater, impor-tance in vivo (Halliwell, 1987). Early events in hu-man cells subjected to oxidative stress include DNAdamage and consequent activation of poly (ADP-ri-bose) synthetase (this enzyme polymerizes ADP-ri-bose residues derived from NAD+ and can lead todepletion of cellular NAD), decreases in ATP con-tent, and increases in intracellular free Ca2+,with

consequent activation of Ca+-stimulated proteasesthat can cause such phenomena as bleb formationon the plasma membrane of the cells (Orrenius et al.,1989).Figure 2 indicates the complexity of the changesthat can occur in mammalian cells subjected to oxida-tive stress; it is very difficult to disentangle the se-quence of events. Increases in intracellular free Ca2+might be particularly damaging to neurons (reviewedby Siesjo, 1990) because several neurotoxins arethought to act in this way (Komulainen and Bondy,1988; Siesjo, 1990). An additional mechanism thathas been suggested to explain the damage producedby ischemia/ reoxygenation in brain is the generationwithin the tissue of excitatory amino acids such asglutamate (reviewed by Meldrum, 1985; Siesjo,

FIG. 2. Interacting mechanisms of cell damage by oxidativestress. Damage may be direct (e.g., if H202oxidizes protein -SHgroups, or reacts with transition metal ions bound to DNA to formOH, which then fragments the DNA) or indirect, e.g., oxidativestress can cause increases in intracellular free Ca+, whichmay activate not only proteases that cleave the cytoskeleton(producing membrane blebs), but also nucleases that fragmentDNA. Uptake of Ca2+ by mitochondria could cause release ofFez+, acilitating oxidative damage (Merryfield and Lardy, 1982).

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1988). t is therefore interesting to note claims thatexcitatory amino acids can be released from rat hip-pocampal slices as a result of exposure to 02*-ndH20z Pellegrini-Giampietro et al., 1988), and thatthe toxicity of the excitatory neurotoxin kainic acid tocerebellar neurons in culture may involve increasedoxygen radical formation (Dykens et al., 1987).Fur-ther work is required in this area.Protein damage is frequently an important conse-quence of oxidative stress (Halliwell, 1987;Orreniuset al., 1989;Cochrane, 1991). Indeed, radicals candamage brain proteins (Oliver et al., 1990) ncludingthe enzyme glutamine synthetase (Schor, 1988).Thus, in gerbil brain, glutamine synthetase, the majorenzyme responsible for glutamate removal, may beinactivated by oxidative stress (Oliver et al., 1990;Carney et al., 199 ). Increased levels of end productsof oxidative damage to proteins, and decreased gluta-mine synthetase activities, have been reported in old(>70 years) human brains when compared withyoung ( -30 years) controls (Smith et al., 1991 .The acidosis resulting from ischemia has been sug-gested to aggravate free radical injury by making re-leased iron more soluble (Siesjo et al., 1985;Bralet etal., 1992); t might also interfere with the activity ofantioxidant defense enzymes (Link, 1988).However,there are also reports that acidosis may protect certaincell types, such as liver endothelial cells, against oxi-dative damage (e.g., Bronk and Gores, 1991).Fur-ther work is needed for clarification.

OXIDATIVE DAMAGE ANDPARKINSONS DISEASEThere has been considerable interest in the possibil-ity that neurotoxins cause or contribute to Parkin-sons disease, especially since the identification of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridineMPTP)as the toxic agent responsible for the sudden onset ofparkinsonian symptoms in a group of heroin addictsin the United States (discussed by Langston et al.,1987;Add, 1991). Suggestions that MPTP and itsmetabolites kill nigral cells by increasing intracellularformation of 02*-nd H,02 have not been rigorouslysubstantiated (e.g., Martinovits et al., 1986; rank etal., 1987) nd it seems more likely that MPTP is oxi-dized to 1-methyl-4-phenylpyridinium,which then in-

hibits mitochondrial electron transport, with conse-quent severe metabolic derangements (Langston etal., 1987;Turski et al., 1991).Of course, such meta-bolic disruption might create a secondary oxidativestress (see Fig. 2 ) that worsens the damage (Adamsand Odunze, 199 ). Consistent with this suggestion,mice transgenic for human CuZn-SOD, which showincreased SOD activity in the brain, are reported to bemore resistant to MPTP than control mice (Przed-borski et al., 199 ). MPTP inhibits the mitochondrialelectron transport chain at complex I, the NADH de-

hydrogenase system (a site known to be sensitive tofree radical attack; Zhang et al., 1990).Defects in thefunction of mitochondrial complex I have recentlybeen claimed in the substantia nigra of parkinsonianpatients, an observation that further encourages thedebate concerning the possibility that Parkinsons dis-ease may be the result of exposure to an environmen-tal toxin acting in a way similar to MPTP (discussedby Shapira et al., 1990;Agid, 199 ).It is also possible that iron-dependent free radicalreactions contribute to nigral cell damage in patientswith Parkinsons disease (Dexter et al., 1989, 1991;Olanow, 1990;Youdim et al., 1990;Ben-Shachar andYoudim, 1991; enner, 1991). Thus, Dexter et al.(1989, 1991 reported increased nigral iron and de-creased brain ferritin levels in parkinsonian patients,and they speculated that increased iron-dependentlipid peroxidation could contribute to cell destruc-tion. Pall et al. ( 1987) ound no change in CSF iron inpatients with Parkinsons disease, but an increase inCSF copper was demonstrable in certain patients; thesignificance of this is uncertain. Riederer et al. ( 1989)and Sofic et al. ( 199 ) reported increased nigral ironand increased ferritin in parkinsonian brains, as wellas decreased GSH. Problems in the assay of brainferritin by antibody-based methods may contribute tothe discrepancy between these results and those ofDexter et al. (1989).Olanow et al. (1990) howedabnormal signal attenuation in the putamen, consis-tent with increased iron, in high-field magnetic reso-nance scans on parkinsonian patients. Cohen (1988)proposed that the compensatory increase in dopa-mine turnover in the remaining dopamine neuronsmight lead to excess generation of H202by mono-amine oxidase, which could interact with iron tocause further cell destruction.Insufficient data are available for the author to de-cide if iron accumulation in parkinsonian brains is anearly or late event in the disease process. Of course, itmust also be remembered that cell injury (by anymechanism) can lead to iron release and more freeradical reactions, even if the cause of the injury hadnothing to do with free radicals. The answer to thequestion, Does any such secondary radical genera-tion contribute significantly to the disease pathol-ogy? (Halliwell and Gutteridge, 1984b) s also un-clear as yet. However, Ben-Shachar and Youdim( 199 ) reported that intranigral injection of ferricchloride into rats produced parkinsonian-type be-havior.

In addition, it must be remembered that somedrugs, such as chlorpromazine and DOPA, havemetal ion-binding capacity and may be capable oftransporting metal ions into and out of the brain(Weiner et al., 1977;Blake et al., 1985).Thus, caremust be used when interpreting changes in the brainsof patients who may have been treated with a widerange of drugs. However, the changes in parkinsonian

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1618 B. HALLIWELLsubstantia nigra seem very distinctive (Dexter et al.,1991; Hirsch et al., 1991), which perhaps suggeststhat they are not nonspecific consequences of drugtreatment.

OXIDATIVE DAMAGE ANDALZHEIMERS DISEASEAlthough it is unlikely (in the authors opinion)that excessive exposure to aluminum is the cause ofAlzheimers disease, interest continues in elucidatingthe precise role played by aluminum in this disorder.Thus, aluminum ions are known to be neurotoxic,aluminosilicates have been identified at the cores ofsenile plaques, and aluminum has been found withinneurons bearing neurofibrillary tangles (discussed byBirchall and Chappell, 1988; Good et al., 1992). Itmay be that Alzheimers disease leads to an impairedblood-brain barrier and thus increased accumulationof aluminum within the brain. Some or all of this

aluminum may enter attached to transfenin, whichbinds aluminum with high affinity. It has been re-ported that iron deposition is increased within orclose to plaques (Youdim, 1988b)and in neurofibril-lary tangles (Good et al., 1991). Gutteridge et al.( 1985)showed that aluminum ions alone cannot pro-mote membrane lipid peroxidation, but that they ac-celerate Fez+ dependent peroxidation under certaincircumstances. Lead (Pb2+) ons also promote Fe2+-dependent peroxidation (Quinlan et al., 1988;Aruoma et al., 1989). Superoxide radicals have beenreported to form a complex with aluminum ions thatis a stronger oxidizing agent than 0- itself (Kong etal., 1992).Aluminum might also interfere with cellu-lar iron metabolism, making iron more available tocatalyze free radical reactions (Abreo et al., 1991).Thus, it is conceivable that coaccumulation of alumi-num and iron (Youdim, 1988b; Good et al., 1991)might facilitate oxidative damage (discussed by Pap-polla et al., 1992),but this has yet to be proved rele-vant to Alzheimers disease.McLachlan et al. ( 1991) reported that intramuscu-lar injection of the powerful iron ion chelator, des-ferrioxamine (which also binds aluminum, but muchmore weakly than it does iron) into patients with Alz-heimers disease led to a significant reduction in therate of decline of daily living skills. Even if theseresults are confirmed, caution should be used in theprolonged administration of a powerful iron-chelat-ing agent to patients who are not iron overloaded(Blake et al., 1985).

ANTIOXIDANT THERAPYThe term antioxidant is frequently used in theliterature to mean a chain-breaking antioxidant inhib-itor of lipid peroxidation. Such antioxidants (writtenA-H below) inhibit the chain reaction of lipid peroxi-dation (Eqs. 5 and 6) by scavenging intermediate per-oxyl radicals:

A-H + l i ~ i d -0 ~ A + lipid-02H (9)This stops the peroxyl radicals from attacking adja-cent fatty acid side chains or membrane proteins. Themost important (Burton and Ingold, 1989),but by nomeans the only (Esterbauer et al., 1989), chain-breaking antioxidant inhibitor of lipid peroxidationin humans is a-tocopherol. Severe and prolonged de-privation of a-tocopherol, as occurs in patients withfat malabsorption syndromes, produces severe neuro-logical derangements (Muller and Goss-Sampson,1990). Hence, a-tocopherol is clearly essential in thehuman brain.However, it takes considerable time (weeks) to in-crease the a-tocopherol content of brain tissue inmammals supplemented with this vitamin (Mullerand Goss-Sampson, 1990). It must also be remem-bered that oxidative damage frequently occurs with-out significant lipid peroxidation (Fig. 2), and, so,inhibitors of lipid peroxidation might be (and oftenare) therapeutically ineffective even in conditionswhere free radicals are important in producing tissueinjury (Halliwell and Gutteridge, 1984b, 1989;Halli-well, 1990). Jenner (personal communication) hasfound no change in a-tocopherol in any brain regionin parkinsonian patients. Some chain-breaking an-tioxidants are capable of reducing Fe3+ o Fez+,andcan exucerbate iron-dependent radical reactions tononlipids. For example, several flavonoids that arepowerful inhibitors of lipid peroxidation can uggru-vale iron ion-dependent radical damage to carbohy-drates or to DNA (Laughton et al., 1989).

Another approach has been to use antioxidant en-zymes, especially as recombinant human SODis nowavailable. Endogenous SO D may have an importantprotective role. However, both negative ( e g , Helfaeret al., 1991) and positive results have been reportedusing administered SOD. For example, SODhas beenclaimed to diminish reperfusion injury after spinalcord ischemia in dogs (Lim et al., 1986) and rabbits(Agee et al., 1991), and edema induced by low braintemperatures in rats (Chan et al., 1987), but muchmore work is required; there is an obvious questionabout the ability of SOD to reach the site of injury.Ebselen, a low molecular mass agent that removesH,O, by a glutathione peroxidase-like activity(Muller et al., 1984) and inhibits lipid peroxidation(Hayashi and Slater, 1986)is also available for evalua-tion, but it probably has other metabolic effects aswell (Muller et al., 1984).Another approach is the use of chelating agents thatprevent iron ions from participating in such reactionsas OH production and lipid peroxidation. The proto-type is desferrioxamine, which inhibits most iron-de-pendent radical reactions and has been used success-fully to diminish oxidative damage in several animalmodel systems of human disease (reviewed by Halli-well, 1989b).However, most antioxidants (includingdesferrioxamine probably do not cross the blood-

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brain barrier. Thus, several scientists have sought al-ternative compounds. For example, a series of 21 -amino steroids with antioxidant activity has been de-veloped (Braughler et al., 1987). One of these,U74006F (generic name tirilazad mesylate), hasgiven interesting results in animal model systems oftraumatic or ischemia/reoxygenation injury to thebrain or spinal cord. Thus, U74006F was observed tominimize the effects of reperfusion injury on thebrain of cats (Hall and Yonkers, 1988), rats (Lesiuket al., 199 1; Haraldseth et al., 1991), or dogs (Perkinset al., 1991), to protect against hemorrhagic shock orposttraumatic spinal cord ischemia in cats (Hall,1988) and to minimize neurological damage afterhead injury in mice (Hall et al., 1988b). Some relatedcompounds, such as U78517F and U74500A, mayalso have protective effects in animal model systemsof ischemia/reperfusion injury (Hall et al., 1990). Al-though they were developed as inhibitors of lipid per-oxidation (Braughler et al., 1987), the 21-amino ste-roids may well inhibit other free radical reactions aswell. Thus, their protective action in some animalmodel systems should not necessarily be taken tomean that they are acting by inhibiting iron-depen-dent lipid peroxidation. Indeed, Sanchez-Ramos et al.( 1992) have suggested that some 21-amino steroidsmight interfere with neuronal dopamine reuptake sys-tems.An exciting area may have been opened up by re-cent studies by Floyd and co-workers (Oliver et al.,1990; Carney et al., 1991; Floyd, 1991; Smith et al.,1991) using N-tert-butyl-a-phenylnitrone (PBN).PBN is a widely used agent that can trap free radicalsin vivo; it reacts with the free radicals to form adductsthat show characteristic spectra when subjected to thetechnique of electron spin resonance (Chen et al.,199 1).Floyd ( 1991) observed that daily administra-tion of PBN to old gerbils caused a decrease in theamount of oxidized brain proteins, an increase inbrain glutamine synthetase activity, and a decline inthe number of errors made in the radial-arm maze testfor memory. PBN had no such effects on young ger-bils. The future will tell if derivatives of PBN will havetherapeutic use.

CONCLUSIONSFree radical reactions are a part of normal humanmetabolism. When produced in excess, radicals cancause tissue injury. However, tissue injury can itselfcause more radical reactions, which may (or may not )contribute to a worsening of the injury. The carefuluse of a range of antioxidants, combined with newmethods for measuring free radical generation in hu-mans (reviewed by Halliwell et al., 1992) is, at longlast, enabling the exact contribution of free radicalreactions to human disease to be evaluated and mayallow the development and effective testing of newtherapeutic agents.

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