Effects of fluoride on rat dental enamel matrix proteinases

Abstract

Enamel fluorosis is characterised by increased porosity and a delay in the removal of enamel matrix proteins as the enamel matures. Amelogenin is the primary matrix protein in secretory-stage dental enamel. As enamel matures, amelogenins are hydrolysed by a number of enamel proteinases, including matrix metalloproteinase-20 (MMP-20 or enamelysin) and serine proteinase. Here, the effect of ingested fluoride on the relative activity of proteinases in the enamel matrix and the specific effect of fluoride on MMP-20 activity were examined. Proteinase activity relative to total enamel matrix protein was measured by fluorescence assay of enamel matrix dissected from rats given 0, 50, or 100 parts per 10 6 fluoride in their drinking water. To determine the specific effect of fluoride on the activity of MMP-20, the hydrolysis of a full-length recombinant human amelogenin by recombinant MMP-20 (rMMP-20) in the presence of 0, 2, 5, 10 or 100 μM fluoride was compared by sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE). In addition, a fluorescent peptide assay was developed to quantify enzyme activity against the tyrosine-rich amelogenin peptide cleavage site. In the late maturation stage, total proteinase activity per unit protein was lower in the fluoride-exposed rats than in the control rats. This in vivo finding indicates that fluoride ingestion can alter the relative amount of active proteinase in mature enamel. Hydrolysis of amelogenin at neutral pH by rMMP-20 was reduced in the presence of 100 μM F. In the peptide assay, rMMP-20 activity was significantly reduced by concentrations of fluoride as low as 2 μM at pH 6, with no significant effect at pH 7.2. These in vitro assays show that micromolar concentrations of fluoride can alter metalloproteinase activity, particularly when the pH is reduced to 6.0. These studies suggest that the effects of fluoride on enamel matrix proteinase secretion or activity could be involved in the aetiology of fluorosis in enamel and other mineralising tissues.