J. Biol. Buccale, vol. 20. pp. 219 à 224, 1992

FLUORIDE INHIBITION OF SCN' AND CL' PEROXIDASE ACTIVITES INWHOLE SALIVA AND OF RECOMBINANT MYELOPEROXIDASE.INFLUENCE OF pH AND HYDROGEN PEROXIDE CONCENTRATION

A. Van Den ABBEELE, M. POURTOIS and P. COURTOIS

Laboratoire de Stomatologie, Université Libre de Bruxelles, Faculté de Médecine, Campus Erasme, CP 622 ; 808, route de Lennik1070 Brussels, Belgium

(Received October27 1992 ; Accepted December 15 1992)

SUMMARY : Floride (F') inhibition of peroxidase activity in whole saliva and of recombinant human myeloperoxidase wasinvestigated using thiocyanate (SCN') and chloride (CI") as substrates. At pH 5.5, SCN'-linked activity in whole saliva reducedto < 40 % of its initial value at F' concentration of 20 mM, while CI' linked activity was maintained at 90 % of ils initial value forthé same F'concentration. Based on this CI" -linked activity, thé contribution of natural MP to thé SCN' -linked activity in wholesaliva can be calculated. This shows a total inhibition of SCN' dépendant activity of salivary peroxidase (SP) for F'concentrations > 10 mM. At a 20 mM F' concentration, recombinant MP activity reduced to 66 % of its initial value with SCN',against 88 % for CI' as substrate. This inhibition of thé SCN' linked SP activity is enhanced at acid pH for a F~ concentrationof 20 mM : 26 % residual activity at pH 5 for whole saliva + SCN' against 93 % for whole saliva + CI' ; 61 % for recombinantMP + SCN' and 88 % for MP + CI'. Calculated activities for SP alone showed a total inhibition at pH 5, while thé inhibition wasabsent at pH 6.5. F~ inhibition in whole saliva could also be suppressed by thé addition of hydrogen peroxide.

KEY WORDS : Salivary peroxidase - Myeloperoxidase - Fluoride

RESUME : L'inhibition de l'activité péroxydase de la salive totale et de la myélopéroxydase (MP) humaine recombinante parl'ion fluorure (F') a été étudiée pour les substrats thiocyanate (SCN') et chlorure (CI"). A pH 5,5, l'activité liée au SCN' dans lasalive tombait à moins de 40 % de sa valeur initiale pour une concentration de 20 mM en F', alors que pour cette même CF~l'activité liée au CI' se maintenait à 90 %. Cette activité liée au CI' permet de calculer la part de la MP dans l'activité liée auSCN' de la salive totale. Ce calcul montre que l'activité liée au SCN' de la péroxydase salivaire pure (SP) est totalementinhibée pour des concentrations en F' supérieures à 10 mM. Pour une CF' de 20 mM, la MP recombinante tombait quant à elleà 66 % de sa valeur initiale avec le SCN', contre 88 % pour le CI' comme substrat. Cette inhibition de l'activité de la SP liée auSCN' par une concentration de 20 mM en F' est exacerbée à pH acide : 26 % d'activité résiduelle à pH 5 pour la salive totale +SCN' contre 93 % pour la salive totale + CI' ; 61 % pour la MP recombinante + SCN'et 88 % pour la MP + CI'. Les activitéscalculées pour la SP pure montrent une inhibition totale à pH 5 et une suppression de l'inhibition à pH 6,5. L'inhibition par F'peut être levée par l'adjonction de peroxyde d'hydrogène.

MOTS-CLES : Péroxydase salivaire - Myélopéroxydase - Fluorure

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ZUSAMMENFASSUNG : Nach Einschrankung der Peroxidase-Aktivitàt des Vollspeichels und der menschlichenMyeloperoxidase (MP) rekombiniert durch das Fluorid ion (F"), sind aïs Substrate Thiocyanat (SCN") und Chlorid (CI")untersucht worden. Bei einem pH-wert von 5,5 sank die mit SCN" im Vollspeichel verbundene Aktivitat unter 40 % desAusgangswertes und dies bei einer Konzentration von 20 mM F-, wohingegen fur dieselbe CF" die mit dem CI" -substratverbundene Aktivitat auf 90 % erhalten blieb. Dièse mit CI" verbundene Aktivitat ermôglicht es, den Anteil des MP in der mit

SCN- des Vollspeichels verbundenen Aktivitat zu berechnen. Dièse Berechnung zeigt dass die SCN" Aktivitat dièse reinenSpeichel-Peroxidase bei Konzentrationen von F- > 10 mM vbllig gehemmt ist. Bei CF" von 20mM sank die rekombinierte MP mitSCN- aïs Substrat auf 66 % ihres Anffangswertes gegenùber 88 % fur CI'. Dièse Unterbindung der SP, verbunden mit SCN",durch eine Konzentration von 20mM in F-, wird bei sauerem pH noch viel deutlicher : 26 % Residualaktivitat bei pH 5 fur denVollspeichel + SCN" verglichen mit 93 % fur den Vollspeichel + CI'; 61 % fur die rekombinierte MP + SCN" und 88 % fur MP + CI".Dièse fur reine SP berechneten Aktivitaten zeigen eine totale Einschrankung bei pH5 und eine Aufhebung der Hemmung beipH 6,5. Die Unterbindung mit F- kann durch Zufiigung von Wasserstoffsuperoxyd aufgehoben werden.

SCHLÙSSELWÔRTER : Speichel-Peroxidase -Myeloperoxidase - Fluorid.

INTRODUCTION

Peroxidase Systems in human saliva are thought toplay an effect ive rôle in defence against invadingmicroorganisms in thé mouth (Oram et al., 1966 ;Carlsson et al., 1984 ; Lumikari et al.,1991). Whole salivacontains both salivary peroxidase (SP) and myeloperoxi-dase (MP). The latter originates from neutrophils inamounts depending on salivary flow rate and gingivalinflammation (Calonius, 1958 ; Tenovuo, 1976 ; Smith etal., 1984 ; Cao et al., 1989). Both SP and MP can cataly-se thé oxidation of thiocyanate (SCN"), bromide (Br) andiodide (I") into hypothiocyanite (OSCN~), hypobromite(OBr) and hypoïodite (Ol") respectively. MP can alsotransform chloride (CI") into hypochlorite (OCI") (Allen,1975 ; Morrison étal., 1976, Pruitt et al., 1985). AH thèsereactions require thé présence of hydrogen peroxide(H2O2). This reagent is produced by neutrophils(Johnston, 1978), oral bacteria (Kraus et al., 1957 ;Carlsson et al., 1983) and salivary glands (Pruitt et al.,1983). Recently, OSCN" -generating enzyme Systemshâve been incorporated in toothpastes, in an attempt toenhance their prophylactic effects. Thèse Systems, byreducing thé acid production of oral bacteria (Tenovuo etal., 1981) are thought to work in synergy with thé well-known bénéficiai effect of fluoride (F"), which not onlyenhances enamel protection against caries but alsoreduces acid production by bacteria (Hamilton, 1977 ;Tenovuo et al., 1981). However, in 1980, Gabier andLeong showed a possibly négative effect of F" on MP.According to thèse authors, F" would inhibit glucosemetabolism, resulting in a réduction of bacterial H2O2

génération. It could thus deprive MP from one of its sub-strates. Moreover, Thibodeau et al., (1985) showed a pH-dependent inhibition of lactoperoxidase and undifferentia-ted salivary peroxidases : F" inhibition was found to beenhanced at lower pH values. For instance, a F' concen-tration of 0.05 mM was enough to produce a réduction ofactivity of 50 % at pH 4, whereas more than 10 mM F"

were necessary to induce a similar inhibition at pH 7.Since dental plaque can contain millimolar amounts of F"(Jenkins et al., 1977), it could be concluded that thé localperoxidase Systems in dental plaque can be inhibited byF". Since SCN" and Cl'concentrations in whole saliva areboth compatible with peroxidase activities, this studyinvestigated thé effect of F" on SCN" and CI" dépendantperoxidase activities of whole saliva and recombinantMP. The influence of raising thé H202concentration wasalso considered.

MATERIAL AND METHODS

Chemicals

Ail chemicals were of reagent grade. H2O2, NaCI, KSCN,NaF, citric acid monohydrate and 2-mercapto-ethanol were pur-chased from Merck (Darmstadt, Germany); di-thio-bis-nitroben-zoic acid (Nbs)2 from Sigma Chemical Co (St Louis, USA).

Sample collection

Unstimulated whole saliva samples were obtained fom sixadults free of pathology. The samples were collected at 9 AM inice chilled glass tubes ; thé subjects were not allowed to eat ordrink 2 hours before collection. The samples were dialysed for12 hours at 4°C against demineralized water to remove ail SCN"and CI". They were kept at 4°C until use.

Enzyme préparation

The human recombinant myeloperoxidase préparation hadan activity of 250 ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) units/ml ; one ABTS unit corresponding to théamount of enzyme catalysing thé oxidation of 1mM ABTS perminute, under thé expérimental conditions described byMansson-Rahemtulla et al., (1986). This préparation was diluted50 times in a 0.1 mM citrate buffer at pH 5.5.

Peroxidase assay

Peroxidase activities were determined by thé method descri-bed by Mansson-Rahemtulla (1986), using (Nbs)2 as a chromo-gen. The oxidation of thé reduced Nbs by OSCN" or by OCI" was

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Fluoride inhibition of salivary peroxidase

monitored on a Beckman DU 65 spectrophotometer at 412 nmand 30 °C. The molar extinction coefficients of (Nbs)2, depen-ding on pH, were also determined according to ManssonRahemtulla et al., (1986) and were used to express thé activitiesin terms of enzyme units, one enzyme unit (U) representing théamount of enzyme catalysing thé oxidation of 1|̂ mole of (Nbs)2

per minute under thé assay conditions. For SCN" -activity, théreaction médium consisted of 44|am (Nbs)2, 63 |aM mercapto-ethanol, 0.2 mM H2O2 and 4 mM KSCN. For CI" -activity, KSCNwas replaced by 150 mM NaCI. Thèse reagents were suspen-ded in a 100 mM citrate buffer at pH 5.5 unless otherwise indi-cated. Concentrated NaF solutions were added to thé cuvettesto obtain thé indicated final concentrations. For each subjectand each concentration or pH, thé resuit represents thé meanvalue between three measurements.

RESULTS

The différent saliva samples used in this study dis-played variations in initial SCIXT - linked peroxidase activi-ty values ranging from 3.37 to 7.42 mU /l. Owing to thisdisparity, thé mean residual activities after F" additionwere calculated in percent of initial values (100 % thusrepresenting thé activity for each unfluoridated salivasample). The same notation principle was also applied tothé enzyme solutions. In ail cases, thé influence of F" onboth SCN' - or CI' -linked activities proved to be dissimi-lar. As shown in Fig. 1, increasing thé fluoride concentra-tions from 0 to 20 mM clearly resulted in a dose dépen-dent inhibition of thé enzyme activities leading to OSCN"production in whole saliva. Indeed, at pH 5.5 and usingSCN" as substrate, ail six peroxidase activities were inhi-bited by a F" concentration as low as 0.5 mM (p<0.05,Wilcoxon's T test). Moreover, for a F" concentration of 20 mM,thé residual activity dropped to < 40 % (ranging from19.3 to 40.9 %). The threshold value for a 50 % inhibitionwas found at a concentration of 7.5 mM F". In contrast,thé CI" -linked activity, only slightly affected by such F"concentrations, always maintained at least 90 % of itsinitial value, and this inhibition was, moreover, not statis-tically significant. The contribution of MP to thé measu-red total SCN" activity can be calculated according to thémethod of Mansson-Rahemtulla et al., (1986) : (6.64 xNbsCI activity) - 60.1, where both activities are expres-sed in uJvl/min. The SCN" -linked activities due to SP aregiven in Table I. A total inhibition was observed at F"concentrations higher than 10 mM, and a 50 % inhibitionat concentrations lower than 2.5 mM. Since CI" -linkedactivity is produced by neutrophils in thé crevicular fluid,it is interesting to examine whether this particular cataly-sis in whole saliva can be compared with that of MPtaken separately. In addition, thé opportunity to measurethé effect of F" on recombinant MP was welcomed. Fig.2. shows that increasing thé F" concentration up to20mM at pH 5.5 slowed MP activity to 66 % when SCN"was given as a substrate. By contrast, using Cl" as asubstrate, thé MP activity fell only to 85 % of its initialvalue.

Fig. 1. Residual Nbs-SCN" and Nbs-CI" activity of wholesaliva depending on F~ concentration. Mean ± SD ; N = 6.

Table I. Residual Nbs-SCN" activities for MP and SP.

CF-(mM)

00.050.10.512.557.51020

Total activity(%)

10099.997.488.176.567.355.251.344

34.5

MP activity(%)

100101.598.595

98.899.393.894.291.688.9

SP activity(%)

10097.695.683.659.141.124.614.73.3<0

Fig. 2. Residual Nbs-SCN" and Nbs-CI" activity ofrecombinant MP. Mean ± SD.

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A. Van Den Abbeele et al.,

Influence of pH

The influence of variations in pH on thé inhibition ofperoxidase activity by a 20 mM F'concentration was tes-ted on both whole saliva and recombinant MP. Figure 3and 4 synthesize both enzyme activities at pH valuesselected from 5 to 7, with or without 20 mM F'. In ail théwhole saliva samples, thé maximal SCN~ activity wasobserved at pH 6, whether F" was présent or not. F"- induced inhibition was most obvious at pH 5 but wascancelled at pH 6.5 (Fig. 3). MP also showed maximalactivity at pH 6, but thé inhibition of this enzyme by F"proved to be weak, although observable from pH rangingfrom 5 to 7 (Fig. 4). For both whole saliva and recombi-nant MP, thé optimal CI" linked activities were also foundat pH 6 (data not shown). Of interest is thé réduction insalivary peroxidase activity at pH 5, which, incidentallymight aiready threaten enamel integrity. At this particularacidity level, thé residual activity for SON" linked activityin whole saliva fell as low as 26 % of its initial value. Atthé same pH however, both recombinant MP and saliva-mixed MP maintened higher residual activities : 61 % forMP with SONT ; 88 % for MP with CI' and 93 % for salivawith CI' (Table II). Table III gives thé calculated SCN"activities for SP after abstraction of MP activities. Itshows a total inhibition at pH 5 and a suppression of théinhibition at ph >6.5.

Effect of increasing thé H2O2 input on fluodridemediated peroxidase inhibition

H2O2 concentration in saliva is known to be thé limi-ting factor for OSCN" synthesis by peroxidase (Pruitt etal., 1983). This parameter can vary following bacterialgrowth or activity. Accordingly, thé effect of H2C>2concentrations on thé fluoride mediated enzyme inhibi-tion was measured. Saliva samples from thé six donorswere supplemented with five différent H2O2 concentra-tions ranging from 0.2 to 1.6 mM. Residual activities afterthé addition of 20 mM F' are plotted in Fig. 5. Underthèse circumstances, thé F" mediated inhibition could becancelled at a H2O2 concentration of 1.6 mM. The resi-dual activities of H2O2 concentrations ranging from 0.2 to1.6 mM show a linear increase (p<0.001 ).

may negatively influence peroxidase activity. This asser-tion was first documented by Thibodeau et al., (1985) on

Fig. 3. Nbs-SCN" peroxidase activity of whole saliva with orwithout 20 mM F" depending on pH. Mean + SD ; N = 6.

Fig. 4. Nbs-SCN" peroxidase activity of MP with or without20 mM F" depending on pH. Mean 1 SD.

DISCUSSION

Salivary peroxidases are thought to be part of thésalivary arsenal that helps to control thé growth of oralmicroorganisms responsible for caries initiation (Qram etal., 1966 ; Carlsson et al., 1983 ; Carlsson et al., 1984 ;Lumikari et al., 1991). Moreover, thé growth of severalanaerobes isolated from periodontal lésions was alsoinhibited in vitro by peroxidase generated OSCN"(Courtois et al., 1989 ; Majerus et al., 1991). Favourableconditions for peroxidase activity in saliva and dentalplaque could therefore be considered a requirement forthé natural préservation of oral health. Fis a factor that

Table II. Residual Nbs-SCN" and Nbs-CI" activities for wholesaliva and recombinant MP depending on pH.

PH Saliva

Nbs-SCN-

5 2617.85.5 37.918.66 61.3166.5 92.3 + 8.7

Saliva

Nbs-CI"

93.419.19519.1

96.213.995.716.5

MP

Nbs-SCN"

MP

Nbs-Cr

103.7111.2 92.8117.1

61.112.5 87.614.1

81.8110.5 88.9 ±6.483.3 + 6.9 99.1114.586.619.9 108114.181.5113.7 99.6115.6

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Fluoride inhibition of salivary peroxidase

Fig. 5. Residual Nbs-SCN~ activity of whole saliva afteraddition of 20 mM F~ depending on H2O2 concentration.

Table III. Residual Nbs-SCN' activity after 20 mM F- additionfor MP and SP depending on pH.

PH

55.566.57

Total activity MP activity SP activity

2637.961.392.3103.7

89.593.696.995.492.6

0018

88.4> 100

rated by thé calculated Nbs-SCIST activities to MP.Evidently, a possible inhibition of SP would account forlittle when compared with thé otherwise positive rôle of F"in caries prévention. However, it must be asked whetherthé heterogenicity of thé oral environment could locallycreate particular and fragile situations. In thé supragingi-val plaque for instance, H202 is produced byStreptococcus Sp. (Carlsson et al., 1983), which couldinitiate peroxidase activity. However, thé well documen-ted F' concentration at thé same site (Jenkins etal.,1977) will act on thé opposite direction. The occasio-nal long lasting pH dépressions in dental plaque can alsobe considered as resting periods for peroxidases. A sub-ject of concern lies in thé fact that anaerobes could takethis opportunity to establish themselves as major coloni-zers into thé deep layers and so accomplish a positivestep towards thé formation of thé so-called infragingivalplaque. MP, little affected by F" concentrations up to 20mM, might be a factor capable of counteractmg tnisinfluence. This enzyme is normally not aboundant in sali-va until inflammation occurs, but Brayer et ai., (1979)showed an increase in leukocyte count during a brushingperiod with a F" containing toothpaste. The linear H2O2

dépendant decrease in percentage inhibition can be rela-ted to thé findings of Adams et al., (1979) and Thibodeauet al., (1985) that thé pH sensitivity seemed to be due tothé opportunity for F' or HF-binding at thé site whereH2O2 reacts. Since F', besides its direct inhibition of SPactivity, also inhibits H202 excrétion by neutrophils(Gabier et al., 1980) and reduces bacterial metabolism(Hamilton, 1977), resulting in decreased bacterial H2O2

sécrétion (Tenovuo et al., 1981), thé total inhibitory effecton peroxidase Systems in whole saliva may be evenmore important than assessed in vitro.

lactoperoxidase and on total peroxidase activity in saliva.It is corroborated by comparable data in this work but théobservations on recombinant MP allow thé distinctionbetween a F" inhibited SP and MP, almost unaffected atsuch F' concentrations. Thèse results were also corrobo-

In thèse conditions, it may be useful to adjust thénatural antibacterial properties of saliva by préparationscontaining enzymes (i.e.glucose-glucose oxidase ; lacto-peroxidase) that enhance thé production of H2O2 andconsequently of OSCN'.

Ackowledgements : The authors thank Prof. A. Bollen (Department of Applied Genetics, Free University of Brussels) for providing théhuman recombinant mye/operoxidase.

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