Effects of Fluoride on Matrix Proteins and Their Properties in Rat Secretory Enamel

Abstract

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 secretory 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 0°C, 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 NaCI 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 secretory stage of rat amelogenesis.