Effects of Fluoride on Matrix Proteins and Their Properties in Rat Secretory Enamel T. AOBA, E.C. MORENO, T. TANABE', and M. FUKAE' Forsyth Dental Center, 140 The Fenway, Boston, Massachusetts 02115; and 1Tsurumi University School of Dental Medicine, Yokohama 230, Japan

This publication concerns the selective adsorption of rat enamel proteins onto hydroxyapatite, their solubility in aqueous solutions, and the effect that systemic fluoride has on these properties. The enamel proteins used as adsorbates were extracted in 0.5 mol/L acetic acid from the secretary enamel of the upper and lower incisors of SD rats (females, 200-220 g body weight). Equilibration of the proteins with hydroxyapatite was performed in two solutions: (i) 50 mmol/L acetate buffer at pH 6.0 and 00C, and (ii) 50 mmol/L Tris buffer containing 4 mol/ L guanidine at pH 7.4 and room temperature. Enamel was dissected from animals, which were given either de-ionized water (control group) or water containing 25, 50, 75, or 100 ppm fluoride as NaF for four weeks. From these enamel samples, the proteins were extracted in sequence with 160 mmol/ L NaCl and 3 mmol/L phosphate (pH 7.3), 50 mmol/L carbonate buffer (pH 10.8), and finally, with 0.5 mol/L acetic acid for dissolution of the enamel mineral. The F, Ca, and P contents of the various enamel samples were determined. The results showed that: (i) The amelogenin having a 26-kD molecular mass on SDS-PAGE displayed the highest adsorption affinity onto the apatite crystals among the amelogenins; (ii) the same selective adsorption onto the crystals was also observed for the amelogenins separated from the teeth of the animals ingesting fluoride; (iii) the amelogenins can be classified into two groups, neutral-soluble moieties (14-15 kD) and alkaline-soluble moieties (19-28 kD), by the use of solutions having ionic strengths of about 160 mmol/L; and (iv) with regard to the electrophoretic patterns, quantities, and amino acid composition of these two groups of amelogenins, no major differences were obtained between the control group and any groups of animals ingesting fluoride, although the fluoride substitution into enamel mineral increased proportionally with an increase in fluoride concentration in the drinking water, and the incisors from animals in the 75 and 100 ppm F treatment were distinctively brittle. All the foregoing results, as well as those previously reported, indicate that possible functional roles of the amelogenins in early enamel mineralization are common to rat and porcine models, and that the fluoride administration at the doses tested, or the resulting incorporation of fluoride in the forming enamel mineral, caused only a marginal effect, if any, on the properties and degradation processes of amelogenins at the secretary stage of rat amelogenesis. J Dent Res 69(6):1248-1255, June, 1990

Introduction. Fluoride plays an important role in stabilizing enamel mineral in the oral environment, but, with ingestion at high doses, it Received for publication October 3, 1989 Accepted for publication January 31, 1990 This investigation was supported in part by USPHS Research Grants DE07623 and DE03187 from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892. 1248

induces anomalous defects (fluorosis). According to the literature, the highest concentrations of fluoride in the enamel tissue appear in the early secretary phase (Hammarstrom, 1971; Weatherell et al., 1975; Speirs, 1975). Although it has been considered that this high influx of fluoride may be associated with the secretion of matrix proteins, the nature of the postulated fluoride-protein association is not yet defined in a satisfactory fashion. Furthermore, Bawden et al. (1981, 1986) reported that the ameloblasts exercise no direct control over fluoride transport into enamel during the secretary phase, but that the cell layer constitutes a diffusion barrier for its passive transport. There are also controversies about possible effects of fluoride on early enamel mineralization. Previous biochemical studies (Patterson et al., 1976; Basford et al., 1976; Drinkard et al., 1983) showed that the fluoride administration in drinking water caused a change in the quantity, amino acid composition, and molecular assembly of enamel matrix proteins in early enamel development in rats. Contrary to these earlier studies, it was reported (DenBesten and Crenshaw, 1984; Bronckers and Woltgens, 1985) that the fluoride administration did not cause any appreciable changes in the quantity and electrophoretic profiles of matrix proteins secreted during the secretory phase of rat amelogenesis. In relation to enzymatic degradation of the secreted matrix proteins (particularly the amelogenins), several investigators (Suga, 1970; Crenshaw and Bawden, 1984; Robinson and Kirkham, 1984) supported the view that fluoride was able to inhibit matrix proteolysis. However, Carter et al. (1989) recently showed that fluoride (even at the high concentration of 70 mmol/L as NaF) had no effect on the activity of the 29-kD proteinase purified from porcine secretary enamel.

Certain evolutionary aspects of amelogenesis suggest that the amelogenins may serve to regulate rates, shape, and order of crystal formation in mammalian enamel (Samuel et al., 1987). In fact, previous studies on porcine secretary enamel (Aoba et al., 1987a, b) gave supportive evidence that the kinetics of the initial precipitation of enamel mineral is most likely controlled in part by the presence of proteinaceous inhibitors, particularly the amelogenins, of apatite crystal growth. An intriguing aspect of this regulatory function by porcine amelogenins is that the secreted parent protein (25-kD molecular mass) was selectively adsorbed onto apatitic surfaces; hence, it displayed the strongest inhibition of apatite crystal growth. The adsorption affinity and inhibitory activities of the amelogenins decreased substantially with enzymatic cleavages of specific protein segments. To our knowledge, it has not yet been ascertained whether a similar regulatory mechanism of early enamel mineralization is present in other species (such as the rat), and whether fluoride administration may disturb the regulatory mechanism of mineralization in the secretary phase by modulating the properties of either matrix proteins, mineral, or both. Thus, the present study was designed to: (i) investigate the properties of rat amelogenins (namely, the selective adsorption onto apatite crystals and their solubility in aqueous solution) relevant to the understanding of the basic mechanism

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of amelogenesis; and (ii) assess possible effects of systemically ingested fluoride on these properties in the secretary phase.

Materials and methods. Experimental animals. -Sprague-Dawley rats (females; total, 95 animals) were obtained from a supplier (Charles River Co., NC). For preparation of the enamel protein samples used as adsorbates, 20 animals (200-220 g body weight) were killed by CO2 inhalation on the arrival day. In experiments studying the effect of fluoride on the properties of amelogenins, animals with initial mean (±+ SD) weights of 100 (±+ 10) g were divided into five groups (each group of five animals). A control group was given de-ionized water, and four experimental groups were given water ad libitum containing 25, 50, 75, and 100 ppm fluoride as NaF. These groups of animals were housed for four weeks on a low-fluoride (2.4 ppm) diet (L-356; TekLad, Madison, WI). The experiment was repeated three times with the same experimental design. Preparation of enamel samples. -Following death of the animals, upper and lower incisors were dissected out of the

bones. The enamel organ was removed, and the exposed enamel surface was carefully wiped with a moistened paper tissue, so that there would be minimum contamination with cellular debris and blood. The secretary enamel, soft in consistency, was scraped with a razor blade. Although special care was taken in the cleaning of the enamel surface, examination of the dissected enamel pieces revealed a small amount of blood contamination. The dissected enamel pieces were pooled and then freeze-dried. Approximately 40 mg of the secretary enamel was collected from the incisors of 20 animals. In the preparation of the enamel samples from the fluoride-ingesting animals (including the control), it was noticed that the incisors of the animals in the 75- and 100-ppm fluoride treatments were brittle (prone to fracture of the erupted portions). The enamel samples dissected from each animal in a group were pooled and then freeze-dried. Finally, the dried enamel was pulverized by hand with an agate mortar and pestle and stored at - 30'C until used. Extraction of enamel proteins. -All the matrix proteins in rat secretary enamel were obtained by dissolution of the mineral in 0.5 mol/L acetic acid containing a mixture of phosphatase-protease inhibitors (50 mmol/L E-amino-n-caproic acid, 5 mmol/L benzamidine, 1 mmol/L phenylmethylsulfonyl fluoride, 0.5 mmol/L N-ethylmaleimide, 1 mmol/L levamisole). In practice, the enamel powder (40 mg) was placed in a centrifuge tube, and 10 mL of the acid solution, cooled at 4TC, was added to the tube. The slurry was stirred at 0C and then separated by centrifugation at 10,000 g for ten min. The supernatant was recovered, and the pellet was re-dispersed in a fresh solution. The same procedure was repeated five times, until no mineral was left in the centrifuge tube. After recovering the last extract, we washed the interior wall of the tube with 0.1% SDS solution to ascertain whether certain fractions of the matrix proteins had been precipitated during the foregoing extraction. The recovered supernatants (i.e., acid extracts) were pooled, de-salted, concentrated with an ultrafiltration membrane (YM-5; cut-off 5 kD M.W., Amicon Co., MA), and then freeze-dried. The total amount of the collected proteins was approximately 11 mg. This batch of the proteins was stored at - 30°C, until used in the required experimentation. Sequential extraction of matrix proteins was performed in duplicate on the enamel sample prepared from each group of the animals ingesting fluoride (including the control group). The three solvents used in sequence were: (1) neutral solution

Fig. 1-SDS electrophoretograms of rat and porcine enamel proteins isolated from the secretary enamel. Lane 1, rat enamel proteins extracted in 0.5 mol/L acetic acid; lane 2, the proteins precipitated during the acid extraction; and lane 3, porcine enamel proteins extracted with use of the same acid solution. Note the apparent differences in the molecular masses between the two species.

containing 160 mmol/L NaCl and 3 mmol/L total phosphate at pH 7.3 [this composition was similar to that found in the enamel fluid (Aoba and Moreno, 1987)]; (2) 50 mmol/L carbonate buffer at pH 10.8; and finally (3) 0.5 mol/L acetic acid for dissolution of the remaining residues. Buffers (1) and (2) have ionic strengths of about 160 mmol/L. All these solvents contained the protease and phosphatase inhibitors. Accurately

weighed enamel samples (9-10 mg) were placed in a microcentrifuge tube (Fisher, NJ). Required volumes of the neutral solution were added to the tube so that a 1 to 20 solid/solution ratio (g/mL) would result. The suspension was homogenized with the aid of a vortex, and then separated by centrifugation. This extraction was repeated twice. After the second neutral extractant was recovered, the alkaline buffer (the same volume used in the preceding extraction) was added to the tube containing the pellet. Protein extraction with this alkaline buffer was repeated four times, so that separation of all extractable proteins would be ensured [most enamel matrix proteins are dissolved in alkaline solutions (Shimizu and Fukae, 1983)]. It was also confirmed with the Bio-Rad micro-protein assay that the protein quantity in the last extraction was substantially lower than those in the first three aliquots. Following the final extraction with the alkaline buffer, the sedimented residues were freeze-dried. Approximately half weights (about 3.5 mg) of the dried residues were used for chemical analyses (see below), while the rest was dissolved completely in the 0.5

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mol/L acetic acid solution, thus solubilizing the remaining proteins. The proteins extracted in each solvent were pooled, neutralized in the case of carbonate buffer, and then de-salted with the YM-5 membrane. The collected samples were freeze-dried and then stored at - 30TC. Adsorption experiments. -Hydroxyapatite, HA, used as adsorbent, was prepared as described previously (Aoba and Moreno, 1984). Its specific surface area was 10.6 m2/g. The experimental procedures used were essentially the same as those reported for the adsorption studies using porcine enamel proteins (Aoba et al., 1987a). Briefly, fractions of the prepared batch of proteins were dissolved with either 50 mmol/L acetate buffer (at pH 6.0 and 0C) or 50 mmol/L Tris buffer (at pH 7.4 and room temperature) containing 4 mol/L guanidine (as a dissociative reagent), to yield 0.1 % wt/v adsorbate solutions. Accurately weighed amounts (30 mg) of HA crystals were added to 2 mL of each adsorbate solution. Equilibration was conducted at the specified temperatures for one h. At the end of equilibration, the slurry was separated by centrifugation at the same temperatures. The supernatant was recovered, and the sedimented crystals were washed three times with the same buffer used in the equilibration. After washings, the crystals were dissolved completely, with 0.5 mol/L acetic acid being used so that the proteins adsorbed onto the crystal surface could be recovered. The proteins extracted in the acid solution, as well as the proteins remaining in the equilibrating solution (i.e., in the first supernatant), were de-salted with use of the YM-5 membrane. This adsorption experiment was repeated twice in each system, and the adsorbed protein samples were pooled for two-dimensional electrophoresis and amino acid analysis. Selective adsorption onto hydroxyapatite of the amelogenins separated from the enamel of animals ingesting fluoride was studied, with the proteins fractionated in the carbonate buffer used as adsorbates. Approximately 1.6 mg of the proteins was dissolved in 2 mL of the acetate buffer (at pH 6.0 and 0°C) and equilibrated with 30 mg of the HA crystals. At the end of the equilibration (30 min), the proteins adsorbed onto the crystals and the proteins remaining in the solution were recovered by the same procedures mentioned above. The initial protein concentration used was much lower than the amelogenin solubility (Shimizu and Fukae, 1983) at the experimental pH value and temperature. Protein analyses. -All the protein samples to be analyzed were freeze-dried and then dissolved in appropriate volumes of 20 mmol/L acetic acid. Aliquots of the protein solution were used for protein assay, electrophoresis, and amino acid analysis. The protein monitoring in extractants or washings was done according to Bio-Rad micro-assay procedure. Quantitative determinations were made based on amino acid analyses. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted with 15% or 18% polyacrylamide gel at 15 mA for four h, according to Laemmli's procedure (1970). The gel was stained with Coomassie Brilliant Blue (CBB). The proteins adsorbed onto HA crystals at pH 6.0 were also characterized by two-dimensional electrophoresis, according to O'Farrell's procedure (1975). The first-dimensional electrophoresis was conducted with Bio-Lytes (Bio-Rad, Richmond, CA) in the ampholyte range of five to seven; the second-dimensional analysis was carried out with 15% acrylamide gel. Details of these analyses were reported previously (Tanabe et al., 1988). Amino acid analysis was performed with an automatic aminoacid analyzer (Model JLC-300, JEOL, Tokyo). Prior to analysis, the proteins were hydrolyzed in 6 mol/L HCl at 110°C for 24 h in evacuated, sealed glass tubes. For determination of the amino acid composition of the proteins separated on SDS gels, the CBB-stained bands corresponding to the proteins

J Dent Res June 1990

concerned were sliced. The protein was recovered (about 85%) from the sliced gel with use of an electrophoretic concentrator (ISCO, Tokyo). The recovered proteins were hydrolyzed in the same way mentioned above. Chemical analyses. -Chemical analyses for F, Ca, and P contents were conducted on the enamel samples originally dissected from the teeth of animals ingesting fluoride (including the control), as well as on the residues recovered after treatment in the alkaline buffer. The latter samples were washed three times with 1 mL of de-ionized water, and then freezedried. Prior to analyses, all the enamel samples were dried overnight in a vacuum desiccator in the presence of 96.5% H2SO4. The fluoride content was determined by a microdiffusion method (Whitford and Reynolds, 1979; Aoba et al., 1989a), in conjunction with an ion-selective electrode (Orion Res., MA). For this purpose, an accurately weighed enamel sample (about 1 mg) was completely dissolved with 2 mL of 3 mol/L HCl04 in a sealed chamber. The acid was pre-saturated with hexamethyldisiloxane (HMDS). The amounts of Ca and P dissolved in the acid solution were determined by atomic absorption spectrophotometry and colorimetrically (Vogel, 1961), respectively. All the measurements of F, Ca, and P were performed in triplicate samples.

Results. Fig. 1 shows electrophoretograms of rat enamel proteins extracted in 0.5 mol/L acetic acid (lane 1), the proteins recovered from (i.e., precipitated onto) the tube wall (lane 2) and, for a comparative purpose, porcine matrix proteins separated from the secretary enamel of slaughtered piglets (Aoba et al., 1987a) (lane 3). On lane 1, several major bands were discernible at 19 kD, 20 kD, 22-24 kD (the most prominent bands), 26 kD, and 28 kD. As reported in the literature (Fincham et al., 1982), these apparent molecular masses of rat matrix proteins were different from those (20 kD, 22-23 kD, and 25 kD) of porcine amelogenins. The molecular masses (15 kD, 18 kD, 18.5 kD, and 19.5 kD) of the proteins that precipitated during the acid extraction were determined by SDSPAGE, as shown on lane 2 of Fig. 1. All these proteins were loaded on this lane, and they were just discernible by CBB staining. Therefore, their maximum quantities were in the order of micrograms, whereas the acid-extractable proteins amounted to more than 10 mg. Fig. 2 shows the results of adsorption experiments conducted in the acetate buffer at pH 6.0 (lanes 2 through 4), and in the Tris-guanidine buffer at pH 7.4 (lanes 5 through 7). In both systems, the same preparation of proteins (i.e., 2 mg of the acid-extracted rat enamel proteins) was used as adsorbate. At the end of equilibration in the acetate buffer, two-thirds of the originally added proteins remained in the solution (lane 3), and the rest (about 0.7 mg) of the proteins was recovered from the crystal surfaces. These adsorbed proteins showed a wide range of molecular masses (major moieties are labeled by A through D), and it was found that the 26-kD moiety (sample C) was most prominently concentrated onto the crystals (lane 4). A two-dimensional electrophoretogram of the adsorbed proteins (Fig. 3) revealed that this 26-kD moiety consisted of at least two charged families around pI = 6.5. It was also seen that the 28-kD moiety, showing marginal adsorption onto the crystals, appeared near pI = 7. In the system that used the Tris-guanidine solution, most of the proteins remained unadsorbed in the solution (lane 6). The total amount of proteins recovered from the crystals was less than 0.1 mg, and, among the adsorbed proteins (lane 7), and 60-kD moiety (labeled by E) became most prominent with a substantial decrease in the adsorption of the 26-kD proteins (labeled by F), by comparison

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A

5

IEF

-, 7

B

Fig. 3-Two-dimensional polyacrylamide gel electrophoresis of the moieties adsorbed onto hydroxyapatite in 50 mmol/L acetate buffer at pH = 6.0 and 0C. (A) The low-molecular-weight standards. (B) Rat enamel proteins recovered from the crystal surface and used for isoelectric focusing. Note that the adsorbed 26-kD moieties consisted of charged families near pI = 6.5, while the 28-kD moiety appeared close to pI = 7.

Fig. 2-SDS electrophoretograms showing the selective adsorption of rat enamel proteins separated from the secretary enamel. Lane 1, lowmolecular-weight standards (Bio-Rad); lanes 2 and 5, the original adsorbates (the same as the proteins shown in lane 1 of Fig. 1); lane 3, the proteins remaining in the equilibrating solution (50 mmol/L acetate buffer at pH = 6.0 and 0C); lane 4, the proteins adsorbed onto hydroxyapatite after equilibration in the foregoing solution; lane 6, the proteins remaining in the solution (50 mmol/L Tris buffer containing 4 mol/L guanidine at pH = 7.4 and room temperature); and lane 7, the proteins adsorbed onto the crystals. The proteins labeled by A through F were eluted from the gel and used for determination of their amino acid composition. The values of molecular weight are in kilo-daltons.

through the experimental period. At the end of the study, there were no significant differences between the weights of animals (303-320 g) in the control group and that in any of the groups ingesting fluoride. Fig. 4 compares the results of sequential extraction of matrix proteins from the secretary enamel of the animals given deionized water (control) and water containing 50 ppm and 100 ppm fluoride. Lanes 2 through 4 correspond to electrophoretograms of the proteins extracted in the neutral solution (control, 50-ppm, and 100-ppm groups, in that order, from left to right). These three SDS-PAGE patterns were identical, showing that the neutral-soluble fraction consisted mainly of the 14-

with their adsorption in the acetate buffer. All the protein species corresponding to the bands labeled A through F were eluted from a separately prepared gel and then used for amino acid analysis. Table 1 gives the amino acid compositions of samples A through F, as well as that of the original adsorbate. The values for samples A through D are averages of two determinations with a maximum deviation of 6%, except for the results for proline that agreed within 13%. The limited quantities of samples E and F were sufficient for one determination only. Samples A and E, which were eluted from the bands corresponding to molecular mass of 60 kD, were very similar in their composition, rich in Glx, Ala, Asx, and Leu. In contrast to this high-molecular-weight protein(s), the compositions of samples B through D were characterized by the high contents of Pro, Glx, Leu, and His, which are common to the composition of amelogenins in various mammalian species. Although sample F at 26 kD on SDS-PAGE also displayed an amino acid composition similar to that of sample C, showing the same molecular weight, it should be pointed out that there were small but appreciable differences between their compositions, namely, the lower contents of Pro and Glx and the higher contents of Gly and Ala in sample F. This may reflect the presence of traces of non-amelogenin(s) concomitantly electrophoresed with the 26-kD amelogenin [this is the case in the porcine model (Fukae and Tanabe, 1987)]. In the experiments giving the animals either de-ionized water (control group) or water containing 25, 50, 75, or 100 ppm fluoride for four weeks, it was verified that the fluoride administration did not influence food consumption or weight gain

AMINO ACID COMPOSITION OF PROTEINS ADSORBED ONTO HYDROXYAPATITE (AMINO ACID RESIDUES/1000 RESIDUES) Solution B Solution A Amino Original C D E F acid A B adsorbate 23 21 104 28 23 20 Asx 102 36 30 Thr 63 33 31 30 58 61 71 65 61 70 68 73 Ser 176 171 199 160 163 200 190 Glx 249 254 262 70 231 Pro 70 267 41 68 45 42 59 59 45 Gly 44 55 40 105 39 106 43 Ala 0 0 0 0 0 4 0 Cys 40 36 Val 63 37 37 37 63 0 28 48 Met 43 46 29 8 37 32 27 38 Ile 37 37 38 94 92 91 95 94 96 94 Leu 38 Tyr 41 23 Phe 18 19 20 46 20 22 61 His 71 72 78 26 78 19 14 84 32 15 84 20 Lys 13 41 11 18 10 37 20 Arg *Solution A, 50 mmol/L acetate buffer at pH 6.0. #Solution B, Tris-guanidine buffer at pH 7.4. @Samples A through F were eluted from bands A through F shown in Fig. 2. !The results obtained from samples A through D and the original adsorbate are represented by the average of two determinations. The amino acid composition of samples E and F is on the basis of single determination because of the limited materials available. Tyr, not determined in the samples eluted from the gel.

TABLE 1

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Fig. 4-SDS electrophoretograms of rat enamel proteins extracted in sequence from the secretary enamel. Lane 1, the low-molecular-weight standards; lanes 2 through 4, the proteins extracted in the presence of 3 mmol/L phosphate and 160 mmol/L NaCl (pH 7.3); lanes 5 through 7, the proteins extracted in 50 mmol/L carbonate buffer at pH 10.8; and lanes 8 through 10, the proteins extracted after dissolution of mineral in 0.5 mol acetic acid. The results shown in lanes 2, 5, and 8 correspond to the proteins obtained from the control animals; the results in 3, 6, and 9, and in 4, 7, and 10 correspond to the proteins, respectively, obtained from the groups of animals ingesting 50 ppm and 100 ppm fluoride.

Fig. 5-SDS electrophoretograms showing the selective adsorption of rat amelogenins onto hydroxyapatite in 50 mmol/L acetate buffer at pH = 6.0 and 0C. The proteins fractionated with the alkaline buffer were used as adsorbates. Lane 1, the low-molecular-weight standards. The results obtained from the control group of animals are shown on lanes 2 through 4; lane 2, the original adsorbate; lane 3, the protein remaining in the solution; and lane 4, the proteins adsorbed onto the crystals. Lanes 5 through 7 and lanes 8 through 10 correspond to lanes 2 through 4, in the same order, in experimentation with the proteins prepared from the groups of animals ingesting 50 ppm and 100 ppm fluoride, respectively. Note that in all the animal groups, the 26-kD amelogenin showed preferential adsorption onto the crystals and, thereby, after equilibration, only traces of this moiety remained in the solution.

15-kD moieties, as well as minor moieties with molecular masses above 60 kD. The amino acid analysis (see below) of the proteins soluble in the neutral solution indicated that this fraction corresponded to about 2-3% of the tissue weight (or 6-9% of the total matrix proteins) in all the animal groups. The protein fraction sparingly soluble in the neutral solution, but soluble in the carbonate buffer, consisted mainly of 19-28-kD amelogenins. Again, their electrophoretic patterns shown in lanes 5 through 7 of Fig. 4 were identical for the three animal groups; these proteins corresponded to about 30% of the tissue weight (or 90% of the total proteins) in all the animal groups. After extraction of these amelogenins, minor constituents (corresponding to 2-3% of the total proteins) were separated by dissolution of the mineral in the acid solution. As shown in lanes 8 through 10, the protein profiles of these minor constituents on SDS-PAGE were also identical for the three animal groups; major constituents of this fraction were discerned as having 20-30-kD molecular masses, but further characterization was not pursued in the present study. Table 2 gives the amino acid composition of the neutraland alkaline-soluble fractions separated from the control and the fluoride-experimental groups of animals. Reproducibility was comparable with that obtained for the samples in Table 1. As anticipated from the identical electrophoretic patterns of each fraction, no appreciable differences were found in the amino acid composition of the proteins separated from the various groups. Both the neutral- and alkaline-soluble fractions were characterized by the high content of Pro, Glx, His, and Leu, typical of the composition of amelogenins. However, the composition of the neutral-soluble species exhibited traces of Tyr and a high content of Gln. Fig. 5 illustrates typical results of the adsorption experiments, in which the alkaline-soluble fractions, prepared from

the fluoride-ingesting animals, were used as adsorbates. As mentioned above, the adsorbate samples consisted mainly of the 19-28-kD amelogenins (lanes 2, 5, and 8 in the control, 50-ppm, and 100-ppm groups, respectively). A prominent finding was that after the adsorbate-HA equilibration, the majority of amelogenins (19-24 kD and 28 kD) remained unadsorbed in the solution (lanes 3, 6, and 9 in the same order of animal groups), while most of the 26-kD amelogenin was concentrated onto the HA crystals (lanes 4, 7, and 10); only traces of this moiety remained in the equilibrating solution. Another finding to be noted was that a broad band at about 50 kD was obtained from the samples recovered from the crystals (lanes 4, 7, and 10), as well as from the acid-extracted samples, as shown on lanes 8 through 10 on Fig. 4. Possible origins of this band will be discussed later. Table 3 gives the Ca, P. and F contents of the enamel samples prepared from all the animal groups. The Ca and P contents (dry weight %) of the dissected enamel were quite similar in all the animal groups. The fluoride content of the enamel increased with increasing fluoride concentration in the drinking water, although the actual total amounts of fluoride ingested by the animals during the whole experimental period is not known. After protein-extraction in the alkaline buffer, the Ca, P. and F contents of the residue increased. The Ca and P contents of the residues were again very similar in all the animal groups, reflecting the fact that no appreciable differences were observed in the amounts of proteins extracted in the alkaline solution. It was also found that most of the fluoride present in the original enamel was retained in the residue; interestingly, the fluoride content per mole of calcium (or phosphorus) was very similar before and after the alkaline treatment in each fluoride-ingesting group, but not in the control group in which the F/Ca values decreased slightly, but

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significantly (p 0.1 in terms of Ca5(P04)3(OH)1- Fx). Such an effect suggested that the fluoride incorporated into the mineral might modulate the content of matrix proteins in the tissue. However, calculations based on the F/Ca ratios given in Table 4 indicate that the expected fluoride substitution in the enamel crystals may be, at the most, x = 0.03 (for animals in the group ingesting 100 ppm fluoride), which corresponds only to onethird of the magnitude of fluoride substitution required for enhancing the protein adsorption onto the crystal surfaces. All the foregoing results suggest that the systemic fluoride ingestion at the doses tested was not sufficient to cause any distinct change in the properties of rat amelogenins and their degradation cascade during the secretary stage. This seems in accord with the view that the pathogenic mechanism of enamel fluorosis is not a severe disturbance in protein synthesis and/ or secretion, but an arrest of maturation (Eastoe and Fejerskov, 1984). Recently, based on results obtained with use of pig enamel, Richards et al. (1986) concluded that enamel fluorosis may be caused by fluoride exposure in the maturation phase only. In this animal, amelogenesis is prolonged, particularly in the maturation stage (Robinson et al., 1987; Kirkham et al., 1988); thus, the fluoride accumulation in the enamel crystals might be increased sufficiently to enhance the interaction of the formed mineral with the remaining matrix proteins. Fluoride may also increase the precipitation of mineral, thus decreasing the activities of ionic species (e.g., calcium) in the liquid phase, which may give abnormal signals for the cyclic modulation of ameloblasts (Smith et al., 1987) observed through the maturation stages. These possibilities are under investigation with use of mini-pigs under fluoride regimes. REFERENCES AOBA, T.; COLLINS, J.; and MORENO, E.C. (1989a): Possible Function of Matrix Proteins in Fluoride Incorporation into Enamel Mineral during Porcine Amelogenesis, J Dent Res 68:1162-1168. AOBA, T.; FUKAE, M.; TANABE, T.; SHIMIZU, M.; and MORENO, E.C. (1987a): Selective Adsorption of Porcine Amelogenins onto Hydroxyapatite and Their Inhibitory Activity on Seeded CIystal Growth of Hydroxyapatite, Calcif Tissue Int 41:281-289. AOBA, T. and MORENO, E.C. (1984): Hydroxyapatite Preparation and Crystal Growth on Hydroxyapatite Seeds, J Dent Res 63:874880. AOBA, T. and MORENO, E.C. (1987): The Enamel Fluid in the

Early Secretory Stage of Porcine Amelogenesis. Chemical Composition and Saturation with Respect to Enamel Mineral, Calcif Tissue Int 41:86-94. AOBA, T.; MORENO, E.C.; KRESAK, M.; and TANABE, T. (1989b): Possible Roles of Partial Sequences at N- and C-termini of Amelogenin in Protein-Enamel Mineral Interaction, J Dent Res 68:1331-1336. AOBA, T.; TANABE, T.; and MORENO, E.C. (1987b): Function of Amelogenins in Porcine Enamel Mineralization during the Secretory Stage of Amelogenesis, Adv Dent Res 1:252-260. AOBA, T.; TANABE, T.; and MORENO, E.C. (1987c): Proteins in the Enamel Fluid of Immature Porcine Teeth, JDent Res 66:17211726. 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, Arch Oral Biol 21:121-129. BAWDEN, J.W. and DEATON, T.G. (1981): In-vitro Study of the Cellular Control of 18F Uptake in the Enamel of Developing Rat Molar Teeth, Arch Oral Biol 26:487-490. BAWDEN, J.W.; McLEAN, P.; and DEATON, T.G. (1986): Fluoride Uptake and Retention at Various Stages of Rat Molar Enamel Development, J Dent Res 65:34-38. BRONCKERS, A.L.J.J. and WOLTGENS, J.H.M. (1985): Shortterm Effects of Fluoride on Biosynthesis of Enamel-matrix Proteins and Dentine Collagens and on Mineralization during Hamster Tooth-germ Development in Organ Culture, Arch Oral Biol 30:181191. CARTER, J.; SMILLIE, A.C.; and SHEPHERD, M.G. (1989): Purification and Properties of a Protease from Developing Porcine Dental Enamel, Arch Oral Biol 34:195-202. CRENSHAW, M.A. and BAWDEN, J.W. (1984): Proteolytic Activity in Embryonic Secretory Enamel. In: Tooth Enamel IV, R.W. Fearnhead and S. Suga, Eds., Amsterdam: Elsevier Science Publishers, pp. 109-113. DenBESTEN, P.K. and CRENSHAW, M.A. (1984): The Effects of Chronic High Fluoride Levels on Forming Enamel in the Rat, Arch Oral Biol 29:675-679. DRINKARD, C.R.; CRENSHAW, M.A.; and BAWDEN, J.W. (1983): The Effect of Fluoride on the Electrophoretic Patterns of Developing Rat Molar Enamel, Arch Oral Biol 12:1131-1134. EASTOE, J.E. and FEJERSKOV, 0. (1984): Composition of Mature Enamel Proteins from Fluorosed Teeth. In: Tooth Enamel IV, R.W. Fearnhead and S. Suga, Eds., Amsterdam: Elsevier Science Publishers, pp. 326-330. FINCHAM, A.G.; BELCOURT, A.B.; LYARUU, D.M.; and TERMINE, J.D. (1982): Comparative Protein Biochemistry of Developing Dental Enamel Matrix from Five Mammalian Species, Calcif Tissue Int 34:182-189. FUKAE, M. and TANABE, T. (1987): Nonamelogenin Components of Porcine Enamel in the Protein Fraction Free from the Enamel Crystals, Calcif Tissue Int 40:286-293. HAMMARSTROM, L. (1971): Distribution in Developing Rat Enamel of Simultaneously Injected Fluoride and Calcium, Scand J Dent Res 79:369-376. KIRKHAM, J.; ROBINSON, C.; WEATHERELL, J.A.; RICHARDS, A.; FEJERSKOV, O.; and JOSEPHSEN, K. (1988): Maturation in Developing Permanent Porcine Enamel, J Dent Res 67:1156-1160. LAEMMLI, U.K. (1970): Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4, Nature 227:680-685. LIMEBACK, H. (1987): Isolation and Characterization of Pig Enamelins, Biochem J 243:385-390. LIMEBACK, H.; SAKARYA, H.; CHU, W.; and MacKINNON, M. (1989): Serum Albumin and Its Acid Hydrolysis Peptides Dominate Preparations of Mineral-Bound Enamel Proteins, J Bone Mineral Res 4:235-241. O'FARRELL, P.H. (1975): High Resolution Two-dimensional Electrophoresis of Proteins, J Biol Chem 250:4007-4021. PATTERSON, C.M.; BASFORD, K.E.; and KRUGER, B.J. (1976): The Effect of Fluoride on the Immature Enamel Matrix Protein of the Rat, Arch Oral Biol 21:131-132. RICHARDS, A.; KRAGSTRUP, J.; JOSEPHSEN, K.; and FEJER-

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SKOV, 0. (1986): Dental Fluorosis Developed in Post-secretory Enamel, J Dent Res 65:1406-1409. ROBINSON, C. and KIRKHAM, J. (1984): Enamel Matrix Components, Alterations during Development and Possible Interactions with the Mineral Phase. In: Tooth Enamel IV, R.W. Fearnhead and S. Suga, Eds., Amsterdam: Elsevier Science Publishers, pp. 261-265. ROBINSON, C.; KIRKHAM, J.; WEATHERELL, J.A.; RICHARDS, A.; JOSEPHSEN, K.; and FEJERSKOV, 0. (1987): Developmental Stages in Permanent Porcine Enamel, Acta Anat 128:110. SAMUEL, N.; BESSEM, C.; BRINGAS, P., Jr.; and SLAVKIN, H.C. (1987): Immunochemical Homology Between Elasmobranch Scale and Tooth Extracellular Matrix Proteins in Cephaloscyllium ventriosum, J Craniofac Genet Dev Biol 7:371-386. SHIMIZU, M. and FUKAE, M. (1983): Enamel Proteins. In: Mechanisms of Tooth Enamel Formation, S. Suga, Ed., Tokyo: Quintessence Pub. Co., Inc., pp. 125-141. SHIMOKAWA, H.; SOBEL, M.E.; SASAKI, S.; TERMINE, J.D.; and YOUNG, M.F. (1987): Heterogeneity of Amelogenin mRNA in the Bovine Tooth Germ, J Biol Chem 262:4042-4047. SMITH, C.E.; McKEE, M.D.; and NANCI, A. (1987): Cyclic Induction and Rapid Movement of Sequential Waves of New Smoothended Ameloblast Modulation Bands in Rat Incisors as Visualized by Polychrome Fluorescent Labeling and GBHA-staining of Maturing Enamel, Adv Dent Res 1:162-175. SNEAD, M.L.; LAU, E.C.; ZEICHNER-DAVID, M.; FINCHAM, A.G.; WOO, S.L.C.; and SLAVKIN, H.C. (1985): DNA Sequence for Cloned cDNA Murine Amelogenin Reveals the Amino

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Acid Sequence for Enamel-specific Protein, Biochem Biophys Res Common 129:812-818. SPEIRS, R.L. (1975): Fluoride Incorporation into Developing Enamel of Permanent Teeth in the Domestic Pig, Arch Oral Biol 20:877883. SUGA, S. (1970): Histochemical Observation of Proteolytic Enzyme Activity in the Developing Dental Hard Tissues of the Rat, Arch Oral Biol 15:555-558. TANABE, T.; AOBA, T.; MORENO, E.C.; and FUKAE, M. (1988): Effect of Fluoride in the Apatitic Lattice on Adsorption of Enamel Proteins onto Calcium Apatites, J Dent Res 67:536-542. VOGEL, A.J. (1961): Quantitative Inorganic Analysis, 3rd ed., New York: John Wiley & Sons, p. 810. WEATHERELL, J.A.; DEUTSCH, D.; ROBINSON, C.; and HALLSWORTH, A.S. (1975): Assimilation of Fluoride by Enamel throughout the Life of the Tooth, Caries Res 11(Suppl.1):85-115. WHITFORD, G.M. and REYNOLDS, K.E. (1979): Plasma and Developing Enamel Fluoride Concentrations during Chronic Acidbase Disturbances, J Dent Res 58:2058-2065. YAMAKOSHI, Y.; TANABE, T.; FUKAE, M.; and SHIMIZU, M. (1989): Amino Acid Sequence of 25 kD Amelogenin. In: Proceedings of Tooth Enamel IV, R.W. Fearnhead, Ed., Yokohama: Florence Pub., pp. 163-168. ZEICHNER-DAVID, M.; MacDOUGALL, M.; VIDES, J.; SNEAD, M.L.; SLAVKIN, H.C.; TURKEL, S.B.; and PAVLOVA, Z. (1987): Immunochemical and Biochemical Studies of Human Enamel Proteins during Neonatal Development,J Dent Res 66:5056.

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