$5.00+ 0.00 0003-9969/92
ArchsoralBiol.Vol. 31,No. 4, pp. 245-248,1992 Copyright
Printed in Great Britain. All rights reserved
0
1992 Pergamon
Press plc
INHIBITION OF HUMAN SUBGINGIVAL PLAQUE PROTEASE ACTIVITY BY CHLORHEXIDINE .l’.R. RADFORD,' K. A. HOMER,*M. N. NA~LOR)and D. BEIGHTON* Department of Conservative Dentistry, UMDS, London Bridge, 2Hunterian Dental Research Unit, LHMC, Whitechapel, London El 2AD and ‘Department of Periodontology and Preventive Dentistry, UMDS, London Bridge, SE1 9RT, U.K. (Accepted 23 October 1991) Summary-Subgingival plaque samples from three discrete sites in each of eight patients with adult chronic periodontitis were used to determine the ability of 0.001, 0.01, 0.1 and 1.0 mM chlorhexidine to inhibit bacterial proteolytic activity. This activity was measured by monitoring the increase in relative fluorescence
(excitation and (emissionwavelengths of 495 and 525 nm, respectively) accompanying the degradation of fluorescein isothiocyanate (FITC)-labelled bovine serum albumin or FITC-labelled transferrin. Chlorhexidine at concentrations of as low as 0.01 mM inhibited the proteolytic degradation of both substrates by more than 50%. As the growth of dental plaque bacteria is dependent upon the liberation of nutrients (amino acids, peptides and carbohydrates) from host-derived macromolecules, similar effectsin viva might explain the ability of chlorhexidine to inhibit plaque formation at subminimal inhibitory concentrations. Key words: chlorhexidine, subgingival plaque, protease, transferrin.
INTRODUCTION Chlorhexidine, when applied to the teeth as a mouthwash, gel or varnish, can reduce the rate of formation of supragingival plaque (Lang and Brecx, 1986) and produces significant long-term reductions in the proportions of mutans streptococci (Emilson, 1981). However, when applied supragingivally, it does not exert a significant eflfect on the presence or composition of subgingivarl plaque. Direct application of chlorhexidine into the gingival crevice did, however, reduce the proportions of motile organisms, including spirochaetes, in subgingival plaque (Khoo and Newman 1983). It is not fully understood how chlorhexidine, after a single application, c#anexert an inhibitory effect on plaque formation (rate of bacterial growth). Recent studies have indicated that its mechanism of action involves an initial, rapid killing of bacteria which is only transient; 6-8 h after the application of a 0.1% chlorhexidine rinse, the number of viable bacteria had recovered to pretreatment levels (Netuschil, Reich and Brecx, 1989). The sustained inhibitory effects of chlorhexidine on plaque formation are probably associated with its adso:rption to, and subsequent slow release from, pellicle-‘coated enamel (Jenkins, Addy and Wade, 1988) or mucosal surfaces (Rolla and Melsen, 1975). Many studies have reported the MIC of chlorhexidine for a wide range of dental plaque bacteria (Emilson et al., 1972; Loesche, 1979; Baker et al., 1987, Stanley, Wilson and Newman, 1989). Thus, for Streptococcus sang& ,these range from 0.39 to 128 pg per ml and for Cupnocytophagu spp. from less than Abbreviations: BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; MIC, minimum inhibitory concentration.
32 to 250 pg per ml. The reason for these wide ranges in reported MIC values is that the composition of the bacteriological culture medium will significantly affect the concentration of unbound chlorhexidine available for interaction with the bacterial cells (Stanley et al., 1989). In saliva, the total concentration of chlorhexidine (measured by [‘4C]-chlorhexidine decreases very rapidly to low levels after the use of a chlorhexidine-containing mouthrinse (Bonesvoll et al., 1974). In addition, the proportion of chlorhexidine unbound and available for interaction with dental plaque bacteria is unknown but will, due to the high affinity of chlorhexidine for proteins and hydroxyapatite (Rolla, Loe and Schiott, 1971), be considerably lower than the total chlorhexidine concentration as measured by Bonesvoll et al. (1974). It is therefore likely that the inhibition of plaque formation observed in vivo occurs at concentrations of chlorhexidine below the MIC. We have previously shown that the growth of dental supragingival plaque bacteria in vivo is dependent upon their ability to degrade hostderived macromolecules into assimilable substrates (Beighton, Smith and Hayday, 1986; Smith and Beighton, 1986, 1987). As it is likely that inhibition of this capability will interfere with bacterial growth and plaque formation we have also studied the inhibition of degradation of synthetic protease substrates by pure cultures of dental plaque bacteria (Beighton, Decker and Homer, 1991). As the ability of bacteria to degrade such substrates does not necessarily mean they can hydrolyse intact, native proteins and glycoproteins we have now sought to determine the effects of chlorhexidine on the proteolytic degradation of human transferrin and bovine serum albumin by pooled subgingival plaque. 245
J. R. RAIXXUJet al.
246 MATERIALS
AND METHODS
Subjects
Eight patients who had been referred to the Department of Periodontology and Preventive Dentistry were enrolled for this study. Each patient was suffering from chronic periodontitis with moderate to severe bone loss as shown by panoramic radiography. None of the patients had received periodontal treatment during the previous 3 months, nor did they suffer from any condition that precluded sampling of subgingival plaque. Collection of subgingival plaque
Plaque samples were collected from three discrete tooth sites in each patient. The sampling sites were isolated with cotton-wool rolls and supragingival plaque was removed with curettes and discarded. Subgingival plaque was taken from each site by gently inserting 3-5, size-60 paper points (Absorbent Dental Points, Johnson and Johnson Ltd, Slough, U.K.) into each periodontal pocket until resistance was felt or the points became soggy as a result of absorption of gingival crevicular fluid. The paper points used to collect plaque from one site were pooled and placed in 1.Oml of 50 mM N-[tris(hydroxymethyl)methyl]aminoethane-sulphonic acid (Sigma, Poole, Dorset, U.K.), pH 7.5 (TES buffer), supplemented with 10 mM dithiothreitol (Sigma), frozen at -20°C within 30 min of collection and stored at this temperature until processed. After plaque collection, the probing depth measurement at each site was recorded; these ranged from 4-9 mm and these pockets were invariably associated with bleeding on probing.
For each sample at each concentration of chlorhexidine, after the appropriate control was subtracted, the percentage inhibition of proteolytic activity was calculated (fluorescence of test/fluorescence of control x 100); thus a reduction in the degree of substrate degradation resulted in an increase in the calculated percentage of inhibition. RESULTS
Each of the 24 subgingival plaque samples degraded FITC-transferrin and FITC-BSA, with the amounts of degradation, after 4 h of incubation, being significantly correlated [(r = 0.8086; p < 0.001) Fig. 11. The kinetics of substrate degradation were approximately linear over a 6-h incubation period, irrespective of the rate of substrate hydrolysis, as shown for five representative plaque samples (Fig. 2). Chlorhexidine inhibited the degradation of both FITC-transferrin and FITC-BSA (Table 1); after 4 h of incubation, 50% inhibition of the proteolytic degradation of both FITC-transferrin and FITCBSA occurred with chlorhexidine concentrations of more than 0.01 mM. The inhibition of degradation of
Inhibition of the protease activity of subgingival plaque
The subgingival plaque samples were thawed and each was dispersed by vortexing in the presence of sterile glass beads for 15 s. The subgingival plaque concentration in each sample was approx. 2OOpg (wet weight) per ml. Reaction mixtures were set up with 50 yl of plaque suspension, 50 ~1 of chlorhexidine solution (as chlorhexidine gluconate, Sigma) in TES buffer and 100 ~1 of 50 pg/l substrate solution. The substrates used were FITC-labelled BSA (Sigma) and FITC-labelled transferrin prepared as previously described (Homer and Beighton, 1990) in TES buffer. Each molecule was labelled with less than 10 molecules of FITC, resulting in labelled molecules with low intrinsic fluorescence. As the FITC-labelled molecules were degraded, so the covalently bound FITC molecules were exposed. When irradiated with light of 495 nm, the FITC molecules have a fluorescence maximum at 525 nm and the relative fluorescence is proportional to the degree of protein degradation (Homer and Beighton, 1990). The final concentrations of chlorhexidine were 0.00 1, 0.0 1, 0.1 and 1.0 mM. Control assays were set up without chlorhexidine, or without both chlorhexidine and plaque suspension. The assays were incubated at 37°C for up to 6 h and the proteolytic degradation of FITC-BSA and FITC-transferrin was monitored by determining the increase in fluorescence recorded with an excitation wavelength of 495 nm and an emission wavelength of 525 nm (Homer and Beighton, 1990).
50
0
150
100
200
250
300
FITC-BSA
Fig. 1. Correlation between proteolytic degradation (measured as relative fluorescence) of FITC-transferrin and FITC-BSA by subgingival plaque samples (n = 24) after 4 h of incubation at 37°C (r = 0.8086; p < 0.001). 350 300 fj
250
5 2
200
_
. Sample 1 Sample2 + Sample 3 A Sample 4 q
E 0 3
150
$
100
=
50
I 0
1
2
3
4
5
6
7
Period of incubation (h) Fig. 2. Relationship between proteolytic degradation (measured as relafive fluorescence) of FITC-BSA and period of incubation at 37°C for five representative subgingival plaque samples.
Chlorhexidine inhibition of protease activity Table 1. Effect of chlorhexidine (CHX) on the mean percentage (f SE) inhibition of proteolytic degradation of FITCtransferrin and FITC-BSA by subgingival plaque samples (n = 24) Concentration of CHX (mM) 0.001 0.01 0.1 1.0
Percentage inhibition* of the degradation ~ FITC-transferrin FITC-BSA 42.2 f 52.8 i 94.3 * 95.5 +
4.8 6.0 1.7 1.8
21.2 f 4.4 69.6 & 2.6 91.8 f 1.0 96.0 f 0.8
Results after 4 h incubat:ion at 37°C. *Percentage inhibition of proteolytic activity was calculated as (100 x fluorescence of test/fluorescence of control).
FITC-transferrin was significantly greater in the presence of 0.001 mM chlorhexidine than it was for FITC-BSA (p < 0.001). DIISCUSSION
We show that subgingival plaque samples degrade two different macromolecules: BSA, a model substrate for the detection of protease activity, and transferrin, a glycosylated serum-derived protein. Transferrin, a major component of gingival crevicular fluid (Curtis et al.., 1988), is available for degradation of subgingival plaque bacteria (Curtis et al., 1988). In vitro investigations by Carlsson et al. (1984) have shown that a range of black-pigmented Bacteroides spp. have the ability to degrade transferrin. As both transferrin and BSA were degraded by subgingival plaque samples and the extent of degradation was significantly correlated, either substrate could be used for studying the proteolytic activity of subgingival plaque samples. However, as FITClabelled BSA is commercially available (from Sigma, Poole, Dorset, U.K.) this would be the substrate of choice. The growth of bacteria in the gingival crevice must depend upon the ability of bacteria to obtain nutrients from host-derived macromolecules entering the crevice from the surrounding host tissues (ter Steeg et al., 1988). The degradation of such macromolecules will yield carbohydrate moieties, short peptides and amino acids, allowing a range of different bacterial species to utilize these components. It is probable therefore that the inhibition of the degradative capacity of subgingival plaque will have a significant effect on the size and composition of this bacterial population. Chlorhexidine applied to supragingival plaque reduces its rate of formation and alters its bacteria.1 composition (Emilson, 1981; Lang and Brecx, 1986) and when introduced into the gingival crevice influences the composition of subgingival plaque (Khoo and Newman, 1983). We show that the proteolytic activity of subgingival plaque collected from discrete periodontal sites in subjects with adult chronic periodontitis is significantly inhibited (>50% inhibition) by chlorhexidine at concentrations of 0.01 mM or greater, with this inhibitory effect being more marked towards transferrin at 0.001 mM chlorhexidine. The origin of the proteolytic activity is not known; but there must be a significant bacterial component, given the method of
247
collection of the samples, although there may be a contribution from host-derived proteases. A number of different mechanisms of action have been described by which chlorhexidine might interfere with bacterial viability and growth (Harold et al., 1969; Marsh et al., 1983; Rogers et al., 1987; Netuschil et al., 1989; Beighton et al., 1991). However, any mechanism proposed to explain the longterm inhibitory effects of chlorhexidine on plaque formation must take into account how such effects can be achieved by subminimal inhibitory concentrations of chlorhexidine, despite its concentration being maintained by its slow release from intraoral surfaces (Rolla and Melson, 1975; Jenkins et al., 1988). The concentrations of chlorhexidine that exerted inhibitory effects in our study were significantly below those reported as MIC for many dental plaque bacteria (Emilson et al., 1972; Loesche, 1979; Baker et al., 1987; Stanley et al., 1989). If similar concentrations of chlorhexidine are attainable in vim, it is possible that chlorhexidine may exert an effect on subgingival plaque by inhibiting bacterial proteolytic enzymes. REFERENCES
Baker P. J., Coburn R. A., Genco R. J. and Evans R. T. (1987) Structural determinants of activity of chlorhexidine and alkyl bisbiguanides against the human oral flora. J. dent Res. 66, 1099-1106. Beighton D., Smith K. and Hayday H. (1986) The growth of bacteria and production of exoglycosidic enzymes in dental plaque of macaque monkeys. Archs oral Biol. 31, 829-835. Beighton D., Decker J. and Homer K. A. (1991) Effects of chlorhexidine on proteolytic and glycosidic enzyme activities of dental plaque bacteria. J. clin. Periodont. 18, 85-89.
Bonesvoll P., Lokken P., Rolla G. and Paus P. N. (1974) Retention of chlorhexidine in the human oral cavity after mouth rinses. Archs oral Biol. 19, 209-212. Carlsson J., Herrmann B. F., Holfling J. F. and Sundqvist G. J. (1984) Degradation of albumin, haemopexin, haptoglobulin and transferrin by black-pigmented Bacteroides species. J. med. Microbial. 18, 3946.
Curtis M. A., Griffiths G. S., Price S. J., Coulhurst S. K. and Johnson N. W. (1988) The total protein concentration of gingival crevicular fluid. Variation with sampling time. J. clin. Periodont. 15, 628-632. Emilson C. G. (1981) Effect of chlorhexidine gel treatment on Streptococcus mutans population in human saliva and dental plaque. Stand. J. dent. Res. 89, 239-246. Emilson C. G., Ericson T., Heyden G. and Lilja J. (1972) Effect of chlorhexidine on human oral stretococci. J. periodont. Res. 7, 189-191.
Harold F. M., Baarda J. R., Baron C. and Abrams A. (1969) Dio 9 and chlorhexidine inhibition of membrane-bound ATPase and cation transport in Streptococcus faecalis. Biochem. biophys. Acta. 183, 129-136. Homer K. A. and Beighton D. (1990) Fluorometric determination of bacterial protease activity using fluorescein isothiocyanate-labeled proteins as substrates Analyt. Biothem. 191, 133-137. Jenkins S., Addy M. and Wade W. (1988) The mechanism of action of chlorhexidine. J. c/in. Periodont. 15, 415-424. Khoo J. G. L. and Newman H. N. (1983) Subgingival plaque control by a simplilied oral hygiene regime plus local chlorhexidine or metronidazole. J. clin. Periodont. 18, 6607-6619.
248
J. R.
RADFQRD
Lang N. P. and Brecx M. C. (1986) Chlorhexidine gluconate-an agent for chemical plaque control and prevention of gingival inflammation. J. periodont. Res. 21, (Suppl. 16), 7489. Loesche W. J. (1979) Clinical and microbiological aspects of chemotherapeutic agents used according to the specific plaque hypothesis. .? dent. Res. 58, 2404-2412. _ Marsh P. D.. Keevil C. W.. McDermid A. S.. Williamson M. I. and Ellwood D.’ C. (1983) Inhibition by the antimicrobial agent chlorhexidine of acid production and sugar transport in oral streptococcal bacteria. Archs oral Biol. 28, 233-240. Netuschil L., Reich E. and Brecx M. (1989) Direct measurement of the bactericidal effect of chlorhexidine on human dental plaque. J. ciin. Periodont. 16, 484-488. Rogers A. J., Zilm P. S., Gully N. J. and Pfennig A. L. (1987) Chlorhexidine affects arginine metabolism as well as glycolysis in a strain of Streptococcus sanguis. Oral microbial. Immunol. 2, 178-182.
Rolla G. and Melsen B. (1975) On the mechanism of plaque
et al.
inhibition by chlorhexidine. J. dent.Rex 54, (Special Issue B), 5762. Rolla G., Loe H. and Schiott C. R. (1971) Retention of chlorhexidine in the human oral cavity. Archs oral Eiol. 16, 1109-1116.
Smith K. and Beighton D. (1986) The effects of the availability of diet on the levels of exoglycosidases in the supragingival plaque of macaque monkeys. J. dent. Res. 65, 1349-1352. Smith K. and Beighton D. (1987) Proteolytic activities in the supragingival plaque of monkeys (Macaca Jascicularis). Archs oral Biol. 32, 473476.
Stanley A., Wilson M. and Newman H. N. (1989) The in vitro effects of chlorhexidine on subgingival plaque bacteria. J. c&t. Periodont. 16. 259-264. ter Steeg P. F., van der Hoeven J. S., de Jong M. H., van Munster and Jansen M. J. H. (1988) Modelling the gingival pocket by enrichment of subgingival microflora in human serum in chemostats. Microbial. ecol. Hlth Disease. 1, 73-84.
ArchsoralBiol.Vol. 31,No. 4, pp. 245-248,1992 Copyright
Printed in Great Britain. All rights reserved
0
1992 Pergamon
Press plc
INHIBITION OF HUMAN SUBGINGIVAL PLAQUE PROTEASE ACTIVITY BY CHLORHEXIDINE .l’.R. RADFORD,' K. A. HOMER,*M. N. NA~LOR)and D. BEIGHTON* Department of Conservative Dentistry, UMDS, London Bridge, 2Hunterian Dental Research Unit, LHMC, Whitechapel, London El 2AD and ‘Department of Periodontology and Preventive Dentistry, UMDS, London Bridge, SE1 9RT, U.K. (Accepted 23 October 1991) Summary-Subgingival plaque samples from three discrete sites in each of eight patients with adult chronic periodontitis were used to determine the ability of 0.001, 0.01, 0.1 and 1.0 mM chlorhexidine to inhibit bacterial proteolytic activity. This activity was measured by monitoring the increase in relative fluorescence
(excitation and (emissionwavelengths of 495 and 525 nm, respectively) accompanying the degradation of fluorescein isothiocyanate (FITC)-labelled bovine serum albumin or FITC-labelled transferrin. Chlorhexidine at concentrations of as low as 0.01 mM inhibited the proteolytic degradation of both substrates by more than 50%. As the growth of dental plaque bacteria is dependent upon the liberation of nutrients (amino acids, peptides and carbohydrates) from host-derived macromolecules, similar effectsin viva might explain the ability of chlorhexidine to inhibit plaque formation at subminimal inhibitory concentrations. Key words: chlorhexidine, subgingival plaque, protease, transferrin.
INTRODUCTION Chlorhexidine, when applied to the teeth as a mouthwash, gel or varnish, can reduce the rate of formation of supragingival plaque (Lang and Brecx, 1986) and produces significant long-term reductions in the proportions of mutans streptococci (Emilson, 1981). However, when applied supragingivally, it does not exert a significant eflfect on the presence or composition of subgingivarl plaque. Direct application of chlorhexidine into the gingival crevice did, however, reduce the proportions of motile organisms, including spirochaetes, in subgingival plaque (Khoo and Newman 1983). It is not fully understood how chlorhexidine, after a single application, c#anexert an inhibitory effect on plaque formation (rate of bacterial growth). Recent studies have indicated that its mechanism of action involves an initial, rapid killing of bacteria which is only transient; 6-8 h after the application of a 0.1% chlorhexidine rinse, the number of viable bacteria had recovered to pretreatment levels (Netuschil, Reich and Brecx, 1989). The sustained inhibitory effects of chlorhexidine on plaque formation are probably associated with its adso:rption to, and subsequent slow release from, pellicle-‘coated enamel (Jenkins, Addy and Wade, 1988) or mucosal surfaces (Rolla and Melsen, 1975). Many studies have reported the MIC of chlorhexidine for a wide range of dental plaque bacteria (Emilson et al., 1972; Loesche, 1979; Baker et al., 1987, Stanley, Wilson and Newman, 1989). Thus, for Streptococcus sang& ,these range from 0.39 to 128 pg per ml and for Cupnocytophagu spp. from less than Abbreviations: BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; MIC, minimum inhibitory concentration.
32 to 250 pg per ml. The reason for these wide ranges in reported MIC values is that the composition of the bacteriological culture medium will significantly affect the concentration of unbound chlorhexidine available for interaction with the bacterial cells (Stanley et al., 1989). In saliva, the total concentration of chlorhexidine (measured by [‘4C]-chlorhexidine decreases very rapidly to low levels after the use of a chlorhexidine-containing mouthrinse (Bonesvoll et al., 1974). In addition, the proportion of chlorhexidine unbound and available for interaction with dental plaque bacteria is unknown but will, due to the high affinity of chlorhexidine for proteins and hydroxyapatite (Rolla, Loe and Schiott, 1971), be considerably lower than the total chlorhexidine concentration as measured by Bonesvoll et al. (1974). It is therefore likely that the inhibition of plaque formation observed in vivo occurs at concentrations of chlorhexidine below the MIC. We have previously shown that the growth of dental supragingival plaque bacteria in vivo is dependent upon their ability to degrade hostderived macromolecules into assimilable substrates (Beighton, Smith and Hayday, 1986; Smith and Beighton, 1986, 1987). As it is likely that inhibition of this capability will interfere with bacterial growth and plaque formation we have also studied the inhibition of degradation of synthetic protease substrates by pure cultures of dental plaque bacteria (Beighton, Decker and Homer, 1991). As the ability of bacteria to degrade such substrates does not necessarily mean they can hydrolyse intact, native proteins and glycoproteins we have now sought to determine the effects of chlorhexidine on the proteolytic degradation of human transferrin and bovine serum albumin by pooled subgingival plaque. 245
J. R. RAIXXUJet al.
246 MATERIALS
AND METHODS
Subjects
Eight patients who had been referred to the Department of Periodontology and Preventive Dentistry were enrolled for this study. Each patient was suffering from chronic periodontitis with moderate to severe bone loss as shown by panoramic radiography. None of the patients had received periodontal treatment during the previous 3 months, nor did they suffer from any condition that precluded sampling of subgingival plaque. Collection of subgingival plaque
Plaque samples were collected from three discrete tooth sites in each patient. The sampling sites were isolated with cotton-wool rolls and supragingival plaque was removed with curettes and discarded. Subgingival plaque was taken from each site by gently inserting 3-5, size-60 paper points (Absorbent Dental Points, Johnson and Johnson Ltd, Slough, U.K.) into each periodontal pocket until resistance was felt or the points became soggy as a result of absorption of gingival crevicular fluid. The paper points used to collect plaque from one site were pooled and placed in 1.Oml of 50 mM N-[tris(hydroxymethyl)methyl]aminoethane-sulphonic acid (Sigma, Poole, Dorset, U.K.), pH 7.5 (TES buffer), supplemented with 10 mM dithiothreitol (Sigma), frozen at -20°C within 30 min of collection and stored at this temperature until processed. After plaque collection, the probing depth measurement at each site was recorded; these ranged from 4-9 mm and these pockets were invariably associated with bleeding on probing.
For each sample at each concentration of chlorhexidine, after the appropriate control was subtracted, the percentage inhibition of proteolytic activity was calculated (fluorescence of test/fluorescence of control x 100); thus a reduction in the degree of substrate degradation resulted in an increase in the calculated percentage of inhibition. RESULTS
Each of the 24 subgingival plaque samples degraded FITC-transferrin and FITC-BSA, with the amounts of degradation, after 4 h of incubation, being significantly correlated [(r = 0.8086; p < 0.001) Fig. 11. The kinetics of substrate degradation were approximately linear over a 6-h incubation period, irrespective of the rate of substrate hydrolysis, as shown for five representative plaque samples (Fig. 2). Chlorhexidine inhibited the degradation of both FITC-transferrin and FITC-BSA (Table 1); after 4 h of incubation, 50% inhibition of the proteolytic degradation of both FITC-transferrin and FITCBSA occurred with chlorhexidine concentrations of more than 0.01 mM. The inhibition of degradation of
Inhibition of the protease activity of subgingival plaque
The subgingival plaque samples were thawed and each was dispersed by vortexing in the presence of sterile glass beads for 15 s. The subgingival plaque concentration in each sample was approx. 2OOpg (wet weight) per ml. Reaction mixtures were set up with 50 yl of plaque suspension, 50 ~1 of chlorhexidine solution (as chlorhexidine gluconate, Sigma) in TES buffer and 100 ~1 of 50 pg/l substrate solution. The substrates used were FITC-labelled BSA (Sigma) and FITC-labelled transferrin prepared as previously described (Homer and Beighton, 1990) in TES buffer. Each molecule was labelled with less than 10 molecules of FITC, resulting in labelled molecules with low intrinsic fluorescence. As the FITC-labelled molecules were degraded, so the covalently bound FITC molecules were exposed. When irradiated with light of 495 nm, the FITC molecules have a fluorescence maximum at 525 nm and the relative fluorescence is proportional to the degree of protein degradation (Homer and Beighton, 1990). The final concentrations of chlorhexidine were 0.00 1, 0.0 1, 0.1 and 1.0 mM. Control assays were set up without chlorhexidine, or without both chlorhexidine and plaque suspension. The assays were incubated at 37°C for up to 6 h and the proteolytic degradation of FITC-BSA and FITC-transferrin was monitored by determining the increase in fluorescence recorded with an excitation wavelength of 495 nm and an emission wavelength of 525 nm (Homer and Beighton, 1990).
50
0
150
100
200
250
300
FITC-BSA
Fig. 1. Correlation between proteolytic degradation (measured as relative fluorescence) of FITC-transferrin and FITC-BSA by subgingival plaque samples (n = 24) after 4 h of incubation at 37°C (r = 0.8086; p < 0.001). 350 300 fj
250
5 2
200
_
. Sample 1 Sample2 + Sample 3 A Sample 4 q
E 0 3
150
$
100
=
50
I 0
1
2
3
4
5
6
7
Period of incubation (h) Fig. 2. Relationship between proteolytic degradation (measured as relafive fluorescence) of FITC-BSA and period of incubation at 37°C for five representative subgingival plaque samples.
Chlorhexidine inhibition of protease activity Table 1. Effect of chlorhexidine (CHX) on the mean percentage (f SE) inhibition of proteolytic degradation of FITCtransferrin and FITC-BSA by subgingival plaque samples (n = 24) Concentration of CHX (mM) 0.001 0.01 0.1 1.0
Percentage inhibition* of the degradation ~ FITC-transferrin FITC-BSA 42.2 f 52.8 i 94.3 * 95.5 +
4.8 6.0 1.7 1.8
21.2 f 4.4 69.6 & 2.6 91.8 f 1.0 96.0 f 0.8
Results after 4 h incubat:ion at 37°C. *Percentage inhibition of proteolytic activity was calculated as (100 x fluorescence of test/fluorescence of control).
FITC-transferrin was significantly greater in the presence of 0.001 mM chlorhexidine than it was for FITC-BSA (p < 0.001). DIISCUSSION
We show that subgingival plaque samples degrade two different macromolecules: BSA, a model substrate for the detection of protease activity, and transferrin, a glycosylated serum-derived protein. Transferrin, a major component of gingival crevicular fluid (Curtis et al.., 1988), is available for degradation of subgingival plaque bacteria (Curtis et al., 1988). In vitro investigations by Carlsson et al. (1984) have shown that a range of black-pigmented Bacteroides spp. have the ability to degrade transferrin. As both transferrin and BSA were degraded by subgingival plaque samples and the extent of degradation was significantly correlated, either substrate could be used for studying the proteolytic activity of subgingival plaque samples. However, as FITClabelled BSA is commercially available (from Sigma, Poole, Dorset, U.K.) this would be the substrate of choice. The growth of bacteria in the gingival crevice must depend upon the ability of bacteria to obtain nutrients from host-derived macromolecules entering the crevice from the surrounding host tissues (ter Steeg et al., 1988). The degradation of such macromolecules will yield carbohydrate moieties, short peptides and amino acids, allowing a range of different bacterial species to utilize these components. It is probable therefore that the inhibition of the degradative capacity of subgingival plaque will have a significant effect on the size and composition of this bacterial population. Chlorhexidine applied to supragingival plaque reduces its rate of formation and alters its bacteria.1 composition (Emilson, 1981; Lang and Brecx, 1986) and when introduced into the gingival crevice influences the composition of subgingival plaque (Khoo and Newman, 1983). We show that the proteolytic activity of subgingival plaque collected from discrete periodontal sites in subjects with adult chronic periodontitis is significantly inhibited (>50% inhibition) by chlorhexidine at concentrations of 0.01 mM or greater, with this inhibitory effect being more marked towards transferrin at 0.001 mM chlorhexidine. The origin of the proteolytic activity is not known; but there must be a significant bacterial component, given the method of
247
collection of the samples, although there may be a contribution from host-derived proteases. A number of different mechanisms of action have been described by which chlorhexidine might interfere with bacterial viability and growth (Harold et al., 1969; Marsh et al., 1983; Rogers et al., 1987; Netuschil et al., 1989; Beighton et al., 1991). However, any mechanism proposed to explain the longterm inhibitory effects of chlorhexidine on plaque formation must take into account how such effects can be achieved by subminimal inhibitory concentrations of chlorhexidine, despite its concentration being maintained by its slow release from intraoral surfaces (Rolla and Melson, 1975; Jenkins et al., 1988). The concentrations of chlorhexidine that exerted inhibitory effects in our study were significantly below those reported as MIC for many dental plaque bacteria (Emilson et al., 1972; Loesche, 1979; Baker et al., 1987; Stanley et al., 1989). If similar concentrations of chlorhexidine are attainable in vim, it is possible that chlorhexidine may exert an effect on subgingival plaque by inhibiting bacterial proteolytic enzymes. REFERENCES
Baker P. J., Coburn R. A., Genco R. J. and Evans R. T. (1987) Structural determinants of activity of chlorhexidine and alkyl bisbiguanides against the human oral flora. J. dent Res. 66, 1099-1106. Beighton D., Smith K. and Hayday H. (1986) The growth of bacteria and production of exoglycosidic enzymes in dental plaque of macaque monkeys. Archs oral Biol. 31, 829-835. Beighton D., Decker J. and Homer K. A. (1991) Effects of chlorhexidine on proteolytic and glycosidic enzyme activities of dental plaque bacteria. J. clin. Periodont. 18, 85-89.
Bonesvoll P., Lokken P., Rolla G. and Paus P. N. (1974) Retention of chlorhexidine in the human oral cavity after mouth rinses. Archs oral Biol. 19, 209-212. Carlsson J., Herrmann B. F., Holfling J. F. and Sundqvist G. J. (1984) Degradation of albumin, haemopexin, haptoglobulin and transferrin by black-pigmented Bacteroides species. J. med. Microbial. 18, 3946.
Curtis M. A., Griffiths G. S., Price S. J., Coulhurst S. K. and Johnson N. W. (1988) The total protein concentration of gingival crevicular fluid. Variation with sampling time. J. clin. Periodont. 15, 628-632. Emilson C. G. (1981) Effect of chlorhexidine gel treatment on Streptococcus mutans population in human saliva and dental plaque. Stand. J. dent. Res. 89, 239-246. Emilson C. G., Ericson T., Heyden G. and Lilja J. (1972) Effect of chlorhexidine on human oral stretococci. J. periodont. Res. 7, 189-191.
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