J Clin Pathol: Mol Pathol 1996;49:M247-M255 Reviews Role of free radicals and antioxidants in the pathogenesis of the inflammatory periodontal diseases Iain L C Chapple Introduction The human inflammatory periodontal diseases are amongst the most common of chronic dis- eases to affect adults. In the UK, 69% of adults have early signs of disease and only 5% are completely free from clinical signs of inflam- mation.' The periodontal complex comprises alveolar bone, periodontal ligament, root ce- mentum, and the overlying gingival (gum) tis- sues (fig 1). Gingivitis may be defined as "an inflammatory lesion, mediated by host- parasite interactions, that is confined to the gingival tissues". The major cause of gingivitis is an accumulation of microbial plaque in and around the dento-gingival complex, which, when removed, results in complete resolution of the inflammatory lesion.2 Periodontitis is regarded as "an inflammatory lesion, mediated by complex host-parasite interactions, that leads to the loss of connective tissue attach- ment to root surface cementum and adjacent alveolar bone". There are many forms of peri- odontitis' and the changes associated with periodontitis are irreversible, resulting in tooth loss and substantial morbidity in medically compromised patients, where a focus of infec- tion and subsequent bacteraemia may present a major risk. The mouth possesses a unique hard/soft tis- sue barrier, that separates the internal systems from the external environment. The barrier (fig 1) is called the junctional epithelium and is permeable to external (bacterial) material passing into the adjacent connective tissues and blood stream, and to products of internal defence systems (leucocytes, complement, an- tibodies, pro-inflammatory cytokines, etc.) passing outwards. To assist this vulnerable bar- rier in protecting the underlying host tissues from damage by products of bacterial plaque, a fluid, gingival crevicular fluid (GCF), is produced from beneath the gingival margin. In health, GCF is a serum transudate containing- all components of serum and also polymor- phonuclear leucocyte cells, but during disease many products of the host-parasite conflict enter the fluid, which becomes a true exudate. GCF may be collected non-invasively on paper strips4 (fig 2), providing an ideal medium in which to study the complex bacterial-host interactions (fig 3) that characterise other similar inflammatory disorders. Substantial data are available in the litera- ture on the role of reactive oxygen species (ROS) and antioxidants in disorders such as the inflammatory lung diseases and in chronic immune mediated conditions such as rheuma- toid arthritis. However, remarkably little infor- mation is available on the periodontal diseases, which show many of the pathological features of other chronic inflammatory diseases. The periodontal tissues also provide an ideal medium within which to study mechanisms of ROS mediated tissue damage and of antioxi- dant defence in response to bacterial colonisa- tion, through the non-invasive collection of GCF. This paper, therefore, attempts to review current knowledge of free radical damage and antioxidant defence systems in inflammatory diseases, and to use the inflammatory perio- dontal diseases as a focus for discussion. Emphasis is placed upon the presence of low molecular weight thiols such as reduced glutathione (GSH) and cysteine in fluids that bathe vulnerable epithelial surfaces, and it is postulated that new therapeutic pathways may be found by using cysteine and GSH preserv- ing drugs-for example, N-acetylcysteine, or indeed, inhibitors of the nuclear transcription factor NF-KB. Inflammatory cells and reactive oxygen species There is good evidence arising from studies of defective neutrophil function in Chediak- Higashi syndrome,5 acatalasia,6 Job's syn- drome,5 and chronic granulomatous disease,7 where profound tissue inflammation can lead to periodontal destruction, that the polymor- phonuclear leucocyte has a protective role in the periodontal environment. However, evi- dence is emerging from several studies'-'0 that in early onset forms of periodontitis, polymor- phonuclear leucocytes are functionally acti- vated and exhibit increased free radical pro- duction as measured by luminol dependent chemiluminescence. Indeed, peripheral blood monocytes from patients with periodontitis demonstrate higher prostaglandin E2 produc- tion upon stimulation than those from patients Periodontal Unit, Birmingham School of Dentistry, Faculty of Medicine and Dentistry, The University of Birmingham Correspondence to: Dr I LC Chapple, Birmingham Dental School, St Chads Queensway, Birmingham B4 6NN. Accepted for publication 4 June 1996 M247 on May 4, 2021 by guest. Protected by copyright. http://mp.bmj.com/ Clin Mol Pathol: first published as 10.1136/mp.49.5.M247 on 1 October 1996. Downloaded from
Transcript
J Clin Pathol: Mol Pathol 1996;49:M247-M255
Reviews
Role of free radicals and antioxidants in thepathogenesis of the inflammatory periodontaldiseases
Iain L C Chapple
IntroductionThe human inflammatory periodontal diseasesare amongst the most common of chronic dis-eases to affect adults. In the UK, 69% of adultshave early signs of disease and only 5% arecompletely free from clinical signs of inflam-mation.' The periodontal complex comprisesalveolar bone, periodontal ligament, root ce-mentum, and the overlying gingival (gum) tis-sues (fig 1). Gingivitis may be defined as "aninflammatory lesion, mediated by host-parasite interactions, that is confined to thegingival tissues". The major cause of gingivitisis an accumulation of microbial plaque in andaround the dento-gingival complex, which,when removed, results in complete resolutionof the inflammatory lesion.2 Periodontitis isregarded as "an inflammatory lesion, mediatedby complex host-parasite interactions, thatleads to the loss of connective tissue attach-ment to root surface cementum and adjacentalveolar bone". There are many forms of peri-odontitis' and the changes associated withperiodontitis are irreversible, resulting in toothloss and substantial morbidity in medicallycompromised patients, where a focus of infec-tion and subsequent bacteraemia may presenta major risk.The mouth possesses a unique hard/soft tis-
sue barrier, that separates the internal systemsfrom the external environment. The barrier(fig 1) is called the junctional epithelium and ispermeable to external (bacterial) materialpassing into the adjacent connective tissuesand blood stream, and to products of internaldefence systems (leucocytes, complement, an-tibodies, pro-inflammatory cytokines, etc.)passing outwards. To assist this vulnerable bar-rier in protecting the underlying host tissuesfrom damage by products of bacterial plaque, afluid, gingival crevicular fluid (GCF), isproduced from beneath the gingival margin. Inhealth, GCF is a serum transudate containing-all components of serum and also polymor-phonuclear leucocyte cells, but during diseasemany products of the host-parasite conflictenter the fluid, which becomes a true exudate.GCF may be collected non-invasively on paperstrips4 (fig 2), providing an ideal medium inwhich to study the complex bacterial-host
interactions (fig 3) that characterise othersimilar inflammatory disorders.
Substantial data are available in the litera-ture on the role of reactive oxygen species(ROS) and antioxidants in disorders such asthe inflammatory lung diseases and in chronicimmune mediated conditions such as rheuma-toid arthritis. However, remarkably little infor-mation is available on the periodontal diseases,which show many of the pathological featuresof other chronic inflammatory diseases. Theperiodontal tissues also provide an idealmedium within which to study mechanisms ofROS mediated tissue damage and of antioxi-dant defence in response to bacterial colonisa-tion, through the non-invasive collection ofGCF. This paper, therefore, attempts to reviewcurrent knowledge of free radical damage andantioxidant defence systems in inflammatorydiseases, and to use the inflammatory perio-dontal diseases as a focus for discussion.Emphasis is placed upon the presence of lowmolecular weight thiols such as reducedglutathione (GSH) and cysteine in fluids thatbathe vulnerable epithelial surfaces, and it ispostulated that new therapeutic pathways maybe found by using cysteine and GSH preserv-ing drugs-for example, N-acetylcysteine, orindeed, inhibitors of the nuclear transcriptionfactor NF-KB.
Inflammatory cells and reactive oxygenspeciesThere is good evidence arising from studies ofdefective neutrophil function in Chediak-Higashi syndrome,5 acatalasia,6 Job's syn-drome,5 and chronic granulomatous disease,7where profound tissue inflammation can leadto periodontal destruction, that the polymor-phonuclear leucocyte has a protective role inthe periodontal environment. However, evi-dence is emerging from several studies'-'0 thatin early onset forms of periodontitis, polymor-phonuclear leucocytes are functionally acti-vated and exhibit increased free radical pro-duction as measured by luminol dependentchemiluminescence. Indeed, peripheral bloodmonocytes from patients with periodontitisdemonstrate higher prostaglandin E2 produc-tion upon stimulation than those from patients
Periodontal Unit,Birmingham School ofDentistry, Faculty ofMedicine andDentistry, TheUniversity ofBirmingham
Correspondence to:Dr I L C Chapple,Birmingham Dental School,St Chads Queensway,Birmingham B4 6NN.
Figure I Longitudinal section through a tooth and adjacent periodontal tissues.
Figure 2 Colour slide illustrating the non-invasivecollection ofgingival crevicularfluid.
without the disease, indicating priming ofperipheral leucocytes by the omnipresentlipopolysaccharide of Gram negative patho-gens, or an underlying immune based suscepti-bility to the disease." There seems, therefore,to be a delicate balance between inflammatoryand immune cell hypofunction, where un-
checked pathogens cause direct tissue damage,and hyperfunction, where host defence cellproducts elaborated in an effort to eliminatepathogens inadvertently cause substantial col-lateral host tissue damage (fig 3). Suchproducts can be categorised broadly into enzy-matic mechanisms and those derived from theoxidative burst.Enzyme based mechanisms arise following
degranulation and include enzymes, such as
collagenase, hyaluronidase, elastase, etc.,which are released in an attempt to destroybacteria, but also cause damage to host tissueas a side effect of their actions.The oxygen dependent pathways of phago-
cyte function give rise to the production of free
radical species, which may be defined as anyspecies capable of independent existence thatcontains one or more unpaired electrons.'2 Inrecent years the term reactive oxygen specieshas been adopted to include molecules such ashydrogen peroxide (H202), hypochlorous acid(HOCI) and singlet oxygen (102), which, whilenot radicals in nature, are capable of radicalformation in the extra- and intracellularenvironments.'3 Reactive oxygen species causetissue damage by a variety of different mecha-nisms:* lipid peroxidation (through activation of
cyclooxygenases and lipoxygenases);* DNA damage (base hydroxylations and
strand breaks);* protein damage, including gingival hy-
aluronic acid and proteoglycans 4;* oxidation of important enzymes for exam-
ple, anti-proteases such as oct-l-antitrypsin";and
* stimulation of pro-inflammatory cytokinerelease by monocytes and macrophages, bydepleting intracellular thiol compounds andactivating nuclear factor KB (NF-KB). "
Most reactive oxygen species have extremelyshort half-lives (10-9 to 10-6 seconds),'7 butthey can cause substantial tissue damage byinitiating free radical chain reactions. There areexogenous sources of ROS such as cigarettesmoke and ionising radiation. Endogenousproduction is, however, more pertinent to thepathogenesis of periodontal disease and ariseseither accidentally due to leakage of electronsfrom their carriers within the respiratory chainof mitochondria passing directly ontooxygen,'8 "' or functionally through the genera-tion of oxygen radicals by phagocytes (fig 4).The latter process is thought to be implicatedin the destruction of the connective tissues ofthe periodontium.
THE SUPEROXIDE ANION
Superoxide (02'-) is formed chemically by theaddition of an extra electron to oxygen (where* signifies an unpaired electron):
02 + e- - * 02Polymorphonuclear leucocytes and macro-
phages (and to a lesser extent eosinophils, lym-phocytes and fibroblasts) are examples of cellsthat produce superoxide as an antibacterialagent.2'22 Production in the polymorphonu-clear leucocyte is the result of the so-calledmembrane bound reduced nicotinamide ad-enine dinucleotide phosphate (NADPH) oxi-dase shunt (or hexose monophosphate shunt).This shunt fails to work in chronic granuloma-tous disease (CGD),2' which is why neu-trophils from patients with CGD can engulfopsonised bacteria, but are unable to killcertain strains which are subsequently releasedin a viable state.
Role offree radicals and antioxidants in the pathogenesis of the inflammatory periodontal diseases
Figure 3 Some examples of the complex host-parasite interactions that may contribute to periodontal tissue destruction.TIMP = tissue inhibitor or matrix metalloproteinases; LTB4 = leukotriene B4; PGE-2 = prostaglandin E-2.
gets of periodontal relevance. It can also spon-taneously dismutate in aqueous solution toform hydrogen peroxide and singlet oxygen,which can in turn cause cell damage. Superox-ide has also recently been localised at theruffled border space of osteoclasts, implying a
role in bone matrix degradation at theosteoclast-bone interface.2302 + 02 + 2H '02 + H202
Superoxide can also be converted to themore potent hydroxyl radical by reaction withhydrogen peroxide, a complex procedure, cata-lysed by metal ions (iron-catalysed Haber-Weiss reaction).
O2 -+ H202 Fe2orCu2ions .0OH + OH- +02
Superoxide is either removed from tissues bysuperoxide dismutase (SOD), a process thatcan also be catalysed to form oxygen andhydrogen peroxide, or by spontaneous dismu-tation to hydrogen peroxide. SOD is a vitalenzyme based antioxidant defence mechanismlargely found intracellularly,24 but may also bepresent in very small amounts in the extracellu-lar environment. The hydrogen peroxide thatforms through its action is then removed by a
second enzyme called catalase, which is againlargely intracellular, and found only in smallamounts, if at all, extracellularly. Red bloodcells contain large amounts of catalase and are
believed to act as a sink for hydrogen peroxideand superoxide removal.
2 02 + 2H+ SOD*-. H202 + 02
2H2O2 catalase 2H20 + 02
The role of catalase in the extracellular envi-ronment is performed by a very importantenzyme called glutathione peroxidase (GSH-Px), which is largely selenium dependent andreduces H202 whilst oxidising reduced glu-tathione (GSH) to its oxidised form (GSSG).
2GSH + H202 G GSSG + 2H20
THE HYDROXYL RADICAL
The hydroxyl radical is the most reactive radi-cal known to man and interacts with most bio-logical molecules. It can be formed fromsuperoxide through the iron catalysed Haber-Weiss reaction, or from hydrogen peroxidethrough another transition metal dependentreaction called the Fenton reaction. This reac-
tion is largely dependent upon Fe2+ and Cu2"ions,25 and thus proteins that sequester iron or
copper (for example, albumin, caeruloplasmin,haptoglobin, lactoferrin, and transferrin) are
extremely important antioxidants.2630
Fe2++ H202 * Fe3+ + 0OH + OH-
The hydroxyl radical can stimulate a classicfree radical chain reaction known as lipidperoxidation. When the hydroxyl radical isgenerated close to membrane phospholipids, itattacks the lipid side chains to form radicalintermediates called peroxyl radicals (RO2 ),hydrogen peroxide and lipid hydroperoxides.Arachadonic acid is a preferential target for thehydroxyl radical. The accumulation of hy-droperoxides can disrupt membrane function'2and the hydroperoxides can decompose toform cytotoxic aldehydes." End products ofsuch lipid peroxidations include a unique class
Endotoxins
Direct tissue Complement activity,damage - inflammatory mediators
H202Formed via spontaneous dismutation or viaaction of intracellular superoxide dismutase
OH-Hydroxyl radical formation via
Fenton reactions with Fe2+ or Cu2+
HOCIHypochlorous acid formationvia action of myeloperoxidase
Figure 4 Production of reactive oxygen species following activation ofpolymorphonuclearleucocytes.
of prostaglandin F2-like compounds,32 33 aprostanoid involved in lymphokine production,vasodilation, and osteoclastic resorption ofbone. Lipid peroxidation end products alsocause a dysfunction of Ca2+-ATPase34 and maycause opening of intracellular pores,34 both ofwhich lead to an accumulation of intracellularcalcium. The Ca2' build up causes activation ofCa2' dependent enzymes (proteases and phos-pholipase), which in turn cause cell damage. Inaddition, protein damage can arise via radicalmediated inactivation of membrane boundreceptors and enzymes36 and damage to glyco-proteins.37
Other mechanisms of hydroxyl mediated tis-sue damage involve DNA strand breaks38 andbase hydroxylations,39 40 leading to ATP deple-tion and gene mutations which can in turnresult in malignant transformation or celldeath.4
Mechanisms of preventing hydroxyl radicalinduced tissue damage include the binding oftransition metal ions by the "preventative anti-oxidants" albumin, caeruloplasmin, hap-toglobin, lactoferrin, and transferrin. Scaven-gers of the hydroxyl radical include vitamin C,uric acid42 and thiols, such as reduced glutath-ione20 and cysteine.
HYDROGEN PEROXIDE
Hydrogen peroxide can be produced by perio-dontal bacteria, by phagocytes from theNADPH oxidase shunt and also after dismuta-tion of superoxide. While only a weak oxidant,it is has high potential to produce damage dueto its ability to diffuse freely across cellmembranes43 and undergo Fenton reactionswith transition metals, thereby giving rise tosite directed or site specific generation of'OH.44 It has been proposed that hydrogen per-oxide may act as a metabolic signal by oxidisingprotein thiol groups and triggering intracellu-lar events.43 An example would be the oxida-tion of an important nuclear regulatory proteinNFKB, a process thought to be responsible forthe expression of HIV genes.45The activity of hydrogen peroxide on NF-KB
is also responsible for the transcription of sev-eral pro-inflammatory cytokines46 of im-portance to periodontal disease pathogenesis,including interleukin-2 (IL-2), IL-6, IL-8,1-interferon, and tumour necrosis factor ct(TNFax).4 NF-KB activation (fig 5) can also becaused by bacterial endotoxins, IL- 1 andTNFa.48-51 The process is rapid, as NF-KBexists in the cytoplasm of most inflammatorycells, in a complex with an inhibited form(I-KB). Cytokines such as TNF-oc and IL-I areable to activate NF-KB via protein kinase Cand other kinases, which phosphorylate theI-KB part of the cytoplasmic complex, therebyreleasing free NF-KB rapidly and without theneed for lengthy protein synthesis. The freeNF-KB diffuses from the cytoplasm, across thenuclear membrane and binds to DNA, stimu-lating the transcription of mRNA for thevarious proinflammatory cytokines. Recently,it has been shown that bacterial lipopolysac-charide (LPS/endotoxin) can also activatemacrophage NF-KB and subsequent cytokinetranscription in a protein kinase C independ-ent manner.5' As IL-1 and TNFoc positivelyregulate their own production through theNF-KB system, it is possible that the additiveeffects of endotoxin mediated cytokine produc-tion and that arising from the respiratory burstof polymorphonuclear leucocytes in responseto the same organisms, could lead to substan-tial periodontal inflammation and subsequenttissue destructionHydrogen peroxide is removed from cells by
the action of antioxidant enzymes for exam-ple, catalase, selenium dependent glutathioneperoxidase and some other peroxidases.52
HYPOCHLOROUS ACIDHypochlorous acid is a powerful antibacterialagent53 and is also capable of causing oxidationof plasma membrane thiol (SH) groups anddisruption of certain protein functions,54 even
Role offree radicals and antioxidants in the pathogenesis of the inflammatory periodontal diseases
Table 1 Important extracellular antioxidants, their modes of action, solubility, and locations
Antioxidant Mode of action Solubility Location
Ascorbic acid (vitamin C) Chain breaking (scavenging) Water soluble Plasma, saliva, GCF, CSF, synovial fluidPreventative (binds metal ions)Regenerates at-tocopherol
Alpha-tocopherol (vitamin E) Chain breaking (scavenging) Lipid soluble Plasma, saliva, GCFCarotenoids (vitamin A) Chain breaking (scavenging) Lipid soluble PlasmaAlbumin Preventative (binds metal ions) Water soluble Plasma, saliva, GCF
Binds bilirubin alsoChain breaking (scavenging)
Bilirubin Chain breaking (binds to and protects albumin) lipid soluble PlasmaCaeruloplasmin Preventative (binds metal ions) Water soluble Plasma, saliva, GCFHaptoglobin Preventative (binds metal ions) Water soluble Plasma, GCFTransferrin Preventative (binds Fe"+ ions) Water soluble Plasma, saliva, GCFUric acid Chain breaking (scavenging) Water soluble Plasma, saliva, GCFReduced glutathione (including Chain breaking (scavenging) Water soluble Plasma, alveolar lining fluid of lungs,
cysteine) Substrate for enzyme GSH-Px ?saliva, ?GCF
CSF = cerebrospinal fluid.
at concentrations as low as 10-20 jM. Suchdisruptions include the inactivation of glucoseand amino acid transport systems and the K+ion pump. Cell lysis occurs at higher hypochlo-rous acid concentrations and hypochlorousacid is capable of oxidising a-1-antitrypsin"and is also reported to activate neutrophil col-lagenase."5 In the presence of amino acidshypochlorous acid reacts to form chloramines,which in turn protect cells from the former.33Hypochlorous acid is removed by reaction withthe scavenging antioxidants albumin andascorbic acid.'3
SINGLET OXYGENSinglet oxygen (102) is not a radical as it doesnot contain an unpaired electron. It is formedby an input of energy to 02, which results inthe reversal of spin direction of one of the out-ermost unpaired electrons, from a parallel spinto an opposing spin direction. This renders thesinglet oxygen molecule unstable and morecapable of oxidising other molecules. It ishighly reactive with membrane lipids toproduce peroxides, but information about itsrole in tissue damage is limited, and its role inperiodontal inflammation is unknown.
Antioxidant mechanismsThe body contains a number of protectiveantioxidant mechanisms, whose specific role isto remove harmful oxidants as they form, or torepair damage caused by ROS in vivo. Antioxi-dants may be regarded as those substanceswhich when present at low concentrations,compared with those of an oxidisable sub-strate, will significantly delay or inhibit oxida-tion of that substrate.43
Antioxidants are classified according to theirmode of action (table 1). Important antioxi-dants include: (1) the chain breaking orscavenging antioxidants: vitamin E (a-toco-pherol), vitamin C (ascorbic acid), vitamin A(,-carotene), urate, bilirubin, and those sub-stances containing thiol groups; (2) preventa-tive antioxidants: these function largely bysequestering transition metal ions and prevent-ing Fenton reactions and are therefore largelyproteins by nature (for example, albumin,transferrin, lactoferrin, caeruloplasmin, hap-toglobin, and ascorbic acid); and (3) enzymeantioxidants: these are enzyme systems thatfunction by catalysing the oxidation of other
molecules (for example, SOD, catalase andglutathione peroxidase).The amount of information available relat-
ing to the importance of the body's antioxidantsystems in protecting against such damage issubstantial"44 and yet only two studies56 57 haveinvestigated total antioxidant defence withinbiological fluids in relation to periodontaldisease. Many antioxidants function by morethan one mechanism. The enzyme basedsystems, including SOD, catalase and glutath-ione peroxidase have been mentioned previ-ously and further review will be limited tothose extracellular antioxidants of perceivedimportance in periodontal disease.
CAROTENOIDS (VITAMIN A)Carotenoids, such as 3-carotene, have longdouble bonds to attract and quench radicalattack,58 but currently little is known about theinteractions of carotenoids and reactive oxygenspecies. Vitamin A deficiency has been impli-cated in periodontal destruction with animalmodels but results have not been reproducedin humans.
It is unlikely that individual vitamin deficien-cies have significant detrimental effects on theperiodontium, but the combined effects ofvitamins A, C and E have potential astherapeutic adjuncts in view of their powerfulantioxidant activities.
ASCORBIC ACID (VITAMIN C)Ascorbate protects against oxidants present incigarette smoke4' and is a powerful scavenger ofhypochlorous acid, superoxide, singlet oxygen,and hydroxyl radicals. It also possesses theability to regenerate a-tocopherol from thetocopherol radical that forms at membranesurfaces. GCF concentrations of ascorbic acidhave been reported to be three times higherthan those of plasma59 and it has been shown toprevent activation of neutrophil derived colla-genase55 in GCF. In rheumatoid arthritis60plasma ascorbate concentrations are low butthere is little evidence for any relation betweenplasma ascorbate concentrations and inflam-matory periodontitis,6' though increased gingi-val bleeding is a common result of ascorbatedepletion.6'""
TOCOPHEROLS (VITAMIN E)Alpha-tocopherol is located within cell mem-brane phospholipids and is a major chainbreaking antioxidant64 but has limited mobility,which restricts its efficacy.65 However, manyreactive oxygen species are generated inaqueous solution, particularly those fromphagocytes and the vascular endothelium, andthe role of x-tocopherol in the pathogenesis ofthe periodontal diseases is thus likely to be aminor one. In one study66 no differences weredetected in plasma vitamin E concentrations inpatients with and without periodontal disease,and in another,67 its prostaglandin inhibitoryproperties were credited for reducing perio-dontal inflammation.
URIC ACIDUric acid is a relatively powerful scavengingantioxidant of water soluble radicals,68 such ashypochlorous acid and singlet oxygen. It canalso bind copper and iron ions and increasedconcentrations of uric acid breakdown prod-ucts are reported in patients with rheumatoidarthritis.69 Reaction of uric acid with someradicals (for example, OH) can produce uricacid radicals, but these are easily removed byascorbate.70 It has recently been reported thaturic acid is the major (>70%) antioxidant insaliva56 and while it is also found in GCF, itsantioxidant contribution to GCF has not beeninvestigated.
CYSTEINE AND REDUCED GLUTATHIONEGlutathione is an essential tripeptide withmany important functions. In its reduced form(GSH) glutathione is an important antioxidant(radical scavenger), a property bestowed uponit by its central thiol containing cysteine aminoacid. It is also regarded as a pivotal molecule tothe immune system,7' especially for regulationof IL-2 dependent T-lymphocyte prolif-
72-74eration.The role of reduced glutathione (GSH) in
the regulation of pro-inflammatory cytokines75is of great potential importance in periodontaldisease. There is evidence that increasingcytosolic cysteine (and thus GSH) concentra-tions of monocytes and macrophages (using asynthetic form of cysteine called N-acetyl-cysteine) blocks hydrogen peroxide mediatedactivation of NF-KB (fig 5), and thus produc-tion of pro-inflammatory cytokines45 by thisroute. Cytokines, such as TNFa, IL-lp andIL-6, are associated with the activation of boneresorbing processes7678 and IL-8 is reported tostimulate polymorphonuclear leucocyte activ-ity.79 Normally, intracellular concentrations ofGSH are high (0.1-10 mM),7' but extracellularfluid concentrations are low (2 jM in humanplasma).' However, local production of GSHhas been reported in alveolar lining fluid atvery high concentrations of 400 jiM,80 withconcentrations being raised in smokers anddeficient in patients with pulmonary fibrosis'and acute respiratory distress syndrome.82
Certain putative periodontopathogens arecapable of metabolising L-cysteine or degrad-ing GSH to form hydrogen sulphide within the
periodontal pocket.83-86 The formation of hy-drogen sulphide within the periodontal pocketis toxic to mammalian cells by inactivatingcytochrome oxidase87 and is also reported toinhibit catalase.88 It can be seen how the degra-dation of cysteine by oral microbiota may alsoprevent the inhibition ofNF-KB mediated pro-duction of pro-inflammatory cytokines in theperiodontal environment, thereby increasingthe risk of cytokine related tissue damage.
THE CONCEPT OF TOTAL ANTIOXIDANT CAPACITYAs ROS and antioxidant defence mechanismsseem to act in concert rather than alone, anexample being the re-cycling of a-tocopherolby vitamin C, studies of individual antioxidantsin relation to inflammatory disease may notnecessarily yield useful information. Researchis now being directed towards assays for totalantioxidant capacities of biological fluids, whilenot forgetting the importance of the constitu-ent antioxidant compounds. Wayner et al89have reported the measurement of totalperoxyl radical trapping parameter (TRAPassay), which is sensitive to all known chainbreaking antioxidants but is a complex andtime consuming assay to perform. Our grouphas recently reported a rapid and simpleenhanced chemiluminescence (ECL) assay57 90for total antioxidant quantification in biologi-cal fluids including serum, saliva and GCF.Initial data have revealed a reduced salivarytotal antioxidant concentration in patients withperiodontitis relative to those with periodontalhealth, with no differences in serum concentra-tions. Guarnieri et al9' investigated GCFsuperoxide scavenging capacity in 14 patientswith adult periodontitis and 16 healthy con-trols. They found no difference between testand control groups. The differences in resultsmay be explained by the fact that in this studyGCF was collected by a crevice washing tech-nique that involved 12 washings with amicrosyringe and samples were centrifugedand stored at -20°C prior to assay. It is likelythat the repeated washing and centrifugationwould have resulted in oxidation of severalantioxidant components by the time of assay.Furthermore, data57 are available demonstrat-ing that storage of fluid samples at -20°Cresults in a loss of antioxidant activity withtime, and that storage should be by immersionin liquid nitrogen to prevent oxidation.A local antioxidant response found in GCF
and also in saliva but not in serum, has beenidentified as a low molecular weight (<10 kilo-daltons) thiol, and this response has beenreproduced from the cytosol of anaerobicallystimulated neutrophils and mimicked using L-cysteine and cysteamine.9" It seems logical thatpolymorphonuclear leucocytes should containpowerful intracellular antioxidants in view oftheir high potential for radical generation.However, the discovery of high GSH concen-trations within alveolar lining fluid80 and thedetection of a low molecular weight thiol inGCF9" raises the possibility that the productionof antioxidant thiols at vulnerable epithelialsurfaces is an important defence mechanism
Role offree radicals and antioxidants in the pathogenesis of the inflammatory periodontal diseases
against unwanted ROS mediated damage(whether polymorphonuclear leucocyte medi-ated or from exogenous sources) that iscommon to several organs. Considerable workis needed in this area to elucidate the trueidentity of the GCF antioxidant reported andto investigate the presence of similar thiol anti-oxidants at high concentrations in anatomicalsites that are exposed to similar sources ofpotential damage, such as the cervical epithe-lium.
ConclusionsThere are many similarities between the host-parasite interactions that characterise perio-dontitis and diseases affecting other areas ofthe body, such as inflammatory lung diseaseand rheumatoid arthritis. In the lungs andperiodontal tissues, the external environmentis separated from the internal connectivetissues by a delicate epithelial barrier, behindwhich large scale polymorphonuclear leuco-cyte responses are seen in reaction to variousbacterial activities.93 While the role of the poly-morphonuclear leucocyte is primarily a protec-tive one, host tissue damage can resultindirectly from over exuberant polymorphonu-clear leucocyte and monocyte responses as wellas directly from the colonising pathogensthemselves. Reactive oxygen species producedby inflammatory and immune cells have beenassociated with significant tissue damage.94 Tocombat such damage the body possesses a
variety of antioxidant defence mechanisms,whose role is to protect vital cell and tissuecomponents from radicals of host cell as well as
parasitic origin. Study of local antioxidant sys-
tems is likely to yield valuable informationabout the pathogenesis of certain diseases, andsuch antioxidants should be studied when act-ing in concert as well as in individual systems.
The balance between antioxidant defenceand repair systems, and pro-oxidant mecha-nisms of cell damage may be tipped in favourof tissue destruction either by increases in radi-cal production or by a lowered antioxidantdefence. While the former situation has beendemonstrated in periodontitis, the latter hasnot, as the technology has not been available to
measure total antioxidant defence in GCFuntil recently. Production of high concentra-
tions of the antioxidant thiol, reduced glutath-ione (GSH), have been demonstrated in areas
of the lung where delicate epithelial tissues are
exposed to free radical attack. GSH, is depend-ent upon the amino acid cysteine for itsantioxidant activity, for IL-2 dependent T-cellfunctions and also for other vital anti-inflammatory mechanisms that rely upon
preventing NF-KB translocation to defence cellnuclei. A local low molecular weight thiol hasalso been detected in GCF, which seems to
contribute substantially to its overall antioxi-dant capacity. The kinetics of light recovery forthis antioxidant, in the enhanced chemilumi-nescence assay used for its detection, can bereproduced by L-cysteine which may be of epi-thelial cell as well as polymorphonuclearleucocyte origin. Considerable work is neededto evaluate further local epithelial defence sys-
tems, such that the important anti-in-flammatory mechanisms, in addition to anti-oxidant activities of thiols such as reducedglutathione and cysteine, can be fully under-stood. If the GCF thiol is cysteine or GSH,these data together with those from alveolarlining fluid8' may lead to the development ofnew therapeutic pathways for inflammatorydisease using cysteine and GSH preservingdrugs such as N-acetylcysteine, or indeed,other NF-KB inhibitors.
I would like to express my sincere thanks to Dr J B Matthews forreading this manuscript and for his very helpful comments.
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