Proteomic Mapping of Dental Enamel Matrix from Inbred Mouse Strains: Unraveling Potential New Players in Enamel.

Enamel formation is a complex 2-step process by which proteins are secreted to form an extracellular matrix, followed by massive protein degradation and subsequent mineralization. Excessive systemic exposure to fluoride can disrupt this process and lead to a condition known as dental fluorosis. The genetic background influences the responses of mineralized tissues to fluoride, such as dental fluorosis, observed in A/J and 129P3/J mice. The aim of the present study was to map the protein profile of enamel matrix from A/J and 129P3/J strains. Enamel matrix samples were obtained from A/J and 129P3/J mice and analyzed by 2-dimensional electrophoresis and liquid chromatography coupled with mass spectrometry. A total of 120 proteins were identified, and 7 of them were classified as putative uncharacterized proteins and analyzed in silico for structural and functional characterization. An interesting finding was the possibility of the uncharacterized sequence Q8BIS2 being an enzyme involved in the degradation of matrix proteins. Thus, the results provide a comprehensive view of the structure and function for putative uncharacterized proteins found in the enamel matrix that could help to elucidate the mechanisms involved in enamel biomineralization and genetic susceptibility to dental fluorosis.

Proteomics of Secretory-Stage and Maturation-Stage Enamel of Genetically Distinct Mice.

The mechanisms by which excessive ingestion of fluoride (F) during amelogenesis leads to dental fluorosis (DF) are still not precisely known. Inbred strains of mice vary in their susceptibility to develop DF, and therefore permit the investigation of underlying molecular events influencing DF severity. We employed a proteomic approach to characterize and evaluate changes in protein expression from secretory-stage and maturation-stage enamel in 2 strains of mice with different susceptibilities to DF (A/J, i.e. 'susceptible' and 129P3/J, i.e. 'resistant'). Weanling male and female susceptible and resistant mice fed a low-F diet were divided into 2 F-water treatment groups. They received water containing 0 (control) or 50 mg F/l for 6 weeks. Plasma and incisor enamel was analyzed for F content. For proteomic analysis, the enamel proteins extracted for each group were separated by 2-dimensional electrophoresis and subsequently characterized by liquid-chromatography electrospray-ionization quadrupole time-of-flight mass spectrometry. F data were analyzed by 2-way ANOVA and Bonferroni's test (p < 0.05). Resistant mice had significantly higher plasma and enamel F concentrations when compared with susceptible mice in the F-treated groups. The proteomic results for mice treated with 0 mg F/l revealed that during the secretory stage, resistant mice had a higher abundance of proteins than their susceptible counterparts, but this was reversed during the maturation stage. Treatment with F greatly increased the number of protein spots detected in both stages. Many proteins not previously described in enamel (e.g. type 1 collagen) as well as some uncharacterized proteins were identified. Our findings reveal new insights regarding amelogenesis and how genetic background and F affect this process.

Bone response to fluoride exposure is influenced by genetics.

Genetic factors influence the effects of fluoride (F) on amelogenesis and bone homeostasis but the underlying molecular mechanisms remain undefined. A label-free proteomics approach was employed to identify and evaluate changes in bone protein expression in two mouse strains having different susceptibilities to develop dental fluorosis and to alter bone quality. In vivo bone formation and histomorphometry after F intake were also evaluated and related to the proteome. Resistant 129P3/J and susceptible A/J mice were assigned to three groups given low-F food and water containing 0, 10 or 50 ppmF for 8 weeks. Plasma was evaluated for alkaline phosphatase activity. Femurs, tibiae and lumbar vertebrae were evaluated using micro-CT analysis and mineral apposition rate (MAR) was measured in cortical bone. For quantitative proteomic analysis, bone proteins were extracted and analyzed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), followed by label-free semi-quantitative differential expression analysis. Alterations in several bone proteins were found among the F treatment groups within each mouse strain and between the strains for each F treatment group (ratio ≥1.5 or ≤0.5; p<0.05). Although F treatment had no significant effects on BMD or bone histomorphometry in either strain, MAR was higher in the 50 ppmF 129P3/J mice than in the 50 ppmF A/J mice treated with 50 ppmF showing that F increased bone formation in a strain-specific manner. Also, F exposure was associated with dose-specific and strain-specific alterations in expression of proteins involved in osteogenesis and osteoclastogenesis. In conclusion, our findings confirm a genetic influence in bone response to F exposure and point to several proteins that may act as targets for the differential F responses in this tissue.

Distribution of fluoride and calcium in plaque biofilms after the use of conventional and low-fluoride dentifrices.

BACKGROUND: The distribution of fluoride and calcium in plaque after the use of fluoride dentifrices has not yet been determined. AIM: To evaluate fluoride and calcium distribution in sections of biofilms generated in situ after the use of conventional and low-fluoride dentifrices. DESIGN: Children (n = 11, 8­10 years old) brushed with placebo (fluoride-free), low-fluoride (513 mgF/kg), and conventional (1072 mgF/kg) dentifrices twice daily for 1 week, following a double-blind, cross-over protocol. Biofilms were generated using Leeds in situ devices, which were collected 1 and 12 h after brushing, and sectioned through their depth. Sections were grouped (10 x 5 µm) for fluoride and calcium analysis. Sections 4 lm thick were used for image analysis and determination of biomass fraction. Results were analysed by ANOVA, Tukey's test, and linear regression analysis (P < 0.05). RESULTS: Fluoride and calcium were mostly located at the outer sections of biofilms for all dentifrices tested, and these ions were directly correlated throughout most of biofilm's sections. Results for conventional dentifrice were significantly higher than for the placebo, but did not differ from those for the low-fluoride dentifrice. CONCLUSIONS: The use of a low-fluoride dentifrice did not promote a higher fluoride uptake in inner biofilms' sections, as hypothesized. As plaque fluoride was significantly elevated only after the use of the conventional dentifrice, the recommendation of low-fluoride formulations should be done with caution, considering both risks and benefits.

Renal proteome in mice with different susceptibilities to fluorosis.

A/J and 129P3/J mouse strains have different susceptibilities to dental fluorosis due to their genetic backgrounds. They also differ with respect to several features of fluoride (F) metabolism and metabolic handling of water. This study was done to determine whether differences in F metabolism could be explained by diversities in the profile of protein expression in kidneys. Weanling, male A/J mice (susceptible to dental fluorosis, n = 18) and 129P3/J mice (resistant, n = 18) were housed in pairs and assigned to three groups given low-F food and drinking water containing 0, 10 or 50 ppm [F] for 7 weeks. Renal proteome profiles were examined using 2D-PAGE and LC-MS/MS. Quantitative intensity analysis detected between A/J and 129P3/J strains 122, 126 and 134 spots differentially expressed in the groups receiving 0, 10 and 50 ppmF, respectively. From these, 25, 30 and 32, respectively, were successfully identified. Most of the proteins were related to metabolic and cellular processes, followed by response to stimuli, development and regulation of cellular processes. In F-treated groups, PDZK-1, a protein involved in the regulation of renal tubular reabsorption capacity was down-modulated in the kidney of 129P3/J mice. A/J and 129P3/J mice exhibited 11 and 3 exclusive proteins, respectively, regardless of F exposure. In conclusion, proteomic analysis was able to identify proteins potentially involved in metabolic handling of F and water that are differentially expressed or even not expressed in the strains evaluated. This can contribute to understanding the molecular mechanisms underlying genetic susceptibility to dental fluorosis, by indicating key-proteins that should be better addressed in future studies.

Fluoride concentrations in dental plaque and saliva after the use of a fluoride dentifrice preceded by a calcium lactate rinse.

Plaque fluoride concentrations ([F]) are directly related to plaque calcium concentrations [Ca]. Attempts to increase plaque F uptake from dentifrices or rinses have used methods designed to increase plaque [Ca] but with inconsistent results. This double-blind, double-crossover study tested the effect of a 150 mM calcium lactate rinse used prior to brushing with placebo or fluoridated dentifrices (1030 p.p.m. as NaF) on plaque and salivary [F] and [Ca]. Sixteen children (8-10 yr of age) were randomly assigned to four different groups according to the four treatments (placebo dentifrice or fluoridated dentifrice preceded by calcium lactate or deionized water prerinses). Plaque and saliva were collected 1 and 12 h after brushing on day 7 after starting to use the dentifrices. F was determined using the electrode and Ca was determined using atomic absorption spectrometry. Plaque and salivary [Ca] were not significantly increased after use of the calcium lactate prerinse, except for plaque [Ca] 1 h after the use of the placebo dentifrice. A significant increase in salivary [F] was associated with the calcium lactate prerinse only at 1 h after the use of the fluoridated dentifrice. The the calcium lactate prerinse did not significantly affect plaque [F] under any condition.

Relationship between daily fluoride intake from diet and the use of dentifrice and human plasma fluoride concentrations.

The literature contains reports of the relationship between the fluoride concentrations in drinking water and human plasma. None of these studies, however, documented individual levels of daily fluoride intake, which can vary considerably among individuals served by the same water supply. Furthermore, while water can be an important source of fluoride, other sources, especially fluoridated dentifrices, also contribute substantially. This 2-day study with five 25-35-year-old subjects in each of three communities (Bauru, 0.6-0.8 ppm F; Domelia, 0.7 ppm F; Floresta, 0.3 ppm F) determined plasma fluoride concentrations and fluoride intake from diet and the use of dentifrice which, together, approximate total daily fluoride intake. The purposes were to determine: (1) the extent to which plasma fluoride concentrations approached levels known to affect the quality and quantity of bone; (2) the relationship between fluoride intake and plasma concentrations. Plasma was collected at 4-h intervals starting at 0800 h and ending at 2000 h each day. Average fluoride intakes from diet and the use of dentifrice in the three communities ranged from 0.16 to 0.82 mg/day and from 0.29 to 3.16 mg/day, respectively. The overall average plasma concentrations in the three communities were 0.44, 0.45 and 0.54 micromol/l (P<0.005). They were directly related to intake from the use of dentifrice (P=0.030) and to total intake (P=0.033), but were not related to dietary intake (P=0.176). In conclusion, despite fluoride intake from various sources, the plasma fluoride concentrations of the study subjects remained at levels far below those associated with effects on bone production.

Bone surface and whole bone as biomarkers for acute fluoride exposure.

This study compares fluoride concentrations ([F]) in surface and whole bone for up to 27 days following an acute oral dose of F. Four groups of rats received single oral F dose (50 mg/kg body weight), and the control group received deionized water (n = 10/group). Groups were euthanized at 1, 3, 9, or 27 days after F administration. Plasma and femurs were collected. F on the femur surface was removed from a circular area (4.52 mm(2)) by immersion in 0.5M HCl for 15 s. The solution was buffered with total ionic strength adjustment buffer and analyzed with an electrode. The subjacent bone was sectioned and ashed at 600 degrees C. Ash and plasma were analyzed for F with the electrode following hexamethyldisiloxane-facilitated diffusion. Data were analyzed by Kruskall-Wallis and Dunn's test and by linear regression (p < 0.05). Peak plasma and bone surface [F] occurred on day 1 (0.26 +/- 0.14 microg/mL and 1801 +/- 888 microg/g, respectively). Bone surface [F] at 3, 9, and 27 were not statistically different from control. A significant increase in whole bone [F] was observed 3 days after F administration and the [F] remained relatively constant thereafter. The mean (+/- SD) surface/whole bone [F] ratios for the control and F groups were 2.45 +/- 0.98, 3.92 +/- 1.32, 1.61 +/- 0.82, 1.73 +/- 0.39, and 1.09 +/- 0.28, respectively. Plasma and bone surface [F]s were positively correlated (r = 0.74). Thus, bone surface was found to be a suitable biomarker for acute, sublethal F exposure 1 day after F administration. Whole bone [F] were significantly increased at 3, 9, and 27 days after F administration.

Nail and bone surface as biomarkers for acute fluoride exposure in rats.

When acute exposure to fluoride is thought to be the cause of death, confirmation often depends on the analysis of some body fluid or tissue. The aim of this study was to evaluate the use of nails and the periosteal surface of bone as indicators of acute exposure to fluoride. Six groups of rats were given a single oral dose of fluoride (50 mg/kg body weight), while the control group was given deionized water. The rats were killed at 2, 4, 8, 16, 24, and 48 h after fluoride administration. Plasma and nails (the proximal halves) were collected and analyzed for fluoride with an ion-specific electrode after hexamethyldisiloxane-facilitated diffusion. A circular area of the femur (4.52 mm(2)) was etched with 0.5M HCl for 15 s, and, after the addition of a buffer, the solution was analyzed with an ion-specific electrode. Peak plasma concentration occurred at 2 h, followed by progressively declining concentrations. Peak nail fluoride concentrations occurred at 8 h. The mean nail concentrations at 8, 16, and 24 h were significantly higher than that of the control group. Bone surface concentrations were significantly higher than that of the control group at 4 h and thereafter. Thus, the proximal portion of nails and bone surface are suitable biomarkers for acute fluoride exposure in rats.

Efeito do tempo de estocagem no flúor detectável na unha de ratos / Effect of storage time on detectable fluoride in rat nails

Estamos realizando estudos para avaliar a viabilidade de se usar unhas como indicadores da exposiçäo ao flúor (F) em ratos. O objetivo do presente estudo foi determinar se a concentraçäo de F em unhas de ratos permanece inalterada em funçäo do tempo após a coleta das mesmas. Ratos Wistar machos recém-desmamados receberam água deionizada e raçäo contendo 25.9 ppm F por 56 dias. O sangue e as unhas foram coletados para análise de F usando o eletrodo, após difusäo facilitada por HMDS. As unhas foram divididas em três grupos que diferiram em relaçäo ao tempo entre a coleta e a análise: imediata (G1), 2 meses (G2) e 3 meses (G3). As concentraçöes plasmáticas de F foram similares entre os grupos (0,017 ± 0,004 ppm). Entretanto, as concentrações de F nas unhas foram muito diferentes entre os grupos: G1, 37,40 ± 10,70 ppm; G2, 2,72 ± 2,70 ppm; G3, 1,38 ± 0,57 ppm. A ANOVA e o teste de Tukey mostraram que a concentraçäo de F do G1 foi significantemente maior em relaçäo ao G2 e ao G3 (p < 0,001). A causa na reduçäo na quantidade de F dependente do tempo ainda näo foi explicada. Pode ser resultado da formaçäo de um composto näo difusível pelo método do HMDS ou da formaçäo de um composto volátil contendo F. Estudos estäo sendo realizados para se avaliar estas possibilidades e para se determinar mais precisamente a cronologia deste fenômeno

Exposiçäo ao flúor e fluorose dental: uma revisäo de literatura / Fluoride exposures and dental fluorosis: a literature review

O declínio na prevalência e incidência de cárie dentária nas duas últimas décadas é considerado ser devido, em grande parte ao amplo uso do flúor. Entretanto, a prevalência de fluorose dentária aumentou simultaneamente. O aumento foi nas formas de fluorose suave e muito suave, tanto em áreas fluoretadas como näo fluoretadas. Uma grande quantidade de dados epidemiológicos mostra que a ocorrência de lesöes fluoróticas está associada à ingestäo excessiva de flúor durante o período de desenvolvimento dental. Muitas fontes de flúor têm sido identificadas. Esta revisäo descreve a condiçäo e sumariza a literatura recente acerca dos fatores de risco para fluorose dentária. Quatro fatores de risco maiores foram consistentemente identificados: uso de água fluoretada, suplemementos de flúor, dentifrícios fluoretados ou fórmulas infantis. Em adiçäo alguns alimentos e bebidas manufaturados podem ser importantes contribuintes para a ingestäo diária total de flúor