A New Sugarcane Cystatin Strongly Binds to Dental Enamel and Reduces Erosion.

Cystatin B was recently identified as an acid-resistant protein in acquired enamel pellicle; it could therefore be included in oral products to protect against caries and erosion. However, human recombinant cystatin is very expensive, and alternatives to its use are necessary. Phytocystatins are reversible inhibitors of cysteine peptidases that are found naturally in plants. In plants, they have several biological and physiological functions, such as the regulation of endogenous processes, defense against pathogens, and response to abiotic stress. Previous studies performed by our research group have reported high inhibitory activity and potential agricultural and medical applications of several sugarcane cystatins, including CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4. In the present study, we report the characterization of a novel sugarcane cystatin, named CaneCPI-5. This cystatin was efficiently expressed in Escherichia coli, and inhibitory assays demonstrated that it was a potent inhibitor of human cathepsins B, K, and L ( Ki = 6.87, 0.49, and 0.34 nM, respectively). The ability of CaneCPI-5 to bind to dental enamel was evaluated using atomic force microscopy. Its capacity to protect against initial enamel erosion was also tested in vitro via changes in surface hardness. CaneCPI-5 showed a very large force of interaction with enamel (e.g., compared with mucin and casein) and significantly reduced initial enamel erosion. These results suggest that the inclusion of CaneCPIs in dental products might confer protection against enamel erosion.

Low-Level Fluoride Exposure Increases Insulin Sensitivity in Experimental Diabetes.

The effect of chronic fluoride (F) exposure from the drinking water on parameters related to glucose homeostasis was investigated. Wistar rats were randomly distributed into 2 groups (diabetic [D] and nondiabetic [ND]; n = 54 each). In D, diabetes was induced with streptozotocin. Each group was further divided into 3 subgroups (0, 10, or 50 mgF/L in drinking water). After 22 days of treatment, plasma and liver samples were collected. No alterations in glycemia, insulinemia, K(ITT), and HOMA2-IR (homeostasis model assessment 2 of insulin resistance) were seen for ND. F-exposure of D rats led to significantly lower insulinemia, without alterations in glycemia (increased %S). Proteomic analysis detected 19, 39, and 16 proteins differentially expressed for the comparisons D0 vs. D10, D0 vs. D50, and D10 vs. D50, respectively. Gene Ontology with the most significant terms in the comparisons D0 vs. D10, D0 vs. D50, and D50 vs. D10 were organic acid metabolic process and carboxylic acid metabolic process, organic acid metabolic process, and cellular ketone metabolic process. Analysis of subnetworks revealed that proteins with fold changes interacted with GLUT4 in comparison D0 vs. D10. Among these proteins, ERj3p was present in D10. Upregulation of this protein in the presence of F might help to explain the higher %S found in these animals. These data suggest that fluoride might enhance glucose homeostasis in diabetes and identify specific biological mechanisms that merit future studies.

Mercury fractionation in dourada (Brachyplatystoma rousseauxii) of the Madeira River in Brazil using metalloproteomic strategies.

This paper presents the results of mercury fractionation in muscle samples of dourada (Brachyplatystoma rousseauxii) from the JIRAU Hydroelectric Power Plant in the Madeira River Basin in the Amazon region of Brazil. The proteome of the dourada muscle was separated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE). The mercury present in the protein spots was determined by graphite furnace atomic absorption spectrometry (GFAAS) after acid mineralisation in an ultrasound bath. The protein spots in which the presence of mercury was detected were characterised by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) after tryptic digestion. The GFAAS determinations indicated that 65% of the mercury was linked to the protein fraction with a molar mass (Mm) of less than 90 kDa. The mercury concentrations in the seven spots in which this protein fraction was present were in the range of 11.40-35.10 µg kg(-1). Based on the mercury concentrations, it was possible to estimate that the protein spots contained approximately 1-3 mercury atoms per protein molecule. The ESI-MS/MS analysis allowed characterisation of the seven protein spots as the following proteins: protein NLRC5 (molar mass=18.10, pI=6.30); 39S ribosomal protein L36 mitochondrial (molar mass=15.40, pI=8.23); N-alpha-acetyltransferase 20 (Mm=15.95, pI=8.80); Mth938 domain-containing protein (Mm=15.01, pI=9.60); ubiquitin-40S ribosomal protein S27a (Mm=9.80, pI=7.60); parvalbumin alpha (Mm=12.40, pI=3.80) and parvalbumin beta (Mm=13.10, pI=3.45).

Effect of dimeticone and pepsin on the bioavailability of metoclopramide in healthy volunteers.

OBJECTIVE: To assess the effect of dimeticone and pepsin on the bioavailability of metoclopramide (CAS 7232-21-5) in healthy volunteers. METHODS: The study was conducted using a randomized, open, 2-period crossover design. The volunteers received single administration of 7-mg conventional metoclopramide capsule and a formulation containing metoclopramide (7 mg) plus dimeticone (40 mg) and pepsin (50 mg), with a 7-day interval between treatments. Serial blood samples were collected before dosing and during 24 h post-treatment. Plasma metoclopramide concentrations were analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The pharmacokinetics parameters AUC(last) and C(max) were obtained from the metoclopramide plasma concentration vs. time curves. RESULTS: Metoclopramide's association was bioequivalent to conventional capsule; 90% CIs for geometric mean treatment ratios of C(max) [108.0% (90% CI, 100.4-116.3%)], AUC(last) [103.3% (90% CI, 99.5-107.4%)] were within the predefined range. The metoclopramide formulations were well tolerated at the administered doses and no significant adverse reactions were observed. Thus, these results confirm the good bioavailability of metoclopramide in the new formulation and rule out any impaired absorption when the drugs are formulated in combination.

Impact of protease inhibitors on dentin matrix degradation by collagenase.

This proof-of-concept study assessed whether the reduction of the degradation of the demineralized organic matrix (DOM) by pre-treatment with protease inhibitors (PI) is effective against dentin matrix loss. Bovine dentin slices were demineralized with 0.87 M citric acid, pH 2.3, for 36 hrs. In sequence, specimens were treated or not (UT, untreated) for 1 min with gels containing epigallocatechin 3-gallate (EGCG, 400 µM), chlorhexidine (CHX, 0.012%), FeSO(4) (1 mM), NaF (1.23%), or no active compound (P, placebo). Specimens were then stored in artificial saliva (5 days, 37°C) with the addition of collagenase (Clostridium histolyticum, 100 U/mL). We analyzed collagen degradation by assaying hydroxyproline (HYP) in the incubation solutions (n = 5) and evaluated the dentin matrix loss by profilometry (n = 12). Data were analyzed by ANOVA and Tukey's test (p < 0.05). Treatment with gels containing EGCG, CHX, or FeSO(4) led to significantly lower HYP concentrations in solution and dentin matrix loss when compared with the other treatments. These results strongly suggest that the preventive effects of the PI tested against dentin erosion are due to their ability to reduce the degradation of the DOM.

Activity of matrix metalloproteinases in bovine versus human dentine.

Metalloproteinases (MMPs) have been implicated with metabolism of collagen in physiological and pathological processes in human dentine. As bovine teeth have been used as a substitute for human teeth in laboratory analysis, this study evaluated the activity of MMP-2 and -9 in bovine versus human dentine. Bovine and human dentine fragments, from crowns and roots, were powderized. Protein extraction was performed by two protocols: a neutral extraction with guanidine-HCl/EDTA (pH 7.4) and an acidic extraction with citric acid (pH 2.3). Gelatinolytic activities of extracts were revealed by zymography. MMP-2 and -9 were detected in crown and root dentine from bovine and human teeth. Total activities of MMP-2 were 11.4 ± 2.2, 14.6 ± 2.0, 9.7 ± 1.2 and 12.4 ± 0.9 ng/ml for bovine root, human root, bovine crown and human crown dentine, respectively. Corresponding activities for MMP-9 were 14.9 ± 2.0, 15.3 ± 1.3, 15.4 ± 1.3 and 15.5 ± 1.3 ng/ml, respectively. Bovine dentine was found to be a reliable substrate for studies involving the activity of MMP-2 and -9.

Biomarkers of fluoride in children exposed to different sources of systemic fluoride.

There has been no comparison between fluoride concentrations in urine and nails of children exposed to different sources of systemic fluoride. The aim of this study was to compare the relationship between fluoride intake with urinary fluoride excretion and fluoride concentrations in fingernails and toenails of children receiving fluoride from artificially fluoridated water (0.6-0.8 mg F/L, n = 25), naturally fluoridated water (0.6-0.9 mg F/L, n = 21), fluoridated salt (180-200 mg F/Kg, n = 26), and fluoridated milk (0.25 mg F, n = 25). A control population was included (no systemic fluoride, n = 24). Fluoride intake from diet and dentifrice, urinary fluoride excretion, and fluoride concentrations in fingernails/toenails were evaluated. Fluoride was analyzed with an ion-selective electrode. Urinary fluoride excretion in the control community was significantly lower when compared with that in the fluoridated cities, except for the naturally fluoridated community. However, the same pattern was not as evident for nails. Both urinary fluoride output and fluoride concentrations in fingernails/toenails were significantly correlated to total fluoride intake. However, the correlation coefficients for fluoride intake and urinary fluoride output were lower (r = 0.28, p < 0.01) than those observed for fingernails/toenails (r = 0.36, p < 0.001), suggesting that nails might be slightly better indicators of fluoride intake at the individual level.

Effect of iron on matrix metalloproteinase inhibition and on the prevention of dentine erosion.

It is known that some metal salts can inhibit matrix metalloproteinase (MMP) activity, but the effect of iron has not been tested yet. On the other hand, it has recently been suggested that MMP inhibition might influence dentine erosion. Based on this, the aims of this study were: (1) to test in vitro the effect of FeSO(4) on MMP-2 and -9 activity, and (2) to evaluate in situ the effect of FeSO(4) gel on dentine erosion. MMP-2 and -9 activities were analysed zymographically in buffers containing FeSO(4) in concentrations ranging between 0.05 and 1.5 mmol/l or not. Volunteers (n = 10) wore devices containing bovine dentine blocks (n = 60) previously treated with the following gel treatments: FeSO(4) (1 mmol/l FeSO(4)), F (NaF 1.23%; positive control) and placebo (negative control). The gels were applied once and removed after 1 min. Erosion was performed extraorally with Coca-Cola 4 times per day for 5 min over 5 days. Dentine wear was evaluated by profilometry. The data were analysed by Kruskal-Wallis and Dunn's tests (p < 0.05). FeSO(4) inhibited both MMP-2 (IC(50) = 0.75 mmol/l) and MMP-9 (IC(50) = 0.50 mmol/l) activities. In the in situ experiment, the mean wear (+/- SD) found for the F gel (0.79 +/- 0.08 microm) was significantly reduced in more than 50% when compared to the placebo gel (1.77 +/- 0.33 microm), but the FeSO(4) gel completely inhibited the wear (0.05 +/- 0.02 mum). Since FeSO(4) was able to inhibit MMP in vitro, it is possible that the prevention of dentine wear by the FeSO(4) gel in situ might be due to MMP inhibition, which should be investigated in further studies.

Gels containing MMP inhibitors prevent dental erosion in situ.

Matrix metalloproteinase (MMP) inhibition has been shown to reduce dentin caries progression, but its role in dental erosion has not yet been assessed. This study tested the hypothesis that gels containing MMP inhibitors (epigallocatechin gallate-EGCG and chlorhexidine) can prevent dental erosion. Volunteers (n = 10) wore palatal devices containing bovine dentin blocks (n = 10/group) treated for 1 min with EGCG at 10 (EGCG10) or 400 microM (EGCG400), chlorhexidine at 0.012%, F at 1.23% (NaF), and no vehicle (placebo). Erosion was performed with Coca-Cola (5 min) 4X/day during 5 days. The wear, assessed by profilometry (mean +/- SD, microm), was significantly reduced by the gels containing MMP inhibitors (0.05 +/- 0.02(a), 0.04 +/- 0.02(a), and 0.05 +/- 0.02(a) for EGCG10, EGCG400, and chlorhexidine, respectively) when compared with NaF (0.79 +/- 0.35(b)) and placebo gels (1.77 +/- 0.35(b)) (Friedman and Dunn's tests, p < 0.01). The use of gels delivering MMP inhibitors was shown to prevent erosion and opens a new perspective for protection against dental erosion.

Influence of genetic background on fluoride metabolism in mice.

A/J and 129P3/J mouse strains have different susceptibilities to dental fluorosis, due to their genetic backgrounds. This study tested whether these differences are due to variations in water intake and/or F metabolism. A/J (susceptible to dental fluorosis) and 129P3/J mice (resistant) received drinking water containing 0, 10, or 50 ppm F. Weekly F intake, excretion and retention, and terminal plasma and femur F levels were determined. Dental fluorosis was evaluated clinically and by quantitative fluorescence (QF). Data were tested by two-way ANOVA. Although F intakes by the strains were similar, excretion by A/J mice was significantly higher due to greater urinary F excretion, which resulted in lower plasma and femur F levels. Compared with 129P3/J mice given 50 ppm F, significantly higher QF scores were recorded for A/J mice. In conclusion, these strains differ with respect to several features of F metabolism, and amelogenesis in the 129P3/J strain seems to be unaffected by high F exposure.

Dietary fluoride intake by children receiving different sources of systemic fluoride.

There has been no comparison of fluoride (F) intake by pre-school children receiving more traditional sources of systemic F. The aim of this study was to estimate the dietary F intake by children receiving F from artificially fluoridated water (AFW-Brazil, 0.6-0.8 mg F/L), naturally fluoridated water (NFW-Brazil, 0.6-0.9 mg F/L), fluoridated salt (FS-Peru, 180-200 mg F/Kg), and fluoridated milk (FM-Peru, 0.25 mg F). Children (n=21-26) aged 4-6 yrs old participated in each community. A non-fluoridated community (NoF) was evaluated as the control population. Dietary F intake was monitored by the "duplicate plate" method, with different constituents (water, other beverages, and solids). F was analyzed with an ion-selective electrode. Data were tested by Kruskall-Wallis and Dunn's tests (p<0.05). Mean (+/- SD) F intake (mg/Kg b.w./day) was 0.04+/-0.01(b), 0.06+/-0.02(a,b), 0.05+/-0.02(a,b), 0.06+/-0.01(a), and 0.01+/-0.00(c) for AFW/NFW/FS/FM/NoF, respectively. The main dietary contributors for AFW/NFW and FS/FM/NoF were water and solids, respectively. The results indicate that the dietary F intake must be considered before a systemic method of fluoridation is implemented.

Kinetics of fluoride removal from plasma and bone of rats after chronic intake of fluoride.

This study evaluated the kinetics of fluoride in plasma, femur surface and the whole femur of rats, after chronic exposure to different water fluoride levels was interrupted. Four groups of Wistar rats received drinking water containing 0, 5, 15 or 50 microg F/ml for 60 days (n = 50/group). The animals were euthanized immediately after exposure to fluoride or after 7, 30, 90 or 180 days (n = 10/subgroup). Plasma and femurs were collected. Fluoride on the femur surface, whole femur and plasma was analyzed with an electrode. Data were analyzed using ANOVA and Tukey's test (p< 0.05). The increase in plasma fluoride levels was significant only for the 50 microg F/ml group at 0 and 7 days. Regarding bone surface and whole bone, for most groups, significant increases in fluoride concentrations were observed with the increase in water fluoride concentrations at each time of euthanasia. For fluoride doses up to 15 microg F/ml, femur surface fluoride levels were reestablished 180 days after the exposure was discontinued, which was not valid for whole femur or for higher fluoride doses. We found a different kinetics of fluoride in plasma, femur surface and the whole femur of rats after chronic exposure to fluoride is interrupted.