Mechanism of cadmium induced crystal defects in developing rat tooth enamel

Abstract

It is well known that exposure to environmental cadmium causes itai-itai (ouch-ouch) disease. However, the exact mechanism underlying this bone disease remains unresolved. By focusing on the calcification mechanism, we examined developing tooth enamel in rats exposed to cadmium to test the hypothesis that cadmium exposure may cause defects in crystal formation. Electron microscopy revealed the presence of perforated crystals in developing tooth enamel, indicating that the process of crystal nucleation may have been interrupted by cadmium exposure. Furthermore, biochemical analyses revealed that the catalytic activity of carbonic anhydrase in the immature enamel matrix declined remarkably despite the fact that quantitative reduction of this enzyme was insignificant, suggesting that the decline of catalytic activity may have resulted from the replacement of zinc with cadmium ions. Therefore, we concluded that the poor catalytic activity of cadmium-binding carbonic anhydrase might hinder the nucleation process, leading to an impairment in mineralization that causes itai-itai disease.

Fig. 1

Electron micrographs of the processes of crystal development in normal enamel. (a) Ribbon-shaped structures and the Tomes’ processes (Tp) of ameloblasts at the secretory stage. Cross-(C) and longitudinal sections (L) of early ribbon-shaped structures. (b) Higher magnification of cross-sections of early ribbon-shaped structures (double-stained sections). (c) Longitudinal sections of early ribbon-shaped structures. A ribbon-shaped structure consists of 2 thin organic layers and an electron-lucent mineral zone (double-stained sections). (d) Higher magnification of cross-sections of early ribbon-shaped structures. Precursor minerals without lattice lines are observed as electron-dense fuzzy images (unstained sections). (e) The appearance of the initial lattice line (arrowheads) within the envelope (double-stained sections). (f) Developing longitudinal sections of enamel crystals with central dark lines (arrows, unstained sections). (g) Developing cross-sections of enamel crystals with central dark lines (arrows, unstained sections). Scale bars: (a) 1 μm, (b–d) 20 nm, (e–g)) 10 nm.

Fig. 2

Electron micrographs of crystals obtained from cadmium-exposed rat tooth enamel. (a) Low magnification of enamel crystals affected by the cadmium exposure for 5 weeks. (b and c) Higher magnification of enamel crystals. The crystal nucleation process seems to be sporadically inhibited. (d and e) Enamel crystals affected by the cadmium exposure for 12 weeks. (d) Perforated crystals at low magnification. (e) Cross-section of perforated and intact crystals at higher magnification. The perforated crystals reveal voids at their centers, while the intact crystals show central dark lines (arrowheads). Scale bars: (a and d) 100 nm, (b, c and e) 10 nm. (a–e) Unstained sections.

Fig. 3

Electrophoretic patterns and immunoblotting analysis of the immature enamel matrix proteins obtained from control, cadmium-exposed and fluoride-exposed rats. (a) Electrophoretic patterns obtained from each sample. (b) Western blot analysis of the immature enamel matrix proteins. Unlike fluoride exposure, cadmium exposure does not affect the synthesis of carbonic anhydrase. LM, low molecular weight standards; C, 8-week-old controls; Cd, 8-week-old cadmium-exposed rats; F, 8-week-old fluoride-exposed rats.

Fig. 4

Carbonic anhydrase activity in immature enamel tissues obtained from each group. Exposure to cadmium or fluoride significantly reduced enzymatic activity. C, 8-week-old controls; Cd, 8-week-old cadmium-exposed rats; F, 8-week-old fluoride-exposed rats. Error bars indicate S.D.

Fig. 5

Schematics of the process of crystal defect formation in developing enamel. (a) Ameloblast Tomes’ process (Tp) and ribbon-shaped structures (long parallel lines). (b) One of the enlarged ribbon-shaped structures under normal conditions. After the formation of the central dark line, the growth of crystal occurs within an organic envelope. (c) Crystallization continues to occur at the peripheral area, which is not affected by Mg ions, despite the failure of the central dark line to form due to the lack of carbonate ions. CDL: Central dark line. Broken lines indicate the organic envelope. CA-Zn: Carbonic anhydrase-Zn. CA-Cd: Carbonic anhydrase-Cd.