ToxSci Advance Access originally published online on July 29, 2008
Toxicological Sciences 2008 106(1):214-222; doi:10.1093/toxsci/kfn156
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Tributyltin Impairs Dentin Mineralization and Enamel Formation in Cultured Mouse Embryonic Molar Teeth
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* Department of Pediatric and Preventive Dentistry, Institute of Dentistry, 00014 University of Helsinki, Helsinki, Finland
Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital, Helsinki, Finland
Department of Oral Pathology, Institute of Dentistry, 00014 University of Helsinki, Helsinki, Finland
Department of Pathology, Helsinki University Central Hospital, Helsinki, Finland
1 To whom correspondence should be addressed at Biomedicum Helsinki, Institute of Dentistry, University of Helsinki, PO Box 63, FI-00014 Helsinki, Finland. Fax: +358-9-1912-5371. E-mail: eija.peltonen{at}helsinki.fi.
Received May 16, 2008; accepted July 23, 2008
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ABSTRACT |
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Tributyltin (TBT), earlier used as an antifouling agent in marine paints, causes damage to the aquatic ecosystem, for example, impaired shell calcification in oysters. TBT affects hard tissue mineralization even in mammals: delayed bone mineralization has been observed in rodents exposed to TBT in utero. To see if TBT interferes with tooth development, especially dental hard tissue formation, we exposed mouse E18 mandibular first and second molars to 0.1, 0.5, 1.0, and 2.0µM TBT chloride in organ culture for 7–12 days. The amount of enamel was assessed and the sizes of the first molars were measured from photographs taken after the culture. TBT concentration dependently impaired enamel formation (p < 0.001) and reduced tooth size (p < 0.001). Histological analysis showed slight arrest of dentin mineralization and enamel formation in first molars exposed to 0.1µM TBT. At the concentration of 1.0µM the effect was overt. The differentiation of ameloblasts in the mesial cusps was retarded but TBT had no effect on odontoblast morphology. The dental epithelium showed enhanced apoptosis. The failure of ameloblasts to form enamel was likely to be secondary to the effect of TBT on dentin mineralization. In the second molars, where predentin deposition had not started, ameloblasts and odontoblasts were nonpolarized and proliferative. The results showed that TBT concentration dependently impairs dental hard tissue formation and reduces tooth size in cultured mouse embryonic molars. The effects depend on the stage of tooth development at the start of exposure and may involve epithelial-mesenchymal interactions.
Key Words: Tributyltin; tooth development; dentin mineralization; enamel; apoptosis; mouse.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Organic tin compounds are composed of tin, which is bound to one to four hydrocarbon groups (mono-, di-, tri-, and tetra-substituted). Organotins are most widely used for stabilization of polyvinyl chloride as mono- or di-substitutes. Nevertheless, a tri-substituted organotin, tributyltin (TBT), causes the main risk for humans (Risk and Policy Analysts limited [RPA], 2005
). Humans are exposed to TBT mainly via seafood in the diet (RPA, 2005). TBT was widely used as an antifouling agent in marine paints until it was observed to accumulate in aquatic animals and cause severe damage to the aquatic ecosystem (Harino et al., 2000; Strand and Jacobsen, 2005). TBT has been shown to cause imposex, that is, development of additional male sex organs in female snails and oysters (Horiguchi et al., 1998; Smith, 1981) and sex reversal also in fish (Shimasaki et al., 2003). Moreover, defective shell calcification and problems in reproduction have been reported in oysters (Alzieu, 2000). Despite restrictions in the use of TBT since the 1980's, high concentrations have been found in coastal areas of harbors and dockyards, heavily used water routes and dredge spoils sites (Antizar-Ladislao, 2008). The aim of the International Maritime Organization has been total prohibition of TBT as an antifouling biocide by the present year (2008).
The average intake of TBT by humans from market-bought seafood has been estimated to vary worldwide between 0.18 and 2.6 µg/day/person (Keithly et al., 1999). Recommendation of the European Food Safety Agency for the highest allowed daily intake of organotins is 0.25 µg/kg body weight (European Food Safety Authority, 2004). However, humans can be exposed to organotins through a wide range of products and other sources. Therefore, the cumulative intake can exceed the tolerable daily intake (TDI) (RPA, 2005). Children especially are estimated to be at risk of being exposed to organotin levels exceeding the TDI values (RPA, 2005). This causes great concern, because TBT has been found to cause developmental defects even in mammals. For example, cleft palate, aberrant gonadal development and delayed ossification of the supraoccipital bone have been observed in rodents exposed to TBT chloride (TBTCl) in utero (Ema et al., 1997; Kishta et al., 2007; Tsukamoto et al., 2004). Experimental studies imply that TBT can transfer to offspring through placenta (Noland et al., 1983). Lactational transfer of organotins has also been reported (Kimura et al., 2005).
Tooth development (Fig. 1) is genetically strictly controlled but susceptible to environmental disturbances. Among chemicals that have been found to interfere with tooth development in clinical and experimental studies are environmental toxicants such as dioxins and nonhalogenated polycyclic aromatic hydrocarbons (PAH compounds) as well as fluoride and certain drugs. The effects not only depend on the chemical concerned and the dose/concentration but also the stage of tooth development at the time of exposure. Because the dental hard tissues are not replaced once they have been formed, tooth is an informative organ model for studying abnormal mineralized tissue formation. TBT has been shown to have an adverse effect on osteoblast differentiation and bone mineralization (Tsukamoto et al., 2004). We therefore tested in vitro if TBT also disturbs tooth development.
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MATERIALS AND METHODS |
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Animals and teeth.
Pregnant mice (NMRI x NMRI) were anesthetized with CO2 and killed by cervical dislocation on embryonic day 18 (Theiler Stage 26; Theiler, 1989
). The vaginal plug was designated day 0 (E0). Mandibular first and second molar tooth germs of E18 mice were dissected in Dulbecco's phosphate-buffered saline (D-PBS) supplemented with penicillin and streptomycin (20 IU penicillin and 0.02 mg streptomycin/ml D-PBS) (Sigma-Aldrich Co., St Louis, MO) under a stereomicroscope. We dissected 190 mandibular first and second molars from 97 pups of 11 dams. The explants were cultured in 11 distinct experiments, four times for 7 days, once for 10 days and six times for 12 days. A total of 28 teeth were cultured for 7 days, 20 teeth were cultured for 10 days and 142 teeth for 12 days. In each culture there were four to eight teeth in a single culture dish. The use of animals was approved by the Institutional Animal Care and Use Committee of the Faculty of Science of the University of Helsinki.
Organ culture.
Dissected teeth were transferred onto polycarbonate Nuclepore filters (pore size 0.1 µm; Corning Inc., NY) on stainless steel grids placed in culture dishes (Trowell-type organ culture). The explants were cultured in Dulbecco's modified Eagle's medium (D-MEM) with GlutaMAX I (GIBCO; Invitrogen Corporation, Carlsbad, CA) supplemented with 10% (vol/vol) fetal calf serum (GIBCO) and ascorbic acid (100 µg/ml; Sigma-Aldrich Co.) (basal medium). For morphological analysis, TBTCl (Sigma-Aldrich Finland/YA-Kemia Oy, Helsinki, Finland) was added to the basal medium at the start of culture. Because the effects of TBT at the cellular level can be expected to become evident after shorter exposure than morphological effects, TBTCI was added after two days of culture to study apoptosis and cell proliferation. TBTCl was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1.0M (TBTCl stock). Working solution was made by diluting TBTCl stock 1:10,000 in distilled water (TBTCl concentration, 0.1mM). Fresh TBTCl dilution was made for each experiment. After a pilot culture with TBTCl at the concentrations of 1.0, 6.0, 12.0, and 60.0µM, the lowest concentrations producing clear effects on dental hard tissue formation were selected for further use. The final TBTCl concentrations in the basal medium were 0.1, 0.5, 1.0, and 2.0µM. Control explants were cultured in the basal medium. DMSO is cytotoxic itself. In our previous study, 0.7% (vol/vol) DMSO, used as a vehicle for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), had no detectable effect on the development of mouse E18 molars cultured for up to 14 days (Partanen et al., 1998). The concentration of DMSO used to dissolve TBTCl was so low (< 0.0002%) that any possible effects of DMSO could be ignored. The explants were cultured in a humidified incubator at 37°C in an atmosphere of 5% CO2 in air for 7, 10, or 12 days. The medium was changed every 2–3 days. At every change, the growth of the explants was monitored under a stereomicroscope. There was no marked difference in the growth of unexposed control teeth or in the effect of TBTCl (hereafter TBT) on tooth development between 10 and 12 days of culture.
Preparation of explants for histological examination.
Provided that there was a thick layer of enamel on the mesial side of the control first molars after 10 or 12 days of culture, the explants were photographed. Explants to be studied histologically were quickly fixed in cold methanol and the fixation was continued in 4% paraformaldehyde in PBS at 4°C overnight. The explants were demineralized with ethylenediaminetetraacetic acid for at least two weeks, dehydrated, embedded in paraffin, serially sectioned at 7 µm, stained with hematoxylin-eosin (HE) and examined histologically. For the analysis of the effect of TBT on the morphology of ameloblasts and odontoblasts of the first molars, mesial cusps were chosen, because the differentiation of the dental cells starts at the tip of the mesial cusp and proceeds in cervical (toward the root) and distal (toward the second molar) directions. The stages of differentiation of ameloblasts and odontoblasts therefore vary with the location. The differentiation is farthest advanced on the mesial side of the mesial cusp.
Detection of apoptotic cells.
Apoptotic cells in tissue sections of teeth cultured for a total of 7 days (2 days in the basal medium followed by 5 days with 1.0µM TBT) were localized by the detection of DNA fragmentation with a digoxigenin-based modification of the original terminal deoxynucleotidyl transferase–mediated deoxy-uridine triphosphate nick end labeling (TUNEL) (Vaahtokari et al., 1996). Briefly, paraffin sections of selected explants were deparaffinized, dehydrated, incubated in ethanol/acetic acid (2:1), 100% methanol, and 0.5% H2O2 in methanol. The sections were rehydrated, washed and incubated in 50mM Tris-HCl/5mM ethylenediaminetetraacetic acid containing 20 mg/ml proteinase K (Finnzymes Oy, Helsinki, Finland). After fixation with 4% paraformaldehyde, the sections were labeled for 1 h at 37°C in a labeling mix. The reaction was stopped and the sections were preblocked. The anti-digoxigenin-AP antibody (alkaline phosphatase-conjugated anti-digoxigenin antibody, Roche Diagnostics GmbH, Mannheim, Germany) was added at 0.05% mixture in preblocking solution and the sections were incubated at 4°C overnight. The color was developed with a mixture of 4-nitro blue tetrazolium chloride (Sigma-Aldrich Co.) and 5-bromo-4-chloro-3-indolyl-phosphate (Sigma-Aldrich Co.) in alkaline phosphatase buffer (NTM). Finally the sections were given a slight background staining with hematoxylin. (For detailed protocol, see Partanen et al., 2004.)
Analysis of cell proliferation.
To analyze cell proliferation, the explants cultured for a total of 7 days (2 days in the basal medium followed by 5 days with 1.0µM TBT) were labeled with 5'-bromo-2'-deoxyuridine (BrdU) (Zymed; Invitrogen Corporation). The tissues were incubated with BrdU 1:100 (from concentrate) in the culture medium for 60 min, fixed and processed for histological examination as described above. BrdU incorporation into DNA was detected by a mouse monoclonal antibody to BrdU (Abcam plc, Cambridge, UK). For the detection of the bound antibody, Vectastain ABC Elite Kit (Vector Laboratories Inc., Burlingame, CA) was used according to the instructions of the manufacturer.
Analysis of the effect of TBT on enamel formation and the size of the tooth after 12 days of culture.
The amount of enamel was estimated from the photographs taken after the culture, before fixation, and the estimation was confirmed by histological analysis of representative explants. For the statistical analysis, each first molar was given a score 0–3 depending on the extent and thickness of the enamel on the mesial side of the mesial cusp (Fig. 2). Score 3 was given to teeth with a thick and even enamel layer extending from the tip of the mesial cusp at least midway along the mesial side. Score 2 was given to teeth showing clearly thinner or less extended enamel. Score 1 was given if there was only a spot of enamel or mineralized dentin mesially and score 0 if there was no enamel or mineralized dentin at all. In teeth scored 0–2 there was no enamel in the medial and distal cusps.
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The sizes of the first molars were measured at the level of the deepest site of the fissure distally to the medial cusp using an image analysis software (analySIS 3.00; Soft Imaging System GmbH, Muenster, Germany; Fig. 2). For the statistical test, the sizes of the molars were grouped into four categories. Differences in mesio-distal widths and the amount of enamel between control and TBT-treated teeth were tested by Pearson's Chi-Square test (SPSS, version 13, SPSS.com). Explants exposed to 2µM TBT were not included in the statistical analysis, because necrosis in the dental tissues was a common finding. The probability value of < 0.05 was considered significant.
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RESULTS |
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Stages of the Mandibular First and Second Molar Development at the Start of Culture
HE-stained sections of the mouse mandibular molar tooth explants at the start of culture at E18 showed that morphogenesis of the three principal cusps of the first molar had been completed (Fig. 1). Ameloblasts were polarized throughout the tooth crown and polarized odontoblasts facing ameloblasts were visible in the region of the mesial cusp and at the tip of the medial cusp but not in the distal cusp. Deposition of predentin had not started (Fig. 1). The second molar was undergoing transition from the cap stage of morphogenesis to the bell stage, which precedes dental cell differentiation (Fig. 1).
The Effect of TBT on the Size and Development of the First Molar
In the unexposed control first molars cultured for 10 or 12 days odontoblasts had deposited a thick layer of predentin throughout the crown. Mineralization of the dentin and formation of the enamel had proceeded from the tip along the mesial side of the mesial cusp where the enamel formed an even layer (Figs. 2A and 3A). The extent of enamel and mineralized dentin varied from one culture experiment to another: in some cultures the enamel layer extended from the tip of the mesial cusp midway along the mesial side, whereas in others enamel was also seen in the medial and distal cusps (Fig. 2A). Enamel had not been formed unless there was mineralized dentin facing it. The thickness of enamel equaled or exceeded the combined thicknesses of the unmineralized predentin and mineralized dentin (Fig. 3A). Mesial ameloblasts were elongated and polarized and they formed a coherent layer (Figs. 3A and 3C). Ameloblasts that had already laid down a thick enamel layer were shortened but still polarized (Fig. 3A). Correspondingly, odontoblasts located on the mesial side of the mesial cusp were polarized and elongated (Fig. 3A) but had lost their elongated shape at the cusp tip upon predentin formation.
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TBT slowed down enamel formation (p < 0.001) and decreased the size of the tooth with the ascending concentration (p < 0.001) (see Figs. 2B–D). At the lowest concentration, 0.1µM, the effect of TBT on predentin mineralization and enamel formation was barely detectable (Fig. 2B). Mineralized dentin and enamel layers in most teeth were as thick as in controls. The majority of teeth exposed to 0.5µM TBT showed a clear reduction in dentin mineralization and enamel formation (Fig. 2C). However, depending on the culture there was variation ranging from reduced thickness of the enamel layer to the absence of mineralized dentin and enamel. The variation was in scale with hard tissue formation in unexposed control teeth. Some teeth were still comparable with the controls. At the concentration of 1.0µM the effect of TBT on predentin mineralization and enamel formation was obvious (Figs. 2D and 3B). In the vast majority of teeth (25/30) dentin had not started to mineralize and no enamel was visible. Only few teeth had a thin layer of mineralized dentin and enamel on the mesial side of the mesial cusp and where present, the enamel did not form a uniform layer but occurred as a spot (see Fig. 2C).
The effect of TBT on predentin formation was less overt. Irrespective of TBT concentration, the thickness of predentin on the mesial side of the mesial cusp in virtually all teeth was comparable to the controls (Fig. 3B). The proportion of teeth showing reduced predentin thickness in the less advanced medial and distal cusps increased with the concentration of TBT (5/12 teeth exposed to 0.1µM, 13/17 teeth exposed to 0.5µM, and 22/24 teeth exposed to 1.0µM TBT). The reduced thickness of predentin consistently coincided with an altered morphology of the stellate reticulum compartment of the epithelial enamel organ between the medial and distal cusps. The stellate reticulum cells had lost their normal star-like shape (Fig. 3E) and the loose-textured tissue had become a dense mass of enlarged eosinophilic cells with round, central nuclei, possibly indicating an incipient cell death (Fig. 3F). Correspondingly, the medial and distal cusps were thin and more or less curly.
At the concentration of 2.0µM the toxic effects of TBT were pronounced: growth of the teeth was markedly retarded or the whole explant had become necrotic. There was no enamel and where predentin was present, its thickness was consistently reduced (data not shown).
Ameloblasts on the mesial side of the mesial cusp of teeth exposed to 0.1µM TBT were polarized and elongated like in the controls. The height of ameloblasts decreased with the ascending TBT concentration. Completely nonpolarized ameloblasts with central nuclei were seen especially in the occlusal third of the mesial side of the mesial cusp (Fig. 3D). Corresponding to the failure of predentin to mineralize morphological changes in ameloblasts were observed in 14/20 teeth exposed to 0.5µM TBT, 22/26 teeth exposed to 1.0µM TBT (Fig. 3D) and in all 10 teeth exposed to 2.0µM TBT. The ameloblastic layer in part of teeth exposed to 1.0 and 2.0µM TBT was slightly disorganized (Fig. 3B) and in some teeth ameloblasts that were undergoing elongation were hyperchromatic (Fig. 3B). TBT had no clear effect on odontoblasts on the mesial side of the mesial cusp (Fig. 3B).
The Effect of TBT on the Development of the Second Molar
After 10 or 12 days of culture, predentin formation was consistently in progress throughout the crown of the unexposed control second molars (Fig. 3G). Varying with the culture, mineralization of dentin and enamel formation were ongoing in some control teeth (8/30) (Fig. 3G). Ameloblasts were polarized and depending on their location also elongated (Fig. 3I). Odontoblasts were polarized and columnar throughout the tooth crown (Fig. 3I).
Of the 12 teeth exposed to TBT at the lowest concentration (0.1µM) two had mineralized dentin and only minor changes in predentin thickness were observed. Of the 19 teeth exposed to 0.5µM TBT one had mineralized dentin. There was no mineralized dentin or enamel in any of the 23 teeth exposed to 1.0µM TBT (Fig. 3H) or of the 10 teeth exposed to 2.0µM. The thickness of predentin was reduced in 14/19, 16/23, and 9/10 teeth exposed to 0.5, 1.0, and 2.0µM TBT, respectively (Fig. 3H). Where predentin deposition had not started, odontoblasts were nonpolarized (Fig. 3J). The proportion of teeth showing altered morphology of ameloblasts increased with TBT concentration. Elongation of ameloblasts was less overt than in control teeth in 15/19 teeth exposed to 0.5µM TBT and in all 23 and 10 teeth exposed to 1.0 and 2.0µM TBT, respectively. Completely nonpolarized ameloblasts were also visible (Fig. 3J). The shape of most teeth exposed to 0.5, 1.0, and 2.0µM TBT was altered: cusps were thin and curly. Cusp tips lacking predentin were disintegrated (Figs. 3H and 3J). The absence of predentin and abnormal shape of cusps coincided with an altered morphology of the stellate reticulum only in few teeth.
The Effect of TBT on Apoptosis and Cell Proliferation
First molar.
In the control first molars cultured for 7 days minimal apoptosis was observed in the enamel organ (Figs. 4A and 4C), the dental papilla and oral epithelium. TBT exposure moderately enhanced apoptotic cell death mainly in epithelial tissues of tooth explants exposed for 5 days to 1.0µM TBT after two days of culture without TBT. Apoptosis was increased in the enamel organ constituents stellate reticulum, stratum intermedium and ameloblasts; in the basal epithelial diaphragma giving rise to Hertwig's epithelial root sheet; and in the basal layer of the oral epithelium (Figs. 4B and 4D). Apoptosis was concentrated in the medial and distal parts of the first molar but was not seen in the mesial cusp.
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In the control first molars cultured for 7 days proliferating cells were seen in the basal parts giving rise to tooth roots. TBT did not alter the rate of cell proliferation (data not shown).
Second molar.
In the control second molars only few apoptotic ameloblasts were seen. TBT had no definite effect on the distribution pattern or frequency of apoptotic cells. Apoptosis was mainly seen in ameloblasts and the epithelial diaphragma (data not shown).
Proliferating cells were mainly seen in the basal parts of the control second molars, and in (pre)ameloblasts and stratum intermedium on the distal side of the distal cusp and in the cervical third of the mesial side of the mesial cusp (Fig. 4E). BrdU labeling was minimal on the mesial side of the distal cusps. TBT exposure increased the frequency of labeled nuclei on the mesial side of the distal cusp and at the deepest site of the fissure between the cusps. Of the eight teeth exposed to TBT, four showed abundant proliferation in (pre)ameloblasts and stratum intermedium in the region of the whole tooth, in (pre)odontoblasts and in cells of the dental papilla (Fig. 4F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The present study showed that the organic tin compound, TBT, disturbs cultured mouse embryonic molar tooth development. In the first molars, started to be exposed at the late bell stage of morphogenesis preceding dental hard tissue formation, TBT arrested mineralization of dentin and enamel formation. Depending on the concentration, the effect ranged from barely detectable to a complete absence of mineralized dentin and enamel. First molars exposed to TBT were smaller than the corresponding control teeth. When predentin was not mineralized and enamel had not been formed in the farthest advanced mesial cusps of the first molars, ameloblasts were not elongated but had remained cuboidal, suggesting arrest of cell differentiation. Odontoblasts, by contrast, showed no morphological change. Reduced or lacking predentin in the less advanced medial and distal cusps of the first molars was accompanied by morphological alteration in the stellate reticulum of the enamel organ and deterioration of the cusps. In the second molars, started to be exposed at the cap-bell stage preceding dental cell, thinner predentin and cusps were observed but the changes in the stellate reticulum were less obvious than in the first molars. When predentin formation had not started in the second molars, ameloblasts and odontoblasts were nonpolarized. TBT increased apoptosis in the epithelial enamel organ of the first molars whereas proliferating cells were more frequent in both the epithelial and mesenchymal tissues of the second molar.
A requirement for the differentiation of the ectomesenchyme-derived odontoblasts is a signal from the epithelial preameloblasts. For the differentiation of ameloblasts, a signal from odontoblasts is needed and ameloblasts do not become secretory until odontoblasts have laid down a thin layer of predentin and it has started to mineralize (Tompkins, 2006).
Possibly by interfering with the reciprocal inductive signalling between the epithelial and mesenchymal cells, precocious and increased apoptosis in the enamel organ of the first molars may have led to the failure of odontoblasts to differentiate and become secretory. The start of dentin matrix mineralization, launching the secretory activity of ameloblasts, represents the final step in the series of epithelial-mesenchymal interactions that instruct early tooth development (Thesleff, 2003; Tompkins, 2006). Therefore, the failure of ameloblasts to become secretory or the impaired secretory capacity of ameloblasts could be secondary to the adverse effect of TBT on predentin mineralization. Even though the reduced size of the first molar can partly be explained by lacking or thin enamel, apoptosis in the enamel organ may have contributed to deterioration of the cusp shape and to smaller tooth size.
Once the dental cells have differentiated, they do no longer proliferate. Therefore, abundant cell proliferation in the second molars is consistent with the morphological and functional signs of retarded differentiation.
Taken together, the effect of TBT on developing tooth is likely to involve epithelial-mesenchymal interactions that are essential for development to proceed. The type and extent of morphological consequences of TBT exposure depend on the point where the sequence of inductive interactions is arrested and vary with the stage of tooth development at the start of exposure.
TCDD and a nonhalogenated PAH, 7,12-dimethylbenz[a]anthracene have been found to interfere with rodent tooth morphogenesis, dental hard tissue formation and tooth size in vivo and in vitro (Alaluusua et al., 1993; Gao et al., 2004; Kattainen et al., 2001; Lukinmaa et al., 2001; Miettinen et al., 2002; Partanen et al., 1998, 2004; Peltonen et al., 2006). TCDD arrested tooth development and altered cusp shape by accelerating and increasing apoptosis in those dental epithelial cells that are predetermined to undergo apoptosis later during normal development (Partanen et al., 2004). Although TBT exposure in the present study was started at a later stage of the first and second molar development, apoptosis was likewise enhanced in the dental epithelium programmed to die apoptotically. Up to 50% of ameloblasts may undergo apoptosis during enamel maturation (Smith and Warshawsky, 1977) and the number of cells of the stratum intermedium and stellate reticulum will significantly reduce by apoptosis during advancing enamel formation (Vaahtokari et al., 1996), to disappear upon tooth eruption. Therefore, those dental epithelial cells that are predestined to undergo apoptosis may respond to a toxic insult by entering apoptotic cell death precociously. The particular cell type is determined by the time window of the toxic effect, which in the present study extended from late morphogenesis to early dental hard tissue formation.
Previous studies show that TBT interferes with biomineralization in different species (Adeeko et al., 2003; Chagot et al., 1990; Suzuki et al., 2006; Tsukamoto et al., 2004). Rodent bones are affected dose-dependently (Adeeko et al., 2003; Tsukamoto et al., 2004). Susceptibility of the process extends from differentiation and activity of osteoblasts to deposition of calcium (Suzuki et al., 2006; Tsukamoto et al., 2004). Likewise, calcium deposition is essential for mineralization of predentin to dentin (Wöltgens et al., 1987). Interference by TBT with predentin mineralization, found here, is in line with the earlier finding that TBT impairs the formation of mineralized nodules and deposition of calcium by cultured rat calvarial osteoblasts (Tsukamoto et al., 2004).
Due to their limitations, organ culture systems do not allow for a comprehensive assessment of developmental defects. Therefore, the clinical relevance of the present study on the effect of TBT on tooth development can only be speculated. A feature common to dioxins, PAH compounds and organotins is that they are fat soluble and can be transferred to offspring through lactation. Because developing teeth are susceptible to each of these compounds, they could interfere with formation of dental hard tissues not only separately but also in combination. Experimental dose response studies in different animal species and clinical observations are needed to find out if there is any risk at prevailing levels.
To conclude, we showed in line with previous findings concerning the effects of TBT on bone that TBT concentration dependently interferes with mouse embryonic tooth hard tissue formation in vitro. The effects are modified by the stage of tooth development at the start of exposure and are likely to involve inductive interactions between epithelial and mesenchymal cells and tissues.
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FUNDING |
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Academy of Finland (206689); and Finnish Dental Society Apollonia to E.S.
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ACKNOWLEDGMENTS |
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The skillful technical assistance of Ms Maarit Hakkarainen, Ms Pirjo Jutila, and Ms Marjatta Kivekäs is gratefully acknowledged.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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