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Fluorosis: A New Model and New InsightsDepartment 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; Correspondence: * corresponding author, tpayne{at}forsyth.org
Fluoride is an effective agent for the prevention of dental caries. However, the mechanism of how excessive fluoride exposure causes fluorosis remains uncertain. Zebrafish (Danio rerio) exhibit periodic tooth replacement throughout their lives, thereby providing continuous access to teeth at developmental stages susceptible to fluoride exposure. Zebrafish teeth do not contain true enamel, but consist of a hard enameloid surface. Therefore, we asked whether zebrafish could be used as a model organism for the study of dental fluorosis. Scanning electron microscopy of fluoride-treated teeth demonstrated that the enameloid was pitted and rough, and FTIR analysis demonstrated that the teeth also contained a significantly higher organic content when compared with untreated controls. Furthermore, we demonstrate for the first time that decreased expression of an important signaling molecule (Alk8) in tooth development may contribute to the observed fluorotic phenotype, and that increased cell apoptosis may also play a role in the mechanism of fluorosis.
Key Words: apoptosis • cell signaling • fluorosis • tooth development • zebrafish
Severe enamel defects often result from genetic dysplasias (Gibson et al., 2001). Ameloblasts, however, are particularly vulnerable to transient perturbations in their environment. Fluoride is the 13th most abundant element in the earth’s crust (Whitford, 1989) and is well-known as an effective caries prophylactic (Castioni et al., 1998). However, acute or chronic exposure to fluoride can result in dental (DenBesten and Crenshaw, 1987) and skeletal fluorosis (Boivin et al, 1989). Fluoride ingestion during tooth formation is manifested in the enamel by an increase in protein content and a decrease in overall mineral content (Bhusshry, 1959; DenBesten, 1986; Wright et al., 1996; Zhou et al., 1996). Fluorosis varies in appearance from white striations to stained pitting of the enamel. Numerous studies have been conducted over several decades in an effort to determine the mechanism by which fluoride induces enamel defects. However, a gap exists in our understanding of the molecular mechanisms underlying the cellular and biochemical changes that occur in developing teeth in the presence of elevated fluoride levels. Mammalian models for tooth development have allowed investigators to determine many key signaling molecules and developmental regulatory processes. To date, most fluorosis studies have been performed in mice and rats. Rodents have continuously erupting incisors that manifest every developmental stage of enamel formation. Although the erupted molars remain relatively unchanged, mice or rats exposed to high doses of fluoride for 3 wks or more will have fluorosed chalky-white incisors. This occurs because developing teeth are the most susceptible to fluorosis. In fact, fluorosis is often more severe in permanent teeth than in primary teeth, because, in utero, the placenta partially blocks access of fluoride to the fetus as the primary teeth begin to develop (Whitford, 1996). Thus, developing teeth, such as the continuously erupting incisor, are necessary for fluorosis studies. In the last decade, the zebrafish (Danio rerio), a small teleost fish, has become a simpler vertebrate model for studies of a variety of developmental processes. Unlike most common laboratory animals, zebrafish are easily maintained and reproduce rapidly, and large numbers of offspring can be raised in a small area. Zebrafish teeth have enameloid rather than true enamel. Enameloid and enamel are both highly mineralized tissues covering the dentin of vertebrate teeth. The inner enamel epithelium (IEE) cells of teleosts elaborate a protein matrix that mineralizes to form this hard covering (Huysseune et al., 1998). The IEE cells of teleosts secrete a collagenous matrix that provides the scaffolding for enameloid, while amelogenin is the major enamel matrix protein. Both the collagen of enameloid and the amelogenin of enamel are broken down and removed as the tissues mineralize, so the resulting product is more than 97% mineral (Prostak and Skobe, 1986).
Zebrafish have 2 sets of pharyngeal teeth, positioned on paired ceratobranchial pharyngeal arches (Fig. 1
Fish Husbandry and Collection of Embryos Zebrafish were maintained at 28.5°C in a 14-hour light/10-hour dark cycle in the Forsyth Zebrafish Facility. Embryos were collected by natural mating and were maintained at 28.5°C as previously described (Payne et al., 2001a). The Animal Protocol was approved under Animal Welfare Assurance (number 305101).
Fluoride Treatment
Scanning Electron Microscopy (SEM)
Compositional Analysis/Fourier Transform Infrared Spectroscopy (FTIR)
Apoptosis Assay
Immunohistochemistry
Fluoride Causes Specific Enameloid Defects We exposed zebrafish to increasing concentrations of ambient fluoride to determine if fluoride exposure caused any growth defects that may non-specifically affect tooth formation. Larval fish, 2 mos old, were exposed to fluoride levels of 1.9 ppm to 95 ppm for 8 wks. Concentrations were selected as a range, based upon reported fluoride levels in fresh water (DenBesten and Crenshaw, 1987; Whitford, 1996; Levy, 2003). No significant difference was observed in zebrafish weight, size, or vigor after fluoride exposure (data not shown).
After 8 wks of fluoride treatment, the fifth ceratobranchial arches (cb5) (Fig. 1
Fluoride Alters the Mineral/Protein Ratio of Zebrafish Teeth
Fluoride Increases Programmed Cell Death in Developing Tooth Germs The similarity between zebrafish fluorotic enameloid and fluorotic enamel in humans led us to examine ameloblast apoptosis upon fluoride exposure. It has been demonstrated that rat ameloblasts in transition to the early-maturation stage undergo apoptosis, decreasing in number by approximately 25% (Smith and Warshawsky, 1977). We asked if zebrafish tooth cells also undergo natural apoptosis, and if the apoptosis could be increased by fluoride exposure. Apoptosis can be assayed with the use of a monoclonal antibody (MAB) specific for ssDNA, which is a result of apoptosis, and is specific for programmed cell death vs. tissue necrosis (Pycroft et al., 2002). There was significant apoptosis in cells of the developing tooth germs, specifically in the ameloblast-like IEE cells (AM) (Fig. 3  ). Panel 3A shows an untreated section of a 4-month-old zebrafish, with a red box highlighting the location of the pharyngeal teeth. The dark brown nuclear staining in Panels 3E' and 3F' indicates apoptosis in the tooth germs exposed to fluoride, and there are more apoptotic nuclei than in the control teeth (Panels 3B, 3C'). The red asterisks mark apoptotic cells, and, on average, there are 12–18% more apoptotic cells in the fluoride-treated tooth germs than in the untreated tooth germs. Eighteen teeth were examined for each condition. The mean % of apoptotic cells in 0-ppm-F teeth was 9.4% (9.4% ± 0.47 SE), and in 38-ppm-F treated teeth, 27.4% (27.4% ± 0.61 SE), p < 0.001 by t test. Furthermore, the apoptosis was restricted to the pharyngeal region of the fish, with the exception of the normal apoptosis observed in the gut, and the chondrocytes of the gill rays below the mouth.
Fluoride Causes Changes in Levels of TGF-beta Family Signaling in Developing Tooth Germs Perturbations in growth factor signals, specifically BMPs, can lead to apoptosis, which prompted us to perform an initial analysis of cell signaling in fluorotic teeth. Alk8 is a zebrafish type II TGF-beta receptor that signals through BMP pathways (Payne et al., 2001a) and is involved in tooth development (Payne et al,, 2001b; Perrino and Yelick, 2004). Upon exposure to fluoride, developing tooth germs exhibited a decreased level of Alk8 when compared with untreated tooth germs (Figs. 4B , 4A). The loss of Alk8 expression was limited to the ameloblast-like IEE cells surrounding the bell-stage tooth germ, whereas Alk8 was expressed normally in the surrounding crypt epithelium, and in other non-tooth tissues. Defects in cell adhesion may also result in apoptosis; however, analysis of the adhesion molecule E-cadherin revealed no change of expression in fluoride-treated tooth germs (Figs. 4C, 4D). Wnt signaling has also been demonstrated to affect apoptosis, so we examined components of the Wnt signaling pathway: beta-catenin and APC (data not shown). Expression of these proteins did not change upon fluoride exposure.
Dental fluorosis specifically affects developing tooth germs, which, in mammalian models, develop slowly and are few. Zebrafish ameloblast-like IEE cells form enameloid, which, when completely mineralized, has properties similar to those of mammalian enamel. To determine if the zebrafish is a good animal model for dental fluorosis, we compared controls and fluoride-treated zebrafish teeth for enameloid defects, enameloid organic content, apoptosis, and expression of cell-signaling molecules. We demonstrate that zebrafish teeth display fluorotic defects similar to those observed in mice, rats, and humans. Compositional analysis revealed that zebrafish fluorosis was concomitant with decreased mineral content and increased protein levels, which is similar to fluorosis observed in mammals. Furthermore, we demonstrate for the first time, by use of the zebrafish model, that decreased expression of cell-signaling molecules in tooth development may contribute to the observed fluorotic phenotype, and, furthermore, that increased apoptosis may also play a role. The major events relevant to early enamel mineralization are: secretion of matrix proteins and proteases into the enamel matrix, and transport of ions and organic matter to and from the enamel matrix (Aoba and Fejerskov, 2002). Transition to the early-maturation stage is the first point at which significant programmed cell death (apoptosis) has been shown to reduce the number of ameloblasts (Smith and Warshawsky, 1977; Nishikawa and Sasaki, 1995). Numerous signaling cascades—including the expression of Bmps, Wnts, Fgfs and Hh—contribute to mammalian tooth development and are localized at sites of epithelial-mesenchymal cell interaction (Thesleff and Nieminen, 1996). Recently, the roles of several Fgfs in zebrafish tooth development have been demonstrated (Jackman et al., 2004), and work in this laboratory has shown that TGF-beta signaling is also required for normal tooth formation (Payne et al., 2001b). The signals relayed by Wnts, BMPs, and TGF-beta have also been shown to be involved in apoptotic events (for review, see Thesleff, 2003). The mechanism by which normal ameloblast apoptosis is controlled is not known. However, recent studies have indicated that tightly regulated gradients of signaling are critical for cell differentiation and apoptotic control. Abnormal levels of BMPs and Wnts, either elevated or reduced, can result in apoptosis, due to a poorly established cell-signal field (Wall and Hogan, 1995; Solloway and Robertson, 1999; Payne-Ferreira and Yelick, 2003). Recently, a transgenic mouse that over-expresses wnt3 in tooth epithelia was shown to have enamel defects that were hypothesized to be the result of elevated ameloblast apoptosis (Millar et al., 2003). Similarly, Alk8, which acts through BMP pathways, when dramatically down-regulated, results in decreased bmp2b-4 activity and increased apoptosis in developing embryos and cartilage elements (Payne-Ferreira and Yelick, 2003). We demonstrate that fluoride treatment of zebrafish teeth results in increased apoptosis and decreased Alk8 expression. This observed apoptosis may reflect a perturbed gradient of BMP signaling and inappropriate growth cues that culminate in programmed cell death. Based on our results—that include analysis of enameloid defects, enameloid organic composition, apoptosis, and Alk8 expression—we propose that the zebrafish is a useful model for the study of fluorosis. The fact that the fluorotic defects in zebrafish collagen-based mineralized tissue are similar to enamel fluorosis suggests that defects in mineralization may be dependent on a ‘common’ event, such as growth factor signaling cascades, which are highly conserved across species. The data obtained from zebrafish can then be used in well-defined mammalian studies to give us a more thorough understanding of the enamel defects caused by excess fluoride. Future studies will address the roles of Alk8 and other TGF-beta signaling molecules during dental fluorosis.
The authors thank Loic Fabricant for expert care of the zebrafish facility, and Dr. Pamela Yelick for the Alk8 antibody. This work was supported by NIDCR grants K22 DE 14683-2 (T.L.F) and DE14084 and DE13237 (J.D.B). Received for publication June 4, 2004. Revision received May 13, 2005. Accepted for publication May 24, 2005.
Journal of Dental Research, Vol. 84, No. 9,
832-836 (2005) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
3 PO4 absorption band was used as a measure of organic content in the teeth. The amide I/PO4 ratio in the untreated teeth (control sample) was 0.33, whereas in the fluoride-treated teeth it was 0.6, indicating that the fluoride-treated teeth had a significantly higher organic content than did the normal teeth. This result is similar to that observed in mammalian fluorosis models (
