Annals ofthe Rheumatic Diseases 1995; 54: 505-5 10 505

Oxygen radicals, nitric oxide and human Ann Rheum Dis: first published as 10.1136/ard.54.6.505 on 1 June 1995. Downloaded from inflammatory joint disease

Barry Halliwell

Interest in the role of free radicals in OH") and important non- derivatives of rheumatoid arthritis (RA) stems from the oxygen such as H202 and hypochlorous acid seminal work of McCord,' who noted the (HOCI). decreased viscosity of synovial fluid in RA patients and showed that a similar decrease could be produced by exposing synovial fluid, in vivo or solutions of hyaluronic acid, to a system The chemical reactivity of oxygen radicals generating superoxide radical, 02-. McCord's varies (table 1). The most reactive is hydroxyl observations led to interest in the use of intra- radical (OH"), which reacts very fast with articular injections of the enzyme almost all molecules in vivo. When OH" is superoxide dismutase as a treatment in RA. formed, it damages whatever it is generated However, the clinical data presented did not next to; it cannot migrate within the cell.4 convince many rheumatologists2 3 and over- enthusiastic interpretations of the data may have led to unwarranted scepticism about the NITRIC OXIDE real role of free radicals in RA. Let us review Whereas OH is probably always harmful, our current knowledge. other (less reactive) free radicals may be useful in vivo. For example, NO is synthesised from L-arginine by many cell types, including Basic definitions chondrocytes.8 However, although human Electrons in atoms and molecules occupy phagocytes can make NO,9 it is not yet clear regions of space termed orbitals, each of which how often they do so in vivo; much of the work holds a maximum of two electrons. A free with NO has investigated rats and mice, both radical is any species capable of independent of which species have phagocytes that make existence that contains one or more unpaired NO much more readily.

electrons-that is, electrons alone in an orbital. http://ard.bmj.com/ Table 1 gives examples. Radicals react with other molecules in a SUPEROXIDE number of ways.4 If two radicals meet, they Superoxide is produced by phagocytes as a can combine their unpaired electrons and killing mechanism.6 Lesser amounts of extra- join to form a covalent bond (a shared pair cellular O2* may be generated, perhaps as an of electrons). An important example is the intercellular signal molecule, by several other

fast reaction of 02- with nitric oxide (also a cell types, including vascular endothelial cells, on September 28, 2021 by guest. Protected copyright. free radical, NO') to form the non-radical osteoclasts, chondrocytes, lymphocytes, and peroxynitrite:5 fibroblasts.""'3 For example, treatment of human fibroblasts with RA synovial fluid O2-- + NO- ONOO- (1) causes 02- secretion. 3 A free radical might donate its unpaired In addition to this 'deliberate' 02 -genera- electron to another molecule. Thus °2- reduces tion, some 02-- iS produced within cells by ferric (Fe3") cytochrome c to ferrous (Fe2+) mitochondria and endoplasmic reticulum, cytochrome c, a reaction often used to assay apparently by the unavoidable 'leakage' of 02- production by activated phagocytes:6 electrons onto oxygen from their correct paths in electron transfer chains and by chemical cyt c (Fe3+) + 02-- ' cyt c (Fe2+) + 02 (2) 'autoxidation' reactions.4 A free radical might take an electron from another molecule, thus oxidising it. For example, 02 oxidises ascorbic acid, a process HYPOCHLOROUS ACID believed to occur in RA:7 Another killing mechanism used by neutro- + - phils (but not macrophages) is the enzyme ascorbate + 02 H+ ascorbate radical myeloperoxidase,14 which uses H202 to oxidise + H202 (3) chloride ions into hypochlorous acid (HOCI), a Left to itself, 02- undergoes the dismutation powerful oxidising and chlorinating agent: reaction: H202 +Cl- - HOCI + OH- (5) 02*- + 02-- +2H+ - H202 + 02 (4) Pharmacology Group, King's College, , H2 02, is not a free University ofLondon, electrons are present). oxide interactions London SW3 6LX, radical (no unpaired Superoxide-nitric United Kingdom The global term reactive oxygen species is often What happens if both 02- and NO are B Halliwell used to include the oxygen radicals (02- and produced at the same site, for example by 506 Halliwell

Table 1 Examples offree radicals Transition metals and hydrogen peroxide Many transition metals have variable oxidation Name Formula Comments numbers: for example iron as Fe2+ or Fe3+, and Ann Rheum Dis: first published as 10.1136/ard.54.6.505 on 1 June 1995. Downloaded from Hydrogen atom *H. The simplest free radical known. copper as Cu+ or Cu24. Changing between Trichloromethyl CC13 A carbon centred radical (the unpaired electron resides on carbon). CC3l is formed during metabolism of these oxidation states transfers single electrons, carbon tetrachloride (CC14) in the liver and for example: contributes to the toxic effects of this solvent. Superoxide 02- An oxygen centred radical. Reacts quickly with a few molecules (such as nitric oxide) but generally not very Fe3+ + e- = Fe2+ (6) reactive. Hydroxyl OH An oxygen centred radical. The most highly reactive Also, for titanium salts: oxygen radical known. When generated in vivo, reacts at its site offormation. Ti4+ + e- = Ti3+ (7) Thiyl RS' General name for a group of radicals with an unpaired electron residing on sulphur. Reactivity varies; often Thus transition metal ions are good react with oxygen to give damaging oxysulphur radicals. promoters of free radical reactions. For Peroxyl, alkoxyl RO2., RO Oxygen centred radicals formed during the breakdown of example, copper, iron and titanium ions react organic peroxides. Oxides ofnitrogen NO, NO2. Both are free radicals. NO is formed in vivo from the with H202 to form OH" radicals:4 amino acid L-arginine. N02 is made when NO reacts with oxygen, and is found in polluted air and smoke from burning organic materials, such as cigarette Cu4+ H202- Cu2+ +OH' + OH- (8) smoke. Ti3+ + H202 -_ Ti4+ + OH + OH- (9) *A superscript dot is used to denote free radical species. H202 is produced in vivo by dismutation of O2- and by several oxidase enzymes, including xanthine oxidase.4 Like 02', H202 can be activated macrophages, or when neutrophils useful in vivo; for example, it is a substrate for adhering to endothelium (a source of NO0) a thyroid peroxidase enzyme that helps make generate 02"-? One possibility is that 02- thyroid hormones."8 It can also regulate gene and NO antagonise each other's bio- expression, for example by activating the logical actions. Inappropriate antagonism of cytoplasmic gene transcription factor NF-KB.'9 NO, by excess 02-, has been suggested H202 is very diffusible within and between to contribute to impaired endothelium cells,4 but if it comes into contact with mediated vasodilatation, for example in transition metal ions, OH will be generated at hypertension. 15 that site and cause immediate damage. The interaction of 02- and NO can also be dangerous.5 The product, peroxynitrite (equation (2)) is not only directly toxic, for Antioxidant defences example by oxidising methionine and protein ENZYMES -SH groups, but also it breaks down to Living organisms have evolved multiple

generate multiple toxic products (figure), in- antioxidant defence systems. Superoxide http://ard.bmj.com/ cluding nitrogen dioxide gas (NO2'), OH' and dismutase (SOD) enzymes remove 02'- by nitronium ion (NO24).5 16 Some of these accelerating its dismutation by about four species will nitrate aromatic amino acids, so orders of magnitude. Human cells have an that formation of nitroaromatics (especially SOD enzyme containing active site manganese nitrotyrosine) is thought to be a 'marker' of (MnSOD) in mitochondria, whereas cytosol peroxynitrite generation.5 17 contains a copper and zinc containing SOD

(CuZnSOD).4 H202 can be destroyed by on September 28, 2021 by guest. Protected copyright. catalases, but the most important H2 02 0; + NO removing enzymes in human cells are gluta- thione peroxidases, which remove H202 by using it to oxidise reduced (GSH) to oxidised glutathione (GSSG): 2GSH + H202- GSSG + 2H20 (10) ONOO- Peroxynitrite

METAL ION SEQUESTRATION H+ Another important antioxidant defence is that iron and copper ions are kept safely protein bound whenever possible, so that OH ONOOH Peroxynitrous acid formation is largely prevented. This is particularly important in extracellular fluids, including synovial fluid, because their levels of antioxidant defence enzymes are low.7 20 21 The value of this sequestration of metal ions is illustrated by an inspection of the severe NO- NO+ NO. Nitrogen dioxide pathology suffered by patients with metal 2 radical overload diseases. For example, in patients Nitrate ion Nitronium with iron overload secondary to idiopathic ion haemochromatosis, transferrin is iron satu- rated and iron ions 'catalytic' for free radical OH- 'OH Hydroxyl radical reactions circulate in the blood.22 Among many Formation and decomposition ofperoxynitrite. other problems, these patients can suffer joint Oxygen radicals, nitric oxide and human inflammatoryjoint disease 507

inflammation,23 illustrating the well known produce 02'-, H202, HOCI, and possibly NO'; relationship between chronic inflammation in excess, these cause damage. Tissue injury and disordered iron metabolism.24 releases iron and copper ions and haem Ann Rheum Dis: first published as 10.1136/ard.54.6.505 on 1 June 1995. Downloaded from proteins (haemoglobin and myoglobin), both catalytic for free radical reactions, from their CI-TOCOPHEROL normal intracellular storage sites;4 29 it also This fat soluble vitamin functions as a chain disrupts electron transport chains in mito- breaking antioxidant in membranes and chondria and endoplasmic reticulum, so that lipoproteins.25 When peroxyl radicals are more electrons leak to oxygen to form 02'- 31 generated during lipid peroxidation (table 1), For such secondary oxidative stress, the key they abstract hydrogen preferentially from the question is 'does it contribute significantly to phenolic -OH group of tocopherol: disease pathology?' -CO2- +TOH---CO2H+TO' (11) This stops peroxyl radicals from attacking an THE CASE OF RA adjacent fatty acid side chain or protein and Following the work of McCord,' I showed that terminates the chain reaction, hence the name hyaluronic acid depolymerisation by 02*- chain breaking antioxidant. The oa-tocopherol generating systems in vitro is caused by iron radical, tocopherol-O, is poorly reactive and dependent formation of OH' from 02- and is widely believed to migrate to the surface H202. 32 The hydroxyl radical causes random of membranes or lipoproteins for conversion fragmentation of hyaluronate, eventually back to a-tocopherol by reaction with ascorbic producing oligosaccharides.33 How could OH' acid. arise in the RA joint? 'Catalytic' copper ions were not detected in fresh synovial fluid,34 but 'catalytic' iron can be measured by the REPAIR SYSTEMS bleomycin assay in about 40% of synovial The antioxidant defences of the human body fluids aspirated from inflamed RA knee are not 100% efficient, so that some free joints,35 and this iron has been directly radical damage occurs in the human body demonstrated to stimulate lipid peroxidation.36 and repair systems are needed. Thus cells In addition, aspiration of synovial fluid from have enzymes that can repair oxidised some RA patients into a solution of phenyl- DNA, degrade free radical damaged proteins, alanine produces a pattern of hydroxylation and remove lipid hydroperoxides from products characteristic of OH' attack upon the membranes.4 aromatic ring,37 suggesting that constituents of RA synovial fluid can lead to OH' formation. The catalytic iron could arise by release from

Oxidative stress dead cells, by H202 mediated degradation of http://ard.bmj.com/ Oxidative stress is said to result when reactive haemoglobin38 (released by traumatic micro- oxygen species are generated in excess in the bleeding in the joint), or by the action of 02'- human body.26 This can occur if antioxidant on synovial fluid ferritin.39 Release ofiron upon concentrations are too low (severe mal- exposure of synovial fluid to 02'-, especially at nutrition, for example, can deplete levels of acidic pH, has been demonstrated.40 The chem- a-tocopherol and ) or if free radical ical pattern of damage to hyaluronate in RA

formation is increased-for example by the synovial fluids (as demonstrated using nuclear on September 28, 2021 by guest. Protected copyright. action of certain toxins.4 magnetic resonance33) is consistent with OH' Cells can tolerate mild oxidative stress, and attack, though hyaluronate may additionally be often respond to it by increased synthesis of secreted as abnormally short chains in RA.4' antioxidant defence enzymes and other protec- Reaction of 02' with NO" is another tive proteins. However, severe oxidative stress potential source of OH', as the synthesis not can cause cell injury or even death; oxidative only of 02'- but also of NO' 42 appears to be damage to DNA, proteins and lipids can increased in RA patients. Demonstration ofthe damage or destroy cells. Cell death induced by presence of nitrotyrosines in patients with oxidative stress can occur by necrosis or apop- active RA43 is consistent with formation of tosis.27 Oxidative stress causes increased intra- peroxynitrite (equation (1)) in vivo.5 cellular free Ca2" 28 and may release intracellular A third source of OH' is reaction of 02' iron to catalyse OH' generation.29 with HOC1,44 both produced by activated phagocytes: + HOCI - + OH- + Cl- Reactive oxygen species in RA 02- 2 (12) GENERAL PRINCIPLES What is the role played by reactive oxygen species in human disease? Some diseases may SOURCES OF OXYGEN DERIVED SPECIES IN RA be caused by oxidative stress, for example the The most discussed source is activated sequelae of overexposure to ionising radiation4 phagocytes. Activated neutrophils liberate and the neurodegeneration produced by 02'-, H202, elastase, HOCI, and eicosanoids, chronic a-tocopherol deficiency.30 and synovial fluid IgG aggregates may activate For most human diseases, however, the neutrophils.45 Hypochlorous acid and 02- oxidative stress is secondary to the primary both react with ascorbate, which may help to disease process.29 For example, tissue injury explain the low levels of ascorbate in RA body recruits and activates neutrophils, which fluids.7 46 Hypochlorous acid inactivates 508 Halliwell

a5-antiproteinase, an important inhibitor of unclear. Hydroxyl radicals degrade isolated serpins (such as elastase), and the amount of proteoglycans' 32 33 and HOC1 fragments active al-antiproteinase is decreased in RA;47 collagen,48 but their effects on intact cartilage Ann Rheum Dis: first published as 10.1136/ard.54.6.505 on 1 June 1995. Downloaded from HOCI also fragments collagen.48 The pannus are probably limited. However, H202 is very contains many macrophage-like cells, pre- diffusible and inhibits cartilage proteoglycan sumably secreting 02*- H202 49 and, possibly, synthesis,72 for example by interfering with NO' (it is not yet clear if neutrophils or macro- ATP synthesis.73 Indeed, intra-articular in- phages in the RA joint make NO"). jection of H2 02 generating systems causes It has also been proposed that the inflamed severe joint damage in animals.74 Hence rheumatoid joint, upon movement and rest, inhibition of cartilage repair systems could undergoes a hypoxia-reperfusion cycle, which aggravate the effects of proteolytic and free may result in free radical generation by several radical mediated cartilage degradation. HOC1 mechanisms.50 It is interesting to note that one can also activate latent forms of neutrophil of these mechanisms is xanthine oxidase,51 52 collagenases and gelatinase, though the extent relating back nicely to the original work of to which this happens in vivo is uncertain.75 McCord.' Chondrocytes are damaged by H202,76 and it has been suggested that low concentrations of H2 02, 02' , or both, accelerate bone DRUG-DERIVED RADICALS resorption by osteoclasts,77 78 whereas NO' A few anti-inflammatory drugs may scavenge inhibits it.79 In addition, ascorbate is essential reactive oxygen species in vivo, but this ability for cartilage function80 and the low concen- is not widespread.53 Indeed, the reverse can be trations found in RA synovial fluid might true: several drugs used in the treatment of RA impair cartilage metabolism. The current might themselves be converted into free interest in the role of tumour necrosis factor ct radicals in vivo. Thus they could suppress the (TNFot) in RA8' may relate to observations signs of RA whilst aggravating oxidative that TNFot causes oxidative stress.82 83 damage. For example, radicals derived from In summary, we do not as yet know the exact penicillamine, phenylbutazone, some fenamic contribution made by reactive oxygen species acids, and the aminosalicylate component of to joint damage in RA. The development of sulphasalazine, might inactivate a1-anti- improved assays of oxidative damage that proteinase, deplete ascorbic acid and acceler- are applicable to humans29 should help to ate lipid peroxidation.5355 address this point and allow a rational selection of for possible therapeutic application.84 Consequences ofoxidative stress in RA I thank the Arthritis and Rheumatism Council for research There is no doubt that oxidative stress occurs support.

in RA patients (table 2).51-71 Lunec et a167 have http://ard.bmj.com/ argued that oxidative damage to IgG generates protein aggregates that can activate neutrophils and set up a 'vicious cycle' of free radical 1 McCord J M. Free radicals and inflammation. Science 1974; 185: 529-31. production. 2 Greenwald R A. Oxygen radicals, inflammation, and Does oxidative stress contribute to cartilage arthritis. Semin Arthritis Rheum 1991; 20: 219-40. 3 Dowling E J, Chander C L, Claxson A W, Lillie C, and bone destruction in RA? The answer is Blake D R. Assessment of a human recombinant

manganese superoxide dismutase in models of inflam- on September 28, 2021 by guest. Protected copyright. mation. Free Radic Res Commun 1993; 18: 291-8. Table 2 Evidence consistent with oxidative stress in rheumatoid disease 4 Halliwell B, Gutteridge J M C. Free radicals in biology and medicine. Oxford: Clarendon Press, 1989. Observation Reference Comment 5 Beckman J S, Chen J, Ischiropoulos H, Crow J P. Oxidative chemistry of peroxynitrite. Methods Enzymol 1994; 233: Increased lipid peroxidation 56 Decreased a-tocopherol (per unit lipid) in 229-40. products in serum and synovial fluid57 is consistent with increased 6 Cumutte J T, Babior B M. Chronic granulomatous disease. synovial fluid lipid peroxidation, as are reports of 'foam cells' Adv Hum Genet 1987; 16: 229-45. containing oxidised low density lipoprotein in 7 Blake D R, Hall N D, Treby D A, Halliwell B, rheumatoid synovium"8 and increased levels of Gutteridge J M C. Protection against superoxide and 4-hydroxy-2-nonenal, a cytotoxic product hydrogen peroxide in synovial fluid from rheumatoid generated by the decomposition oflipid patients. Clin Sci 1981; 61: 483-6. peroxides, in RA.59 8 Palmer R M J, Hickery M S, Charles I G, Moncada S, Depletion of ascorbate in See text Presumably results from oxidation of ascorbate Bayliss M T. Induction of nitric oxide synthase in human serum and synovial fluid during its antioxidant action. Activated chondrocytes. Biochem Biophys Res Commun 1993; 193: neutrophils also take up oxidised ascorbate 398-405. rapidly.60 9 Carreras M C, Pargament G A, Catz S D, Poderoso J J, Increased exhalation of 61 A putative endproduct of lipid peroxidation,4 Boveris A. Kinetics ofnitric oxide and hydrogen peroxide pentane although its validity as an assay is debated.62 production and formation of peroxynitrite during the Increased concentrations of 63 Products measured appear to be endproducts of respiratory burst of human neutrophils. FEBS Lett 1994; uric acid oxidation free radical attack upon uric acid. 341: 65-8. products 10 Rathakrishnan C, Tiku K, Raghavan A, Tiku M L. Release Formation of 2,3-dihydroxy- 65 2,3-DHB appears to be a product of attack of ofoxygen radicals by articular chondrocytes. J Bone Miner benzoate from OH' in Res 1992; 7: 1139-48. (2,3-DHB) upon salicylate patients taking 11 Tiku M L, Liesch J B, Roberston F M. Production of salicylate in increased aspirin.'6 hydrogen peroxide by rabbit articular chondrocytes. amounts Jlmmunol 145: 690-6. Degradation ofhyaluronic - See text. 1990; acid by free radical 12 Key LL Jr, Ries W L, Glasscock H, Rodriguiz R, Jaffe H. mechanisms Osteoclastic superoxide generation. Int J Tissue React Formation of 'fluorescent' 67,68 Fluorescence probably caused by oxidative 1992; XIV: 295-8. proteins damage to amino acid residues in proteins. 13 Meier B, Radeke H H, Selle S, et al. Human fibroblasts Increased steady state levels 69,70 80HdG is a major product ofoxidative damage release reactive oxygen species in response to treatment (in cellular DNA) and to DNA.4 with synovial fluids from patients suffering from arthritis. increased urinary excretion Free Radic Res Commun 1990; 8: 149-60. of 8-hydroxy-deoxyguano- 14 Weiss S J. Tissue destruction by neutrophils. N Engl J' Med sine 1989; 320: 365-76. (8OHdG) 15 Nakazono K, Watanabe N, Matsumo K, et al. Does Increased levels of 'protein 71 Protein carbonyls are an endproduct of oxidative superoxide underly the pathogenesis ofhypertension? Proc carbonyls' in synovial fluid damage to proteins. NatlAcad Sci USA 1991; 88: 10045-8. Oxygen radicals, nitric oxide and human inflammatoryjoint disease 509

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