The effect of amoxicillin on dental enamel development in vivo.

The exposure to amoxicillin has been associated with molar incisor hypomineralization. This study aimed to determine if amoxicillin disturbs the enamel mineralization in in vivo experiments. Fifteen pregnant rats were randomly assigned into three groups to received daily phosphatase-buffered saline or amoxicillin as either 100 or 500 mg/kg. Mice received treatment from day 13 of pregnancy to day 40 postnatal. After birth, the offsprings from each litter continued to receive the same treatment according to their respective group. Calcium (Ca) and phosphorus (P) content in the dental hard tissues were analyzed from 60 upper first molars and 60 upper incisors by the complexometric titration method and colorimetric analysis using a spectrophotometer at 680 nm, respectively. Lower incisors were analyzed by X-ray microtomography, it was measured the electron density of lingual and buccal enamel, and the enamel and dentin thickness. Differences in Ca and P content and electron density among the groups were analyzed by one-way ANOVA. There was no significant difference on enamel electron density and thickness among the groups (p > 0.05). However, in incisors, the higher dose of amoxicillin decreased markedly the electron density in some rats. There were no statistically significant differences in Ca (p = 0.180) or P content (p = 0.054), although the higher dose of amoxicillin could affect the enamel in some animals. The amoxicillin did not significantly alter the enamel mineralization and thickness in rats.

The effect of amoxicillin on dental enamel development in vivo

Abstract The exposure to amoxicillin has been associated with molar incisor hypomineralization. This study aimed to determine if amoxicillin disturbs the enamel mineralization in in vivo experiments. Fifteen pregnant rats were randomly assigned into three groups to received daily phosphatase-buffered saline or amoxicillin as either 100 or 500 mg/kg. Mice received treatment from day 13 of pregnancy to day 40 postnatal. After birth, the offsprings from each litter continued to receive the same treatment according to their respective group. Calcium (Ca) and phosphorus (P) content in the dental hard tissues were analyzed from 60 upper first molars and 60 upper incisors by the complexometric titration method and colorimetric analysis using a spectrophotometer at 680 nm, respectively. Lower incisors were analyzed by X-ray microtomography, it was measured the electron density of lingual and buccal enamel, and the enamel and dentin thickness. Differences in Ca and P content and electron density among the groups were analyzed by one-way ANOVA. There was no significant difference on enamel electron density and thickness among the groups (p > 0.05). However, in incisors, the higher dose of amoxicillin decreased markedly the electron density in some rats. There were no statistically significant differences in Ca (p = 0.180) or P content (p = 0.054), although the higher dose of amoxicillin could affect the enamel in some animals. The amoxicillin did not significantly alter the enamel mineralization and thickness in rats.

The effect of high temperature on the development of mouse dental enamel in vitro.

OBJECTIVE: Our aim was to study the effect of high temperature (39°C) corresponding to fever on the development of enamel in cultured mouse molars. DESIGN: For morphological studies mandibular molar blocks from E18 mice were cultured for 11 days. After three days at 37°C the teeth were exposed to 39°C for three or five days and returned to 37°C. At the end of culture, the tooth explants were photographed. The heights of the enamel and the crown of the first molars were measured and the enamel/crown height ratio was calculated. The ratios were compared between the groups using the Mann-Whitney test. To analyze gene expression in ameloblasts and odontoblasts with RT-qPCR and in situ hybridization, part of the test explants were cultured for three days at 37°C and then five days at 39°C. Control explants were kept at 37°C for 11 or eight days. RESULTS: The enamel/crown height ratio of the first molars cultured for five days at 39°C was smaller than that of the unexposed (P<0.001). Tooth morphology did not differ between controls and exposed teeth. In qPCR-analysis, dentine sialophosphoprotein showed clearly decreased expression. In situ hybridization showed no dentine sialophosphoprotein expression in preameloblasts. The expressions of bone morphogenic protein 4, matrix metalloproteinase 20, dentine matrix protein 1, amelogenin and osteocalcin showed a trend of downregulation. CONCLUSIONS: High culturing temperature interferes with enamel formation of mouse molars and alters the expression of some genes essential for normal enamel development.

Combined effect of amoxicillin and sodium fluoride on the structure of developing mouse enamel in vitro.

OBJECTIVE: Excess fluoride intake during tooth development is known to cause dental fluorosis. It has also been suggested that amoxicillin use in early childhood is associated with enamel hypomineralization. The aim was to investigate separate and combined effects of sodium fluoride (NaF) and amoxicillin on enamel formation in vitro. DESIGN: Mandibular molar tooth germs of E18 mouse embryos were cultured for 10 days in a medium containing NaF (10, 12 or 15µM) and/or amoxicillin (0.5, 1, 2 or 3.6mg/mL) or sodium clavulanate (0.07mg/mL) alone or in combination with 0.5mg/mL of amoxicillin. Morphological changes were studied from the whole tooth photographs and histological tissue sections with light microscope. RESULTS: Only with the highest concentrations of NaF or amoxicillin alone the extent of enamel in the first molars measured as the vertical enamel height/crown height ratio was reduced (p<0.01, p<0.001, respectively). At lower concentrations, combination of NaF (12µM) and amoxicillin (2mg/mL) significantly reduced enamel extent compared with the controls (p<0.001). Histologically, the ameloblasts were still columnar but poorly organized and the nascent enamel was often non-homogeneous. Enamel formation was not seen in any second molars exposed to 12µM NaF and 2mg/mL of amoxicillin (or higher concentrations) compared with the presence of enamel in half of the controls (p<0.001). CONCLUSIONS: Amoxicillin and NaF dose dependently affect developing enamel of mouse molars in vitro and the effects are potentiative. The clinical significance of the results remains to be studied.

Tributyltin alters osteocalcin, matrix metalloproteinase 20 and dentin sialophosphoprotein gene expression in mineralizing mouse embryonic tooth in vitro.

We showed in a previous in vitro study that tributyltin (TBT) arrests dentin mineralization and enamel formation in developing mouse tooth. The present aim was to investigate the effect of TBT on the expression of genes associated with mineralization of dental hard tissues. Embryonic day 18 mouse mandibular first molars were cultured for 3, 5 or 7 days and exposed to 1.0 µM TBT and studied by real-time quantitative polymerase chain reaction (RT-QPCR) for the expressions of osteocalcin (Ocn), alkaline phosphatase (Alpl), dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp) and matrix metalloproteinase 20 (Mmp-20).Ocn, Mmp-20 and Dspp, whose expressions showed changes in RT- QPCR, were further analyzed by in situ hybridization of tissue sections. In situ hybridization showed that TBT decreased Ocn expression in odontoblasts but increased the expression in the epithelial tooth compartment. In QPCR assays, the net effect in the whole tooth was increased expression. TBT also reduced Mmp-20 expression in ameloblasts and odontoblasts. Dspp expression varied but both QPCR assays and in situ hybridization showed a decreasing trend. TBT exposure had no clear effect on Alpl and Dmp1 expressions. Increased Ocn expression by epithelial enamel organ may inhibit dentin mineralization and enamel formation. Decreased Ocn, Mmp-20 and Dspp expressions in odontoblasts may indicate delayed cell differentiation, or TBT may specifically decrease the expression of genes involved in dentin mineralization. While decreased Mmp-20 expression by TBT in ameloblasts may impair enamel mineralization, the coincident reduction in Mmp-20 and Dspp expressions in odontoblasts may potentiate the delay of dentin mineralization.

Combined effect of fluoride and 2,3,7,8-tetrachlorodibenzo-p-dioxin on mouse dental hard tissue formation in vitro.

Fluoride interferes with enamel matrix secretion and mineralization and dentin mineralization. The most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), also impairs dental hard tissue formation and mineralization in vitro and in vivo. Our aim was to investigate in vitro whether the combined effect of sodium fluoride (NaF) and TCDD on dental hard tissue formation is potentiative. For this purpose, mandibular first and second molar tooth germs of E18 mouse embryos were cultured for 5-12 days with NaF and TCDD alone at various concentrations (2.5, 5, 10, 12.5, 15, and 20 µM and 5, 10, 12.5, and 15 nM, respectively) to determine the highest concentrations, which alone cause no or negligible effects. Morphological changes were studied from the whole tooth photographs and histological tissue sections. The concentrations found were 15 µM for NaF and 10 nM for TCDD. While at these concentrations, the effects of NaF and TCDD alone were barely detectable, the effect of simultaneous exposure on dentin and enamel formation was overt; mineralization of predentin to dentin and enamel matrix secretion and mineralization were impaired. Immunohistochemical analysis revealed that the combined exposure modified amelogenin expression by odontoblasts. Morphology of ameloblasts and the expression of amelogenin indicated that ameloblasts were still secretory. The results show that NaF and TCDD have potentiative, harmful effects on the formation of dental hard tissues. Since children can be exposed to subclinical levels of fluoride and dioxins during early childhood, coincidently with mineralization of the first permanent teeth, this finding may have clinical significance.

Tributyltin impairs dentin mineralization and enamel formation in cultured mouse embryonic molar teeth.

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 2.0 microM 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 0.1 microM TBT. At the concentration of 1.0 microM 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.

2,3,7,8-tetrachlorodibenzo-p-dioxin specifically reduces mRNA for the mineralization-related dentin sialophosphoprotein in cultured mouse embryonic molar teeth.

Previous studies show that the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), interferes with mineralization of the dental matrices in developing mouse and rat teeth. Culture of mouse embryonic molar teeth with TCDD leads to the failure of enamel to be deposited and dentin to undergo mineralization. Lactationally exposed rats show defectively matured enamel and retardation of dentin mineralization. To see if the impaired mineralization is associated with changes in the expression of dentin sialophosphoprotein (Dspp), Bono1 and/or matrix metalloproteinase-20 (MMP-20), thought to be involved in mineralization of the dental hard tissues, we cultured mouse (NMRI) E18 mandibular molars for 3, 5 or 7 days and exposed them to 1 microM TCDD after 2 days of culture. As detected by in situ hybridization of tissue sections, localization and intensity of Bono1 and MMP-20 expression showed no definite difference between the control and exposed tooth explants, suggesting that TCDD does not affect their expression. On the contrary, TCDD reduced or prevented the expression of Dspp in secretory odontoblasts and decreased it in presecretory ameloblasts. The results suggest that the retardation of dentin mineralization by TCDD in mouse molar teeth involves specific interference with Dspp expression.

7,12-dimethylbenz[a]anthracene interferes with the development of cultured mouse mandibular molars.

Clinical studies suggest that maternal smoking during pregnancy can reduce the crown size of the child's teeth. Delayed dental age compared with chronological age has also been reported in children whose parents smoke. Among the main components of tobacco smoke are nonhalogenated polycyclic aromatic hydrocarbons (PAHs), many of which are highly toxic. Humans are exposed to PAH compounds mainly via tobacco smoke and diet. The aim of our study was to investigate the effect of PAHs on tooth formation and the function of tooth-forming cells. We exposed mouse (NMRI) E18 mandibular first and second molar explants to 7,12-dimethylbenz[a]anthracene (DMBA), a toxic PAH compound, in organ culture for 7 or 12 days. DMBA concentrations used were 0.1, 0.5, 1, and 2 microM. The mesiodistal width of each first molar (12-day culture) was measured in stereomicroscopic images, and the teeth were analysed histologically. DMBA exposure significantly reduced the mesiodistal width of the first molars. DMBA impaired or delayed amelogenesis and dentinogenesis in both molars at the lowest concentration of 0.1 microM. DMBA affected enamel formation more severely than dentin formation and occasionally prevented amelogenesis completely. Elongation and polarization of ameloblasts were impaired, and blood vessel architecture of the dental papilla (future pulp) was altered. Cusps were thin and sharp. In line with the finding that maternal smoking during pregnancy has an adverse effect on child's tooth development, this study shows the toxic influence of PAHs on tooth development in vitro.

Interference by 2,3,7,8-tetrachlorodibenzo-p-dioxin with cultured mouse submandibular gland branching morphogenesis involves reduced epidermal growth factor receptor signaling.

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to mouse embryonic teeth, sharing features of early development with salivary glands in common, involves enhanced apoptosis and depends on the expression of epidermal growth factor (EGF) receptor. EGF receptor signaling, on the other hand, is essential for salivary gland branching morphogenesis. To see if TCDD impairs salivary gland morphogenesis and if the impairment is associated with EGF receptor signaling, we cultured mouse (NMRI) E13 submandibular glands with TCDD or TCDD in combination with EGF or fibronectin (FN), both previously found to enhance branching morphogenesis. Explants were examined stereo-microscopically and processed to paraffin sections. TCDD exposure impaired epithelial branching and cleft formation, resulting in enlarged buds. The glands were smaller than normal. EGF and FN alone concentration-dependently stimulated or inhibited branching morphogenesis but when co-administered with TCDD, failed to compensate for its effect. TCDD induced cytochrome P4501A1 expression in the glandular epithelium, indicating activation of the aryl hydrocarbon receptor. TCDD somewhat increased epithelial apoptosis as observed by terminal deoxynucleotidyl transferase (TdT)-mediated nick end-labeling method but the increase could not be correlated with morphological changes. The frequency of proliferating cells was not altered. Corresponding to the reduced cleft sites in TCDD-exposed explants, FN immunoreactivity in the epithelium was reduced. The results show that TCDD, comparably with EGF and FN at morphogenesis-inhibiting concentrations, impaired salivary gland branching morphogenesis in vitro. Together with the failure of EGF and FN at morphogenesis-stimulating concentrations to compensate for the effect of TCDD this implies that TCDD toxicity to developing salivary gland involves reduced EGF receptor signaling.

Developmental toxicity of dioxin to mouse embryonic teeth in vitro: arrest of tooth morphogenesis involves stimulation of apoptotic program in the dental epithelium.

Previous studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can arrest molar tooth development in rats after in utero and lactational exposure, and that the sensitive stage is temporally restricted. To define the stage in which TCDD is able to arrest tooth development and the cellular background of the effect, mouse embryonic molar tooth explants including various early developmental stages from initiation to late cap stage were exposed to TCDD in organ culture. TCDD did not inhibit morphogenesis of the first molar teeth including the early bud-staged E12 first molars, but the teeth were smaller than in control cultures. Accordingly, the second molars underwent morphogenesis in the presence of TCDD when explanted at E15 when they were at the bud stage. TCDD arrested their development when explanted at E14 when they had not yet reached the early bud stage. Immunohistochemical localization of incorporated bromodeoxyuridine in cultured E14 teeth showed that TCDD did not affect cell proliferation. Localization of apoptosis by terminal deoxynucleotidyl transferase (TdT)-mediated nick end labeling (TUNEL) method revealed that TCDD enhanced apoptosis of dental epithelial cells, especially in the dental lamina of both the first and second molars, and in the inner dental epithelium at the cusp tips of the first molars. Thus, TCDD can arrest tooth development in vitro if the exposure starts at the initiation stage, whereas exposure at later stages leads to smaller tooth size and deformation of cuspal morphology. TCDD interferes with tooth development by stimulating apoptosis in those cells of the dental epithelium, which are predetermined to undergo apoptosis during normal development.

Response of the incisor tooth to 2,3,7,8-tetrachlorodibenzo-p-dioxin in a dioxin-resistant and a dioxin-sensitive rat strain.

Dioxins are ubiquitous environmental pollutants that afflict developing teeth. To find out if the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the continuously erupting rat incisor is associated with the sensitivity to TCDD acute lethality and to see the histological basis for any macroscopic findings, we exposed 25 resistant Han/Wistar (Kuopio; H/W) and 20 sensitive Long-Evans (Turku/AB; L-E) female rats to total doses of 0.17, 1.7, 17, and 170 (only H/W rats) micro g/kg TCDD. Each dose group comprised five animals. The treatment was started when the rats were 10 weeks old and continued for 20 weeks. The exposure time covered two life cycles of the incisor. Stereomicroscopic examination of the dissected mandibles showed color defects and pulpal perforation of the lower incisors at 17 and 170 micro g/kg TCDD. Tissue sections revealed odontoblastic and pulpal cell death and the consequent arrest of dentin formation at the incisal tooth end at the same doses. H/W rat incisors were affected closer to the germinative tooth end at 170 than at 17 micro g/kg TCDD, resulting in a larger perforation. In accordance with the enamel discoloration, the postsecretory enamel organ underwent, albeit inconsistently, precocious squamous metaplasia with pronounced proliferation. Thus, both the mesenchymal and, to a lesser extent, epithelial elements of the forming tooth were affected dose-dependently at relatively high doses of TCDD. Similar responses in both strains implied that the impaired formation of the incisor tooth, at least of its mesenchymal elements, is not associated with the differential resistance of H/W and L-E rats to TCDD acute lethality.

Expression of the mediators of dioxin toxicity, aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT), is developmentally regulated in mouse teeth.

Dioxins are persistent and ubiquitous environmental poisons that become enriched in the food chain. Besides being acutely lethal, the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is developmentally toxic to many animal species. We have previously found that developing teeth of children may be sensitive to environmental dioxins via their mother's milk and that rat and mouse teeth are dioxin-sensitive throughout their development. The aryl hydrocarbon receptor (AHR) together with the AHR nuclear translocator (ARNT) protein is believed to mediate the toxic effects of dioxins. To study the potential involvement of the AHR-ARNT pathway in the dental toxicity of TCDD, we analysed the expression of AHR and ARNT by in situ hybridization and immunohistochemistry in developing mouse teeth. AHR mRNA first appeared in the epithelium of E12 first molar tooth buds and both proteins were weakly expressed in the bud. After cytodifferentiation the expression was up regulated and became intense in secretory odontoblasts and ameloblasts. The coexpression of AHR and ARNT during early tooth development as well as during the information and mineralization of the dental matrices is suggestive of the AHR-ARNT pathway as a mediator of dental toxicity of TCDD.