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Fluorosis: A New Model and New Insights

Department of Biomineralization and Department of Cytokine Biology, The Forsyth Institute, and Department of Oral and Developmental Biology, Harvard School of Dental Medicine, 140 The Fenway, Boston, MA 02115, USA;

View larger version (106K): [in a new window]   Figure 1. Phenotypic effect of fluoride treatment. (A) Ventral view of Alcian-blue-stained cartilages of a 7-dpf zebrafish larva. The first few teeth are located on cb5. (B-D) Adult alizarin-red-stained cb5 arches and higher-magnification images of ankylosed teeth. (E) SEM tooth images from 2-month-old fish that were exposed to the indicated fluoride concentrations for 2 mos prior to tooth removal. Characteristic enameloid pitting is seen at concentrations of 38 ppm F and higher. Top, 20,000X; bottom, 2000X magnification. Abbreviations: cb, ceratobranchial; d, dentin; D, dorsal tooth; dpf, days post-fertilization; e, enameloid; M, Meckel’s cartilage; MD, mediodorsal tooth; p, pulp; V, ventral tooth.

View larger version (25K): [in a new window]   Figure 2. FTIR spectra of the control (solid line) and fluoride-treated (dashed line) zebrafish teeth. As a measure of mineral content, the ratios between the maximum height of an amide I absorption band (a major protein absorption) at ~ 1645 cm–1 and a phosphate absorption band at ~ 1020 cm–1 were calculated. The amide I/3 PO4 ratio in the control sample was 0.33, whereas this ratio in fluoride-treated teeth was 0.6. These results suggest that the fluoride-treated teeth have a much higher organic content than do normal teeth. No significant differences in the mineral phase of the control and experimental teeth were observed.

View larger version (145K): [in a new window]   Figure 3. Apoptosis assay. Two-month-old fish were placed in either 0-ppm-F water (A,B,C) or 38-ppm-F water (D,E,F) for 4 wks. After 4 wks, ssDNA MAB was used to detect apoptotic nuclei (red asterisks) in the paraffin sections. (Panels A, D) Low magnification of ssDNA-stained paraffin sections. Red box indicates cb5 and its associated pharyngeal teeth. Dark brown nuclear staining indicates apoptosis. (Panels B-C, E-F) 20x magnification of control and NaF-treated fish, respectively. (Panels B'-C') 100x magnification of control (0 ppm) tooth germs. (Panels E'-F') 100x magnification of 38-ppm-F-treated tooth germs. Abbreviations: AM, ameloblast-like IEE cell; d, dentin; G, gut; GR, gill rays; PT, pharyngeal teeth.

View larger version (167K): [in a new window]   Figure 4. Paraffin sections of 4-month-old zebrafish pharyngeal teeth stained with anti-Alk8 polyclonal antibody, and E-cadherin monoclonal antibody. (A,C) Untreated. (B,D) 38-ppm-fluoride-treated. Alk8 is present on the surfaces of the IEE cells, as is E-cadherin. The arrowhead in panels A and B indicates later-stage IEE cells, and the asterisks in the E-cadherin sections (C and D) indicate early-stage tooth germs and more uniform staining of the less mature IEE cells. Abbreviations: am, ameloblast-like IEE cell; ce, crypt epithelium; D, dentin; T, bell-stage tooth.