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Intermittent sucrose feeding and caries in hamsters
Arrhs oral Bid. Vol. 17, pp. 215-217,
INTERMITTENT
1972. Pergamon
Press. Printed in Great Britain.
SUCROSE FEEDING IN HAMSTERS
AND CARIES
DOROTHEAB. FITZGERALDand R. J. FITZGERALD Institute of Oral Biology, University of Miami, Miami, Florida 33152 and Veterans Administration Hospital, Miami, Florida 33125, U.S.A. SEVERALstudies have compared the effects of sucrose and other carbohydrates on caries activity in rats receiving modifications of cariogenic diet 2000 of KEY= and JORDAN(1964) and it has been concluded that complete or intermittent replacement of dietary sucrose with other carbohydrates results in greater inhibition of caries on the molar smooth surfaces of rats than in the deep molar fissures (LARJEand LARSON 1970; FROSTELL,KEYESand LARSON,1967; GUGGENHEIM et al., 1966). In the hamsters, where the predominant type of caries observed resembles smooth surface caries in rats, major reductions in caries have also resulted when sucrose was completely replaced in Diet 2000 by other carbohydrates, (CAMPBELL and ZINNER,1970; FROSTELL, KEYESand LARSON,1967; KRASSE,1965). Because of the implied relationship between dietary sucrose and smooth surface caries activity in these test systems, it was desirable to determine the effects of intermittent substitution of sucrose with other carbohydrates on caries activity in the hamster since evidence of this particular aspect of the sucrose caries relationship in hamsters was not available. In the present study, glucose or corn starch was used as the sucrose substitute according to the schedule shown in Table 1. Theexperimentalfeeding programmes were started 7 days after weanling hamsters, 19-21 days of age, were infected orally with Streptococcus mutans strain Kl-R and provided with Diet 2000, which contained 56 per cent powdered sucrose, to facilitate implantation of Strep. mutans and initiation of plaque formation. One week prior to the termination of the experiment, oral swabs of 3 animals in each group were incubated overnight at 37°C in Todd-Hewitt broth and the cultures were examined by the fluorescent antibody technique (JABLONand ZINNER,1966) for the detection of the following serotypes : Strep. mutans strains AHT, BHT and GS-5, which correspond to serologic groups a, b, and c of BRATTHALL (1970); Streptococcus sdivarius strain HHT and Actinomyces viscosus strain T-6. The study was terminated after 6 weeks of the experimental dietary programme and the defleshed jaws were stained with 5 per cent aqueous (w/v) Procion Brilliant Red 3 BNS (I.C.I. Organics, Providence, Rhode Island) according to ZINNER, DUANY and GOLAND(1971). Plaque extent was expressed as the number of molar surface units (KEYES, 1959) containing adherent deposits; caries was scored by the method of KEYES(1959). Table 1 shows that complete replacement of sucrose by glucose or corn starch after the first week of the experiment resulted in drastic reductions in caries scores in the 215
216
DOROTHEAB. FITZGERALDAND R. J. FITZGERALD
hamsters, as expected from previously cited reports (Group I vs. Groups II, III and VI). Animals which received sucrose in the diet for only 2 days of each week and glucose or corn starch for the remaining five days of the week showed reductions of approximately 30 and 50 per cent respectively in caries scores, compared to the groups which received the sucrose diet every day (Group I vs. Groups IV and V). Plaque scores of animals receiving any of the carbohydrates on a full time basis (Groups I, II, III) were approximately double those of groups in which two carbohydrates were alternated. Fluorescent antibody studies indicated some qualitative differences in the types of organisms present in cultures of oral swabs taken from the various dietary groups. For example, only cultures from animals which received corn starch in the diet, either alone or with glucose, showed the presence of organisms reacting with T-6 antiserum and most of these same cultures contained rod-shaped organisms which cross-reacted with antiserum to Strep. mutans strain BHT. All cultures were positive for HHT-reacting cocci. Streptococci which reacted with Strep. mutans strain AHT antiserum were present in all animals infected with strain Kl-R but were far more numerous in animals receiving sucrose alone than in any of the other groups. Strain Kl-R belongs to the AHT serological group. The uninfected controls were consistently negative for AHT serotypes. GS-5 serotypes were not found in any of the groups. The lowered plaque scores in animals receiving alternate feedings of two carbohydrates considered together with the results of the fluorescent antibody studies suggest that each carbohydrate supported development of a distinctive plaque microflora which began to be replaced when the alternate carbohydrate was substituted in the diet. Apparently with the cyclical alternation of substrates neither type of plaque was capable of developing to its fullest extent. TABLE 1. PLAQUEAND CARIESSCORESOP HAM.WERS INFECTFDWITH Strep. mutans DIETARYREGIMES
Group I ::I IV V VI Uninfected controls VII VIII IX
Diet
Proportion week
AND
ON
VARIOUS
of No. animals
Plaque score
Caries score 117.3 f 11.5 8.5 + 2.2 3.3 + o-5
Sucrose Glucose Starch Sucrose + glucose Sucrose + starch Starch + glucose
7 7 7 2 5 2 5 2 5
days days days days days days days days days
9 9 8
22.3 f 1*3* 15.2 f 1.8 26-O f 2-4
9
8.8 f 2.0
82.3 f
9
9.4 f
1.3
60.8 + Il.3
9
9-o f
1-o
12.3 i
Sucrose Glucose Starch
7 days 7 days 7 days
5 5 5
* Standard error of mean.
15.0 + 4.0 15.8 + 1-O 18.0 & 5.0
11.5
2.0
13-o f 4.0 5.1 f 1.1 0.8 f O-2
INTERMITTENT SUCROSEFEEDINGAND CARIESIN HAMSTERS
217
Corn starch alone is virtually non-cariogenic in this test system (Group III) and glucose alone is only slightly more cariogenic than starch (Group II), thus most of the caries observed when sucrose was fed for two days of the week must have been due primarily to this sugar. However, the added increment in caries in the sucrose-glucose group over the sucrose-starch group (Group IV vs. Group V), although not statistically significant (p > 0*05), suggests that the cariogenic plaque flora which developed during sucrose feeding may have been able to utilize glucose also to produce added cariogenic effects. The ability of cariogenic plaque streptococci to form intracellular polysaccharides from glucose and metabolize them to lactic acid (GIBBONS and SOCRANSKY, 1962; BERMAN and GIBBONS,1966) and the fact that plaques formed in Strep. mutans infected hamsters receiving Diet 2000 produce acid at the same rate and to the same extent from either glucose or sucrose (CHARLTON,FITZGERALD,and KEYES, 1971) also support this interpretation. The results of these experiments in hamsters and the previously cited studies in rats indicate that effective control even of smooth surface caries, by the use of substitute carbohydrates may be difficult to achieve as long as some sucrose remains in the diet. Acknowledgement-Supported in part by Grant DE-O 2552 the National Institute of Dental Research and by a grant from Warner-Lambert Research Institute.
BERMAN, K. S. and GIBBONS, R. J.
REFERENCES 1966.Iodophilic polysaccharide synthesis by human and rodent
oral bacteria. Archs oral Biol. 11, 533-542. BRAITHALL, D. 1970. Demonstration of five serological groups of streptococci strains resembling Streptococcus mutans. Odont. Revy 21, 143-152. CAMPBELL,R. G. and ZINNER, D. D. 1970. Effect of certain dietary sugars on hamster caries. J. Nutr. 100, 11-20. CHARLTON,G., FITZGERALD, D. B., and KEYES, P. H. 1971. Hydrogen ion activity in dental plaques of hamsters during metabolism
of sucrose, glucose and fructose. Archs oral Biof. 16, 655-661.
FROSTELL, G., KEYES, P. H. and LARSON,R. H. 1967. Effect of various sugars and sugar substitutes on dental caries in hamsters and rats. J. Nutr. 93, 65-76. GIBBONS, R. J. and SOCRANSKY,S. S. 1962. Intracellular polysaccharide storage by organisms in dental plaque. Its relations to dental caries and microbial ecology of oral cavity. Archs oral
Biol. 7, 73-90. GUOGENHEIM,B., KOENIG,K. G., HERZOG, E., and M~~HLEMANN, H. R. 1966. The cariogenicity of different dietary carbohydrates tested on rats in relative gnotobiosis with a streptococcus producing extracellular polysaccharide. Hefv. odont. Acta 10, 101-113. JABLON, J. M. and ZINNER, D. D. 1966. Differentiation of cariogenic streptococci by fluorescent antibody. J. Bact. 92, 1590-1596. KEYES, P. H. 1959. Dental caries in the Syrian hamster. VIII. The induction of rampant caries
activity in albino and golden hamsters. J. dent. Res. 28, 523-533. KEYES, P. H. and JORDAN,H. V. 1964. Periodontal lesions in the Syrian hamster. III. Findings related to an infectious and transmissible component. Archs oral Biol. 9, 377400. KRASSE, B. 1965. The effect of caries-inducing streptococci in hamsters fed diets with sucrose or glucose. Archs oral Biol. 10, 233-238. LARJE, 0. and LARSON, R. H. 1970. Reduction of dental caries in rats by intermittent feeding of sucrose. Archs oral Biol. 15, 805-816. ZINNER, D. D. DUANY, L. F. and GOLAND, P. 1971. Efficacy of a dichloro-S-triazinyl marker in the identification of demineralized enamel in the molar teeth of Syrian hamsters. J.Phar. Therap. Dent.
INTERMITTENT
1972. Pergamon
Press. Printed in Great Britain.
SUCROSE FEEDING IN HAMSTERS
AND CARIES
DOROTHEAB. FITZGERALDand R. J. FITZGERALD Institute of Oral Biology, University of Miami, Miami, Florida 33152 and Veterans Administration Hospital, Miami, Florida 33125, U.S.A. SEVERALstudies have compared the effects of sucrose and other carbohydrates on caries activity in rats receiving modifications of cariogenic diet 2000 of KEY= and JORDAN(1964) and it has been concluded that complete or intermittent replacement of dietary sucrose with other carbohydrates results in greater inhibition of caries on the molar smooth surfaces of rats than in the deep molar fissures (LARJEand LARSON 1970; FROSTELL,KEYESand LARSON,1967; GUGGENHEIM et al., 1966). In the hamsters, where the predominant type of caries observed resembles smooth surface caries in rats, major reductions in caries have also resulted when sucrose was completely replaced in Diet 2000 by other carbohydrates, (CAMPBELL and ZINNER,1970; FROSTELL, KEYESand LARSON,1967; KRASSE,1965). Because of the implied relationship between dietary sucrose and smooth surface caries activity in these test systems, it was desirable to determine the effects of intermittent substitution of sucrose with other carbohydrates on caries activity in the hamster since evidence of this particular aspect of the sucrose caries relationship in hamsters was not available. In the present study, glucose or corn starch was used as the sucrose substitute according to the schedule shown in Table 1. Theexperimentalfeeding programmes were started 7 days after weanling hamsters, 19-21 days of age, were infected orally with Streptococcus mutans strain Kl-R and provided with Diet 2000, which contained 56 per cent powdered sucrose, to facilitate implantation of Strep. mutans and initiation of plaque formation. One week prior to the termination of the experiment, oral swabs of 3 animals in each group were incubated overnight at 37°C in Todd-Hewitt broth and the cultures were examined by the fluorescent antibody technique (JABLONand ZINNER,1966) for the detection of the following serotypes : Strep. mutans strains AHT, BHT and GS-5, which correspond to serologic groups a, b, and c of BRATTHALL (1970); Streptococcus sdivarius strain HHT and Actinomyces viscosus strain T-6. The study was terminated after 6 weeks of the experimental dietary programme and the defleshed jaws were stained with 5 per cent aqueous (w/v) Procion Brilliant Red 3 BNS (I.C.I. Organics, Providence, Rhode Island) according to ZINNER, DUANY and GOLAND(1971). Plaque extent was expressed as the number of molar surface units (KEYES, 1959) containing adherent deposits; caries was scored by the method of KEYES(1959). Table 1 shows that complete replacement of sucrose by glucose or corn starch after the first week of the experiment resulted in drastic reductions in caries scores in the 215
216
DOROTHEAB. FITZGERALDAND R. J. FITZGERALD
hamsters, as expected from previously cited reports (Group I vs. Groups II, III and VI). Animals which received sucrose in the diet for only 2 days of each week and glucose or corn starch for the remaining five days of the week showed reductions of approximately 30 and 50 per cent respectively in caries scores, compared to the groups which received the sucrose diet every day (Group I vs. Groups IV and V). Plaque scores of animals receiving any of the carbohydrates on a full time basis (Groups I, II, III) were approximately double those of groups in which two carbohydrates were alternated. Fluorescent antibody studies indicated some qualitative differences in the types of organisms present in cultures of oral swabs taken from the various dietary groups. For example, only cultures from animals which received corn starch in the diet, either alone or with glucose, showed the presence of organisms reacting with T-6 antiserum and most of these same cultures contained rod-shaped organisms which cross-reacted with antiserum to Strep. mutans strain BHT. All cultures were positive for HHT-reacting cocci. Streptococci which reacted with Strep. mutans strain AHT antiserum were present in all animals infected with strain Kl-R but were far more numerous in animals receiving sucrose alone than in any of the other groups. Strain Kl-R belongs to the AHT serological group. The uninfected controls were consistently negative for AHT serotypes. GS-5 serotypes were not found in any of the groups. The lowered plaque scores in animals receiving alternate feedings of two carbohydrates considered together with the results of the fluorescent antibody studies suggest that each carbohydrate supported development of a distinctive plaque microflora which began to be replaced when the alternate carbohydrate was substituted in the diet. Apparently with the cyclical alternation of substrates neither type of plaque was capable of developing to its fullest extent. TABLE 1. PLAQUEAND CARIESSCORESOP HAM.WERS INFECTFDWITH Strep. mutans DIETARYREGIMES
Group I ::I IV V VI Uninfected controls VII VIII IX
Diet
Proportion week
AND
ON
VARIOUS
of No. animals
Plaque score
Caries score 117.3 f 11.5 8.5 + 2.2 3.3 + o-5
Sucrose Glucose Starch Sucrose + glucose Sucrose + starch Starch + glucose
7 7 7 2 5 2 5 2 5
days days days days days days days days days
9 9 8
22.3 f 1*3* 15.2 f 1.8 26-O f 2-4
9
8.8 f 2.0
82.3 f
9
9.4 f
1.3
60.8 + Il.3
9
9-o f
1-o
12.3 i
Sucrose Glucose Starch
7 days 7 days 7 days
5 5 5
* Standard error of mean.
15.0 + 4.0 15.8 + 1-O 18.0 & 5.0
11.5
2.0
13-o f 4.0 5.1 f 1.1 0.8 f O-2
INTERMITTENT SUCROSEFEEDINGAND CARIESIN HAMSTERS
217
Corn starch alone is virtually non-cariogenic in this test system (Group III) and glucose alone is only slightly more cariogenic than starch (Group II), thus most of the caries observed when sucrose was fed for two days of the week must have been due primarily to this sugar. However, the added increment in caries in the sucrose-glucose group over the sucrose-starch group (Group IV vs. Group V), although not statistically significant (p > 0*05), suggests that the cariogenic plaque flora which developed during sucrose feeding may have been able to utilize glucose also to produce added cariogenic effects. The ability of cariogenic plaque streptococci to form intracellular polysaccharides from glucose and metabolize them to lactic acid (GIBBONS and SOCRANSKY, 1962; BERMAN and GIBBONS,1966) and the fact that plaques formed in Strep. mutans infected hamsters receiving Diet 2000 produce acid at the same rate and to the same extent from either glucose or sucrose (CHARLTON,FITZGERALD,and KEYES, 1971) also support this interpretation. The results of these experiments in hamsters and the previously cited studies in rats indicate that effective control even of smooth surface caries, by the use of substitute carbohydrates may be difficult to achieve as long as some sucrose remains in the diet. Acknowledgement-Supported in part by Grant DE-O 2552 the National Institute of Dental Research and by a grant from Warner-Lambert Research Institute.
BERMAN, K. S. and GIBBONS, R. J.
REFERENCES 1966.Iodophilic polysaccharide synthesis by human and rodent
oral bacteria. Archs oral Biol. 11, 533-542. BRAITHALL, D. 1970. Demonstration of five serological groups of streptococci strains resembling Streptococcus mutans. Odont. Revy 21, 143-152. CAMPBELL,R. G. and ZINNER, D. D. 1970. Effect of certain dietary sugars on hamster caries. J. Nutr. 100, 11-20. CHARLTON,G., FITZGERALD, D. B., and KEYES, P. H. 1971. Hydrogen ion activity in dental plaques of hamsters during metabolism
of sucrose, glucose and fructose. Archs oral Biof. 16, 655-661.
FROSTELL, G., KEYES, P. H. and LARSON,R. H. 1967. Effect of various sugars and sugar substitutes on dental caries in hamsters and rats. J. Nutr. 93, 65-76. GIBBONS, R. J. and SOCRANSKY,S. S. 1962. Intracellular polysaccharide storage by organisms in dental plaque. Its relations to dental caries and microbial ecology of oral cavity. Archs oral
Biol. 7, 73-90. GUOGENHEIM,B., KOENIG,K. G., HERZOG, E., and M~~HLEMANN, H. R. 1966. The cariogenicity of different dietary carbohydrates tested on rats in relative gnotobiosis with a streptococcus producing extracellular polysaccharide. Hefv. odont. Acta 10, 101-113. JABLON, J. M. and ZINNER, D. D. 1966. Differentiation of cariogenic streptococci by fluorescent antibody. J. Bact. 92, 1590-1596. KEYES, P. H. 1959. Dental caries in the Syrian hamster. VIII. The induction of rampant caries
activity in albino and golden hamsters. J. dent. Res. 28, 523-533. KEYES, P. H. and JORDAN,H. V. 1964. Periodontal lesions in the Syrian hamster. III. Findings related to an infectious and transmissible component. Archs oral Biol. 9, 377400. KRASSE, B. 1965. The effect of caries-inducing streptococci in hamsters fed diets with sucrose or glucose. Archs oral Biol. 10, 233-238. LARJE, 0. and LARSON, R. H. 1970. Reduction of dental caries in rats by intermittent feeding of sucrose. Archs oral Biol. 15, 805-816. ZINNER, D. D. DUANY, L. F. and GOLAND, P. 1971. Efficacy of a dichloro-S-triazinyl marker in the identification of demineralized enamel in the molar teeth of Syrian hamsters. J.Phar. Therap. Dent.