4rchs oral Bid.
Vol.21.pp.703to704 Pergamon Press 1976. Printed m GreatBrltam.
SHORT COMMUNICATIONS EFFECT OF FLUORIDE ON MOLECULAR SIZE OF PROTEOGLYCANS IN THE RAT INCISOR TOOTH J. W. SMALLEY and G. EMBERY Deps.rtment of Dental Sciences, University of Liverpool, P.O. Box 147, Liverpool, England Summary-Proteoglycans were extracted from the incisors of fluorotic rats and examined by gel-filiration on Sepharose 2B. In contrast to the proteoglycans isolated from the pulp, those isolated from the hard tissue elements exhibited a broad range of molecular size.
Although the literature contains much information about the relationship between fluoride and the inorganic phase of the tooth relatively little is known concerning the influence of fluoride on the organic constituents. In an electron microscope study, Walton and Eisenmann (1975) suggested that the organic matrix of rat incisor dentine undergoes extensive alteration during fluorosis. Lemperg and Rosenquist (1974) found decreased acidic mucopolysaccharides in fluorotic rat cortical bone and, in an autoradiographic study, Belanger et al. (1957) reported increase in [35S]-sulphate uptake into the mineralised tissues of bones and teeth. There is little biochemical evidence to explain these changes, particularly in teeth. We have attempted to determine the biochemical changes which occur in rat teeth during fluorosis directing particular attention at the metabolism of the sulphated glycosaminoglycans. During this study, we made an interesting observation on the metabolism of the proteoglycans of the rat incisor, a finding which was clearly related to the fluorotic condition. Proteoglycans are present in dentine (Jones and Leaver, 1974) and dental pulp (G. Embery, unpublished) and are glycosamino,glycans bound covalently to a protein core; they thus feature as high molecular-weight carbohydrate-protein complexes. They contain hexuronic acid which may be used as a convenient marker for these compounds during analysis. Their role as aggregate units has been shown in cartilage (Muir and Jacobs, 196’7; Sadjera and Hascall, 1969) and we were interested to observe if the proteoglycans obtained from the teeth of fluorotic rats had undergone any alteration in molecular size as determined by their gel-filtration properties, an approach which has been used extensively to investigate their metabolism in cartilage (Hardingham and Muir, 1972). Two groups of Harvard albino rats aged eight weeks were allowed free access to drinking water containing NaF at dose levels of 22 and 45 parts/lo6 (ppm) F- respectively. A further group of eight animals were given unfluoridated water and served as controls. The three groups were fed a standard diet (Spillers Ltd.) ad libitum. The 22 and 45 parts/lo6 fed groups became fluorotic after four and three weeks respectively, as evidenced by the degree of enamel mottling during incisor fluorosis (Lindemann, 1967). The animals were killed at this stage
by chloroform overdose and the upper and lower incisors were removed and cleared of adhering connective tissue. The teeth were split longitudinally and the pulps were isolated and stored at -4°C until sufficient material for analysis was obtained. The remainder of the tooth, termed the hard tissue elements, was treated separately from the pulp. To overcome the practical difficulties involved and to conserve the small amounts of proteoglycans available, no attempt was made to separate the enamel from the dentine as earlier studies (Embery, 1974) showed absence of sulphate or uranic acid in rat enamel. The pulps and hard tissue samples were then treated separately as follows: Samples were placed in dialysis sacs (Visking) and immersed in 7.5 per cent (w/v) EDTA for four days at 4°C. Excess EDTA was removed by exhaustive dialysis against distilled water and proteoglycans were extracted from the demineralised material by placing the dialysis sacs in 2 M CaCl, solution for 48 hr at 4°C (Gregory et al., 1970). Residual CaCl, was removed by dialysis against distilled water and the proteoglycans were precipitated by the addition of cetyl pyridinium chloride (CPC). The precipitate was recovered by centrifugation at 31,000 g for 30 min. The CPCproteoglycan complex was dialysed against 2 M NaCl to remove the dissociated CPC. Further exhaustive dialysis against distilled water removed the 2 M NaCl and the proteoglycan was finally recovered by lyophilisation. Proteoglycan samples were dissolved in 0.5 M sodium acetate (pH 6.8) and applied to a column of Sepharose 2B (30 x 0.9 cm; Pharmacia Products) previously equilibriated in 0.5 M sodium acetate at 4°C. The column was eluted using the same buffer at a flow rate of 5 ml/hr and fractions (1 ml) were collected and analysed for hexuronic acid (Bitter and Muir, 1962) and protein by ultra violet absorption at 280 nm (Fig. 1). The results represent the changes occurring in the 45 parts/lo6 F- rats, although substantially the same result was found at the 22 parts/lo6 F- dose level, as did a repeat of the whole experiment. Examination of the pulp extract revealed no apparent differences between the test and control animals as evidenced by the high molecular-weight nature of proteoglycans obtained from each group and thus suggested that fluoride has no apparent influence on 703
.I. W. Smalley
704 Protein A280nm
lkonic acid A530nm _---_
mation of incompletely formed proteoglycan subunits, it is evident that fluoride hinders the normal synthetic events in the mineralised elements of the rat incisor. We have established that the glycosaminoglycan component of the proteoglycan is chondroitin 4’ sulphate (Smalley and Embery, 1976).
(1. Pulp Control 02
A
t
and G. Embery
A :: I ,
Acknow(e~~ements-This investigation was supported by financial award to J.W.S. from the Borrow Dental Milk Foundation.
b. Pulptest
REFERENCES
c. Hord tissue Control
d. HardtissueteSt
Vb
Fig. 1. Gel filtration
Eluate,
ml
profiles of proteoglycans ose 2B.
on Sephar-
the metabolism
of the proteoglycans present in the dental pulp (Fig. la, b). In contrast, the results obtained from the hard tissue extract showed that the proteoglycans isolated from the fluorotic teeth had undergone a major alteration in molecular size as indicated by the presence in the elution profile of
a range of lower molecular weight components (Fig. Id). The reason for this effect is not yet apparent although the findings closely parallel those described for cartilage by Rokosova, Hanson and Bentley (1973) who demonstrated the presence of proteoglycan subunits following removal of a glycoprotein-aggregating factor. Although it is difficult to equate our results with those obtained for cartilage, it is tempting to speculate that fluoride interferes with the aggregation of the sub-units into a larger complex. The effect could presumably occur during the anabolic events of tissue turnover rather than leading to a breakdown of established tissue. On the other hand, Lin, Mosteller and Hardesty (1966) have shown that fluoride inhibits protein synthesis during the initiation of new protein chains and Bleiberg, Zauderer and Baglioni (1972) have indicated that fluoride is involved in the disaggregation of ribosome sub-units. Moreover, Basford, Patterson and Kruger (1976) showed that fluoride inhibits the synthesis of enamel matrix protein in rat incisor ameloblasts. It is possible, therefore, that fluoride influences the structure of the protein core of proteoglycans, leading to their inability to associate into the native proteoglycan complex. Whether the effect is ultimately the physical hindrance of the sub-unit aggregation or causes the for-
Basford K. E., Patterson C. M. and Kruger B. J. 1976. Multivariate analyses of the influence of mottling doses of fluoride on the amino acids of enamel matrix protein of rat incisors. Archs oral Biol. 21, 121-129. Belanger L. F., Visek W. J., Lotz W. E. and Comar C. L. 1957. The effects of fluoride feeding on the organic matrix of bones and teeth of pigs as observed by autoradiography after in oitro uptake of 45Ca and 35S. J. biophys. biochem. Cytol. 3, 559-566. Bitter T. and Muir H. M. 1962. A modified uranic acid carbazole reaction. Analyt. Biochem. 4, 33&334. Bleiberg I., Zauderer M. and Baglioni C. 1972. Reversible disaggregation by NaF of membrane-bound polyribosomes of mouse myeloma cells in tissue culture. Biochim. biophys. Acta 249, 453464. Embery G. 1974. The isolation of chondroitin 4-35 sulphate from the molar teeth of young rats receiving sodium %lphate. Cnlc. 7?ss. Res. 14, 5945. Gregory J. D., Sadjera S. W., Hascall V. C. and Dziewiatkowski D. D. 1970. The proteoglycans of bovine nasal cartilage: dissociative extraction. In: Chemistry and Molecular Biology of the Intercellular Matrix, (Edited by Balazs E. A.) Vol. 2. pp. 843-849. Academic Press, London. Hardingham T. and Muir H. M. 1972. Biosynthesis of glycosaminoglycans in cartilage slices. Fractionation by gelchromatography and equilibrium density gradient centrifugation. Biochem. J. 126, 791-803. Jones I. L. and Leaver A. G. 1974. Studies on the minor components of the organic matrix of human dentine. Archs oral Biol. 19, 371-380. Lemperg R. K. and Rosenquist J. B. 1974. Effects of supply and withdrawal of fluoride. 3. Concentration of acid glycosaminoglycans and hydroxyproline in cortical bone. Acta path. microbial. stand. 82, 435444. Lin S., Mosteller R. and Hardesty B. 1966. The mechanism of sodium fluoride and cycloheximide inhibition on haemoglobin biosynthesis in the cell-free reticulocyte system. J. molec. Biol. 21, 51-69. Lindemann G. 1967. Pigment alterations and other disturbances in the rat incisor enamel in chronic fluorosis and in recovery. Acta odont. stand. 25, 525-530. Muir H. M. and Jacobs S. 1967. Protein-polysaccharides of pig laryngeal cartilage. Biochem. J. 103, 3677374. Rokosova B., Hanson A. N. and Bentley J. P. 1973. Evidence for the presence of a low molecular weight proteoglycan aggregation factor in rabbit ear cartilage. Biochim. biophys. Acta 320, 442452. Sadjera S. W. and Hascall V. C. 1969. Protein-polysaccharide complex from bovine nasal cartilage. A comparison of low and high shear extraction procedures. J. biol. Chem. 244, 77-87. Smalley J. W. and Embery G. 1976. Chemical studies on the influence of fluoride on the organic matrix of rat teeth. J. dent. Res. (in press). Walton R. E. and Eisenmann D. R. 1975. Ultrastructural examination of dentine formation in rat incisors following multiple fluoride injections. Archs oral Biol. 20, 485+x.
Vol.21.pp.703to704 Pergamon Press 1976. Printed m GreatBrltam.
SHORT COMMUNICATIONS EFFECT OF FLUORIDE ON MOLECULAR SIZE OF PROTEOGLYCANS IN THE RAT INCISOR TOOTH J. W. SMALLEY and G. EMBERY Deps.rtment of Dental Sciences, University of Liverpool, P.O. Box 147, Liverpool, England Summary-Proteoglycans were extracted from the incisors of fluorotic rats and examined by gel-filiration on Sepharose 2B. In contrast to the proteoglycans isolated from the pulp, those isolated from the hard tissue elements exhibited a broad range of molecular size.
Although the literature contains much information about the relationship between fluoride and the inorganic phase of the tooth relatively little is known concerning the influence of fluoride on the organic constituents. In an electron microscope study, Walton and Eisenmann (1975) suggested that the organic matrix of rat incisor dentine undergoes extensive alteration during fluorosis. Lemperg and Rosenquist (1974) found decreased acidic mucopolysaccharides in fluorotic rat cortical bone and, in an autoradiographic study, Belanger et al. (1957) reported increase in [35S]-sulphate uptake into the mineralised tissues of bones and teeth. There is little biochemical evidence to explain these changes, particularly in teeth. We have attempted to determine the biochemical changes which occur in rat teeth during fluorosis directing particular attention at the metabolism of the sulphated glycosaminoglycans. During this study, we made an interesting observation on the metabolism of the proteoglycans of the rat incisor, a finding which was clearly related to the fluorotic condition. Proteoglycans are present in dentine (Jones and Leaver, 1974) and dental pulp (G. Embery, unpublished) and are glycosamino,glycans bound covalently to a protein core; they thus feature as high molecular-weight carbohydrate-protein complexes. They contain hexuronic acid which may be used as a convenient marker for these compounds during analysis. Their role as aggregate units has been shown in cartilage (Muir and Jacobs, 196’7; Sadjera and Hascall, 1969) and we were interested to observe if the proteoglycans obtained from the teeth of fluorotic rats had undergone any alteration in molecular size as determined by their gel-filtration properties, an approach which has been used extensively to investigate their metabolism in cartilage (Hardingham and Muir, 1972). Two groups of Harvard albino rats aged eight weeks were allowed free access to drinking water containing NaF at dose levels of 22 and 45 parts/lo6 (ppm) F- respectively. A further group of eight animals were given unfluoridated water and served as controls. The three groups were fed a standard diet (Spillers Ltd.) ad libitum. The 22 and 45 parts/lo6 fed groups became fluorotic after four and three weeks respectively, as evidenced by the degree of enamel mottling during incisor fluorosis (Lindemann, 1967). The animals were killed at this stage
by chloroform overdose and the upper and lower incisors were removed and cleared of adhering connective tissue. The teeth were split longitudinally and the pulps were isolated and stored at -4°C until sufficient material for analysis was obtained. The remainder of the tooth, termed the hard tissue elements, was treated separately from the pulp. To overcome the practical difficulties involved and to conserve the small amounts of proteoglycans available, no attempt was made to separate the enamel from the dentine as earlier studies (Embery, 1974) showed absence of sulphate or uranic acid in rat enamel. The pulps and hard tissue samples were then treated separately as follows: Samples were placed in dialysis sacs (Visking) and immersed in 7.5 per cent (w/v) EDTA for four days at 4°C. Excess EDTA was removed by exhaustive dialysis against distilled water and proteoglycans were extracted from the demineralised material by placing the dialysis sacs in 2 M CaCl, solution for 48 hr at 4°C (Gregory et al., 1970). Residual CaCl, was removed by dialysis against distilled water and the proteoglycans were precipitated by the addition of cetyl pyridinium chloride (CPC). The precipitate was recovered by centrifugation at 31,000 g for 30 min. The CPCproteoglycan complex was dialysed against 2 M NaCl to remove the dissociated CPC. Further exhaustive dialysis against distilled water removed the 2 M NaCl and the proteoglycan was finally recovered by lyophilisation. Proteoglycan samples were dissolved in 0.5 M sodium acetate (pH 6.8) and applied to a column of Sepharose 2B (30 x 0.9 cm; Pharmacia Products) previously equilibriated in 0.5 M sodium acetate at 4°C. The column was eluted using the same buffer at a flow rate of 5 ml/hr and fractions (1 ml) were collected and analysed for hexuronic acid (Bitter and Muir, 1962) and protein by ultra violet absorption at 280 nm (Fig. 1). The results represent the changes occurring in the 45 parts/lo6 F- rats, although substantially the same result was found at the 22 parts/lo6 F- dose level, as did a repeat of the whole experiment. Examination of the pulp extract revealed no apparent differences between the test and control animals as evidenced by the high molecular-weight nature of proteoglycans obtained from each group and thus suggested that fluoride has no apparent influence on 703
.I. W. Smalley
704 Protein A280nm
lkonic acid A530nm _---_
mation of incompletely formed proteoglycan subunits, it is evident that fluoride hinders the normal synthetic events in the mineralised elements of the rat incisor. We have established that the glycosaminoglycan component of the proteoglycan is chondroitin 4’ sulphate (Smalley and Embery, 1976).
(1. Pulp Control 02
A
t
and G. Embery
A :: I ,
Acknow(e~~ements-This investigation was supported by financial award to J.W.S. from the Borrow Dental Milk Foundation.
b. Pulptest
REFERENCES
c. Hord tissue Control
d. HardtissueteSt
Vb
Fig. 1. Gel filtration
Eluate,
ml
profiles of proteoglycans ose 2B.
on Sephar-
the metabolism
of the proteoglycans present in the dental pulp (Fig. la, b). In contrast, the results obtained from the hard tissue extract showed that the proteoglycans isolated from the fluorotic teeth had undergone a major alteration in molecular size as indicated by the presence in the elution profile of
a range of lower molecular weight components (Fig. Id). The reason for this effect is not yet apparent although the findings closely parallel those described for cartilage by Rokosova, Hanson and Bentley (1973) who demonstrated the presence of proteoglycan subunits following removal of a glycoprotein-aggregating factor. Although it is difficult to equate our results with those obtained for cartilage, it is tempting to speculate that fluoride interferes with the aggregation of the sub-units into a larger complex. The effect could presumably occur during the anabolic events of tissue turnover rather than leading to a breakdown of established tissue. On the other hand, Lin, Mosteller and Hardesty (1966) have shown that fluoride inhibits protein synthesis during the initiation of new protein chains and Bleiberg, Zauderer and Baglioni (1972) have indicated that fluoride is involved in the disaggregation of ribosome sub-units. Moreover, Basford, Patterson and Kruger (1976) showed that fluoride inhibits the synthesis of enamel matrix protein in rat incisor ameloblasts. It is possible, therefore, that fluoride influences the structure of the protein core of proteoglycans, leading to their inability to associate into the native proteoglycan complex. Whether the effect is ultimately the physical hindrance of the sub-unit aggregation or causes the for-
Basford K. E., Patterson C. M. and Kruger B. J. 1976. Multivariate analyses of the influence of mottling doses of fluoride on the amino acids of enamel matrix protein of rat incisors. Archs oral Biol. 21, 121-129. Belanger L. F., Visek W. J., Lotz W. E. and Comar C. L. 1957. The effects of fluoride feeding on the organic matrix of bones and teeth of pigs as observed by autoradiography after in oitro uptake of 45Ca and 35S. J. biophys. biochem. Cytol. 3, 559-566. Bitter T. and Muir H. M. 1962. A modified uranic acid carbazole reaction. Analyt. Biochem. 4, 33&334. Bleiberg I., Zauderer M. and Baglioni C. 1972. Reversible disaggregation by NaF of membrane-bound polyribosomes of mouse myeloma cells in tissue culture. Biochim. biophys. Acta 249, 453464. Embery G. 1974. The isolation of chondroitin 4-35 sulphate from the molar teeth of young rats receiving sodium %lphate. Cnlc. 7?ss. Res. 14, 5945. Gregory J. D., Sadjera S. W., Hascall V. C. and Dziewiatkowski D. D. 1970. The proteoglycans of bovine nasal cartilage: dissociative extraction. In: Chemistry and Molecular Biology of the Intercellular Matrix, (Edited by Balazs E. A.) Vol. 2. pp. 843-849. Academic Press, London. Hardingham T. and Muir H. M. 1972. Biosynthesis of glycosaminoglycans in cartilage slices. Fractionation by gelchromatography and equilibrium density gradient centrifugation. Biochem. J. 126, 791-803. Jones I. L. and Leaver A. G. 1974. Studies on the minor components of the organic matrix of human dentine. Archs oral Biol. 19, 371-380. Lemperg R. K. and Rosenquist J. B. 1974. Effects of supply and withdrawal of fluoride. 3. Concentration of acid glycosaminoglycans and hydroxyproline in cortical bone. Acta path. microbial. stand. 82, 435444. Lin S., Mosteller R. and Hardesty B. 1966. The mechanism of sodium fluoride and cycloheximide inhibition on haemoglobin biosynthesis in the cell-free reticulocyte system. J. molec. Biol. 21, 51-69. Lindemann G. 1967. Pigment alterations and other disturbances in the rat incisor enamel in chronic fluorosis and in recovery. Acta odont. stand. 25, 525-530. Muir H. M. and Jacobs S. 1967. Protein-polysaccharides of pig laryngeal cartilage. Biochem. J. 103, 3677374. Rokosova B., Hanson A. N. and Bentley J. P. 1973. Evidence for the presence of a low molecular weight proteoglycan aggregation factor in rabbit ear cartilage. Biochim. biophys. Acta 320, 442452. Sadjera S. W. and Hascall V. C. 1969. Protein-polysaccharide complex from bovine nasal cartilage. A comparison of low and high shear extraction procedures. J. biol. Chem. 244, 77-87. Smalley J. W. and Embery G. 1976. Chemical studies on the influence of fluoride on the organic matrix of rat teeth. J. dent. Res. (in press). Walton R. E. and Eisenmann D. R. 1975. Ultrastructural examination of dentine formation in rat incisors following multiple fluoride injections. Archs oral Biol. 20, 485+x.
