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The effect of fluoride on the immature enamel matrix protein of the rat
4rchs orul BiolVol.21.pp.131to132. Pergamon Press 1976. Printed m GreatBritain.
SHORT THE EFFECT ENAMEL
COMMUNICATION
OF FLUORIDE ON THE IMMATURE MATRIX PROTEIN OF THE RAT
C. M. PATTERSON, K. E. BASFORD and B. J. KRUGER Oral
Biology,
Physiology Building, University St. Lucia, Australia, 4067
of Queensland,
Summary-Fluoride in drinking water decreased the total quantity of enamel matrix protein formed in rat incisors and altered the relative proportions of individual amino acids of the matrix.
Fluoride is l&own to affect the uptake of certain amino acids by ameloblasts (Kruger, 1970, 1972), but lias been shown not to affect their ability to synthesize RNA from orotate (Patterson and Kruger, 1975). -“here exist at least two possible explanations of the effect of fluoride on the uptake of amino acids by ameloblasts: an altered total rate of synthesis of the matrix or an alteration to the actual chemistry of the secreted matrix. This paper compares the amino acid composition of the total soluble protein from the enamel matrix of fluoride-treated rats with that of untreated controls. Mature female Wistar rats were given 50 ppm fluoride in their drinking water for 10 days and the formilg enamel matrix from the incisors of the maxilla and the mandible was collected by micro-dissection. The matrix was then decalcified in 0.3 M EDTA IH 7.4 at 4°C for 4-5 days, dialysed, centrifuged, a sample of the retentate taken for protein estimation (‘r’olin-Ciocalteu) and the remainder freeze-dried. Five such samples, with nor-leucine added as an internal standard, were hydrolysed in 3.0ml of constant B.P. HCl at 110°C f 1°C for 20 hr. Following hydrolysis, tire solvent was evaporated and the residue taken up in 1.0 ml of 0.1 M citrate buffer, pH 2.00 and the amino acid analysis carried out on a Technicon P.utoanalyser modified for amino acid analysis. Five csntrol samples were treated in the same way and the amino acid contents of both groups were calculated using duplicate amino acid standards. Checks were made for the presence of hydroxyproline using a different buffer system (none was found) and corrections made for the destruction of serine and threonine during the 20-hr hydrolysis. The controls yielded between l&1.3 mg of soluble protein, but more variable yields were obtained from the “fluoride” samples (0.3GO.7mg). However, for all Table
Table 1. Amounts of protein in each original sample of enamel matrix collected--expressed as mg of protein for control and fluoride-treated rats Replication 1 2 3 4 5 * Some sample
of variation
Between treatments Sampling error Residual error
dF 1 10 8
Fluoride
1.08 1.20 1.00 1.05 1.30
0.33 *0.27* 0.48 0.70 0.66
lost.
samples there was a close relationship between the amount of protein and the corresponding total amount of amino acids (expressed in nanomoles) found in the hydrolysates, indicating that there had not been a differential loss of protein in the samples during the experiment. An analysis of variance on the total n moles of amino acids (Table 2) showed that there was a significant difference between the controls and the fluoride-treated samples. The low samplingerror mean-square supports the view that the experimental technique was satisfactory. The values for individual amino acids were subjected to statistical analysis (Table 3). In the raw data, it will be seen that fluoride treatment significantly altered the concentration of all amino acids. Of greater interest, however, are the significant differences seen in the percentage data: increases were recorded for aspartate, threonine, serine, glycine. valine, tyrosine and arginine. It would seem that the effect of fluoride cannot be due solely to a depression in synthesis or secretion of the matrix. Enamel matrix is known to contain a number of protein species
2. Analysis of variance to determine differences for fluoride and control
Source
Control
Mean
significance of between treatment total amino-acid content s.sqs.
284521.00 18.10 16804.30 131
F
Significance
16.931
co.01
C. M. Patterson,
132 Table
3. Univariate
Amino acids
asp thr ser g’u pro gly ala val meth i-leu leu tyr phe lys his arg * and ** significant
K. E. Basford
and B. J. Kruger
analysis of percentage data from amino fluoride and control samples Table of means-Percentage data fluoride control 3.92 3.66 7.14 15.14 19.99 5.59 4.52 4.51 4.61 3.89 9.60 4.02 2.61 2.82 5.68 2.05
acid analysis
for
Significant difference
3.37 3.40 7.22
** ** *
15.41 21.99 5.19
*
4.35 4.34
*
4.93 3.84 9.15 3.80
*
2.19 2.34 6.08 1.I9
**
at 5% and 1% level respectively.
which differ in amino acid composition. A likely effect of fluoride would seem to be that it changes the relative rates of synthesis of these protein species. Acknowledgements-This work was supported by a grant from the National Health and Medical Research Council of Australia. We thank Mr. T. Daley and Dr. K. Scott for their technical assistance.
REFERENCES Kruger B. J. 1970. An autoradiographic assessment of the effect of fluoride on the uptake of C3H]-proline by ameloblasts. Archs oral Biol. 15. 1033108. Kruger B. J. 1972. Utilization of [sH]-serine by ameloblasts of rats receiving sub-mottling doses of fluoride. Archs oral Biol. 17, 1389-1394. Patterson C. M. and Kruger B. J. 1975. An autoradiographic study of the effect of a range of fluoride doses on the utilization of [3H]-orotate by ameloblasts in the rat. Archs oral Biol. 20, 749-151
SHORT THE EFFECT ENAMEL
COMMUNICATION
OF FLUORIDE ON THE IMMATURE MATRIX PROTEIN OF THE RAT
C. M. PATTERSON, K. E. BASFORD and B. J. KRUGER Oral
Biology,
Physiology Building, University St. Lucia, Australia, 4067
of Queensland,
Summary-Fluoride in drinking water decreased the total quantity of enamel matrix protein formed in rat incisors and altered the relative proportions of individual amino acids of the matrix.
Fluoride is l&own to affect the uptake of certain amino acids by ameloblasts (Kruger, 1970, 1972), but lias been shown not to affect their ability to synthesize RNA from orotate (Patterson and Kruger, 1975). -“here exist at least two possible explanations of the effect of fluoride on the uptake of amino acids by ameloblasts: an altered total rate of synthesis of the matrix or an alteration to the actual chemistry of the secreted matrix. This paper compares the amino acid composition of the total soluble protein from the enamel matrix of fluoride-treated rats with that of untreated controls. Mature female Wistar rats were given 50 ppm fluoride in their drinking water for 10 days and the formilg enamel matrix from the incisors of the maxilla and the mandible was collected by micro-dissection. The matrix was then decalcified in 0.3 M EDTA IH 7.4 at 4°C for 4-5 days, dialysed, centrifuged, a sample of the retentate taken for protein estimation (‘r’olin-Ciocalteu) and the remainder freeze-dried. Five such samples, with nor-leucine added as an internal standard, were hydrolysed in 3.0ml of constant B.P. HCl at 110°C f 1°C for 20 hr. Following hydrolysis, tire solvent was evaporated and the residue taken up in 1.0 ml of 0.1 M citrate buffer, pH 2.00 and the amino acid analysis carried out on a Technicon P.utoanalyser modified for amino acid analysis. Five csntrol samples were treated in the same way and the amino acid contents of both groups were calculated using duplicate amino acid standards. Checks were made for the presence of hydroxyproline using a different buffer system (none was found) and corrections made for the destruction of serine and threonine during the 20-hr hydrolysis. The controls yielded between l&1.3 mg of soluble protein, but more variable yields were obtained from the “fluoride” samples (0.3GO.7mg). However, for all Table
Table 1. Amounts of protein in each original sample of enamel matrix collected--expressed as mg of protein for control and fluoride-treated rats Replication 1 2 3 4 5 * Some sample
of variation
Between treatments Sampling error Residual error
dF 1 10 8
Fluoride
1.08 1.20 1.00 1.05 1.30
0.33 *0.27* 0.48 0.70 0.66
lost.
samples there was a close relationship between the amount of protein and the corresponding total amount of amino acids (expressed in nanomoles) found in the hydrolysates, indicating that there had not been a differential loss of protein in the samples during the experiment. An analysis of variance on the total n moles of amino acids (Table 2) showed that there was a significant difference between the controls and the fluoride-treated samples. The low samplingerror mean-square supports the view that the experimental technique was satisfactory. The values for individual amino acids were subjected to statistical analysis (Table 3). In the raw data, it will be seen that fluoride treatment significantly altered the concentration of all amino acids. Of greater interest, however, are the significant differences seen in the percentage data: increases were recorded for aspartate, threonine, serine, glycine. valine, tyrosine and arginine. It would seem that the effect of fluoride cannot be due solely to a depression in synthesis or secretion of the matrix. Enamel matrix is known to contain a number of protein species
2. Analysis of variance to determine differences for fluoride and control
Source
Control
Mean
significance of between treatment total amino-acid content s.sqs.
284521.00 18.10 16804.30 131
F
Significance
16.931
co.01
C. M. Patterson,
132 Table
3. Univariate
Amino acids
asp thr ser g’u pro gly ala val meth i-leu leu tyr phe lys his arg * and ** significant
K. E. Basford
and B. J. Kruger
analysis of percentage data from amino fluoride and control samples Table of means-Percentage data fluoride control 3.92 3.66 7.14 15.14 19.99 5.59 4.52 4.51 4.61 3.89 9.60 4.02 2.61 2.82 5.68 2.05
acid analysis
for
Significant difference
3.37 3.40 7.22
** ** *
15.41 21.99 5.19
*
4.35 4.34
*
4.93 3.84 9.15 3.80
*
2.19 2.34 6.08 1.I9
**
at 5% and 1% level respectively.
which differ in amino acid composition. A likely effect of fluoride would seem to be that it changes the relative rates of synthesis of these protein species. Acknowledgements-This work was supported by a grant from the National Health and Medical Research Council of Australia. We thank Mr. T. Daley and Dr. K. Scott for their technical assistance.
REFERENCES Kruger B. J. 1970. An autoradiographic assessment of the effect of fluoride on the uptake of C3H]-proline by ameloblasts. Archs oral Biol. 15. 1033108. Kruger B. J. 1972. Utilization of [sH]-serine by ameloblasts of rats receiving sub-mottling doses of fluoride. Archs oral Biol. 17, 1389-1394. Patterson C. M. and Kruger B. J. 1975. An autoradiographic study of the effect of a range of fluoride doses on the utilization of [3H]-orotate by ameloblasts in the rat. Archs oral Biol. 20, 749-151