Metalloproteomic approach to liver tissue of rats exposed to mercury.

The aims of this study were to identify mercury-associated protein spots in the liver tissue of rats exposed to low concentrations of mercury and to elucidate the physiological and functional aspects of the proteins identified in the protein spots. Therefore, proteomic analysis of the liver tissue of Wistar rats exposed to mercury chloride (4.60 µg kg-1 in Hg2+) was performed for thirty days (Hg-30 group) and sixty days (Hg-60 group). The proteomic profile of the liver tissue of the rats was obtained by two-dimensional electrophoresis (2D-PAGE), and the determinations of total mercury in the liver tissue, pellets and protein spots were performed by graphite furnace atomic absorption spectrometry (GFAAS). ImageMaster 2D Platinum 7.0 software was used to identify the differentially expressed mercury-associated protein spots, which were then characterized by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The determinations by GFAAS indicated a total mercury bioaccumulation of 2812% in the Hg-30 group and 3298% in the Hg-60 group and 10 mercury-associated protein spots with a concentration range of 51 ± 1.0 to 412 ± 6.00 mg kg-1 in the 2D PAGE gels from the liver tissue of the Hg-60 group. The LC-MS/MS analyses allowed the identification of 11 metal binding proteins in mercury-associated protein spots that presented fold change with upregulation >1.5, downregulation < -1.7 or that were expressed only in the Hg-60 group. Using the FASTA sequences of the proteins identified in the mercury-associated protein spots, bioinformatics analyses were performed to elucidate the physiological and functional aspects of the metal binding proteins, allowing us to infer that enzymes such as GSTM2 presented greater mercury concentrations and downregulation < -3; Acaa2 and Bhmt, which showed expression only in the Hg-60 group, among others, may act as potential mercury exposure biomarkers.

Effects of Fluoride Long-Term Exposure over the Cerebellum: Global Proteomic Profile, Oxidative Biochemistry, Cell Density, and Motor Behavior Evaluation.

Although the literature does not provide evidence of health risks from exposure to fluoride (F) in therapeutic doses, questions remain about the effects of long-term and high-dose use on the function of the central nervous system. The objective of this study was to investigate the effect of long-term exposure to F at levels similar to those found in areas of artificial water fluoridation and in areas of endemic fluorosis on biochemical, proteomic, cell density, and functional parameters associated with the cerebellum. For this, mice were exposed to water containing 10 mg F/L or 50 mg F/L (as sodium fluoride) for 60 days. After the exposure period, the animals were submitted to motor tests and the cerebellum was evaluated for fluoride levels, antioxidant capacity against peroxyl radicals (ACAP), lipid peroxidation (MDA), and nitrite levels (NO). The proteomic profile and morphological integrity were also evaluated. The results showed that the 10 mg F/L dose was able to decrease the ACAP levels, and the animals exposed to 50 mg F/L presented lower levels of ACAP and higher levels of MDA and NO. The cerebellar proteomic profile in both groups was modulated, highlighting proteins related to the antioxidant system, energy production, and cell death, however no neuronal density change in cerebellum was observed. Functionally, the horizontal exploratory activity of both exposed groups was impaired, while only the 50 mg F/L group showed significant changes in postural stability. No motor coordination and balance impairments were observed in both groups. Our results suggest that fluoride may impair the cerebellar oxidative biochemistry, which is associated with the proteomic modulation and, although no morphological impairment was observed, only the highest concentration of fluoride was able to impair some cerebellar motor functions.

Changes in energy metabolism induced by fluoride: Insights from inside the mitochondria.

The mechanisms involved in changes in energy metabolism caused by excessive exposure to fluoride (F) are not completely understood. The present study employed proteomic tools to investigate the molecular mechanisms underlying the dose- and time-dependency of the effects of F in the rat liver mitochondria. Thirty-six male Wistar rats received water containing 0, 15 or 50 mgF/L (as NaF) for 20 or 60 days. Rat liver mitochondria were isolated and the proteome profiles were examined using label-free quantitative nLC-MS/MS. PLGS software was used to detect changes in protein expression among the different groups. The bioinformatics analysis was done using the software CYTOSCAPE® 3.0.7 (Java®) with the aid of ClueGo plugin. The dose of 15 mgF/L, when administered for 20 days, reduced glycolysis, which was counterbalanced by an increase in other energetic pathways. At 60 days, however, an increase in all energy pathways was observed. On the other hand, the dose of 50 mgF/L, when administered for 20 days, reduced the enzymes involved in all energetic pathways, indicating a lower rate of energy production, with less generation of ROS and consequent reduction of antioxidant enzymes. However, when the 50 mgF/L dose was administered for 60 days, an increase in energy metabolism was seen but in general no changes were observed in the antioxidant enzymes. Except for the group treated with 50 mgF/L for 20 days, all the other groups had alterations in proteins in attempt to maintain calcium homeostasis and avoid apoptosis. The results suggest that the organism seems to adapt to the effects of F over time, activating pathways to reduce the toxicity of this ion. Ultimately, our findings corroborate the safety of the use of fluoride for caries control.

Physiological and functional aspects of metal-binding protein associated with mercury in the liver tissue of pirarucu (Arapaima gigas) from the Brazilian Amazon.

High concentrations of mercury found in soils, sediments, fish, and humans of the Amazon region have gained prominence in scientific studies during the last decade. However, studies related to the elucidation of mercury toxicity mechanisms in ichthyofauna at the molecular and metallomic levels that seek to elucidate physiological and functional aspects, as well as the search for biomarkers of mercury exposure, are still sparse. In the search for these answers, the present study analyzed the hepatic tissue proteome of the Arapaima gigas (pirarucu) fish species collected in the Jirau hydroelectric power plant reservoir in the state of Rondônia state, Brazil, in order to identify mercury-related metal-binding proteins and to elucidate their physiological and functional aspects. The proteomic profile of the hepatic tissue of Arapaima gigas was obtained by two-dimensional electrophoresis (2D-PAGE) and the presence of mercury was mapped in the protein SPOTS by graphite furnace atomic absorption spectrometry(GFAAS). Mercury was detected in 18 protein SPOTS with concentrations ranging from 0.13 ±â€¯0.003 to 131.00 ±â€¯3 mg kg-1. The characterization of the protein SPOTS associated with mercury was performed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS), and 10 proteins were identified. Bioinformatics analyses showed that most of the proteins found linked to mercury were involved in cellular component processes and biological processes. For the most part, protein sequences have cellular functions comprising catalytic, binding, sense of localization, and metabolic processes.

Use of sodium trimetaphosphate in the inhibition of dentin matrix metalloproteinases and as a remineralizing agent.

OBJECTIVES: Because of its ability to act as an antiproteolytic agent, the effect of sodium trimetaphosphate (STMP) against specific enzymes extracted from sound dentin and its performance under acidic challenge on demineralized dentin were investigated. METHODS: The antiproteolytic potential of STMP (0.5%, 1.0%, and 1.5%) was assessed in triplicate by zymography. For the evaluation of remineralization activity, 50 bovine-root dentin specimens were selected and randomly divided into 5 groups (n=10). Three areas were determined for each specimen: 1) control (no treatment); 2) demineralized (artificial caries-like challenge); 3) treated (demineralized and subjected to pH-cycling for 7days, and treated for 10min with 1.5% STMP, 1.5% STMP+calcium hydroxide (Ca[OH]2), 1.5% STMP+sodium fluoride (NaF), NaF, or deionized H2O). The dentin specimens were analyzed for superficial hardness (SH) and cross-sectional hardness (CSH) at different depths (10, 30, 50, 70, 90, 110, and 220µm) using a Knoop penetrator (10g/10s). Statistical analyses were performed with analysis of variance (ANOVA) and Tukey tests (p<0.05). RESULTS: The zymographic analysis showed that 1.5% STMP promoted complete inhibition of gelatinolytic activity. Therefore, 1.5% STMP was investigated in association with supplemented calcium or fluoride; a combination of 1.5% STMP and Ca(OH)2 significantly increased the mechanical properties of the treated dentin. CONCLUSION: 1.5% STMP serves as an antiproteolytic agent against matrix metalloproteinases extracted from human dentin. Furthermore, when supplemented with Ca(OH)2, 1.5% STMP may potentially induce remineralization. CLINICAL SIGNIFICANCE: STMP can be introduced as a novel strategy that combines enzymatic inhibition and remineralizing potential, which can serve to strengthen dentin and improve stability. STMP may have potential in the treatment of demineralized dentin lesions, especially when supplemented with calcium.

Effect of different concentrations and application times of proanthocyanidin gels on dentin erosion.

PURPOSE: To analyze the effect of different concentrations and application times of proanthocyanidin gels on dentin before an erosive challenge in order to evaluate if there is a dose-response or application time-response relationship in the use of these gels for erosion prevention. METHODS: 80 bovine root dentin blocks were randomly and equally divided into 10 groups and treated according to the two factors under study (purified grape seed proanthocyanidin gel concentration and time of application): 0.05P1: 0.05% proanthocyanidin gel during 1 minute; 0.05P5: 0.05% proanthocyanidin gel during 5 minutes; 1P1: 1% proanthocyanidin gel during 1 minute; 1P5: 1% proanthocyanidin gel during 5 minutes; 5P1: 5% proanthocyanidin gel during 1 minute; 5P5: 5% proantho-cyanidin gel during 5 minutes; 10P1: 10% proanthocyanidin gel during 1 minute; 10P5: 10% proanthocyanidin gel during 5 minutes; Control 1: placebo gel during 1 minute; and Control 5: placebo gel during 5 minutes. The gels were applied over dentin blocks once before the first erosive challenge. After that, the blocks were subjected to three erosive cycles per day, during 5 days. Profilometry was used to quantify the dentin loss (µm). Data were analyzed by two-way ANOVA and Fisher's test (P< 0.05). RESULTS: Statistical analysis showed that there was no significant difference between the application times. The different concentrations of proanthocianidin gels presented similar results (P> 0.05). All tested gels resulted in significantly less wear when compared to the placebo gel. CLINICAL SIGNIFICANCE: Grape seed proanthocyanidin gels could be considered as a promising therapy to diminish erosive dentin wear because it may interact with the exposed collagen, enhancing the demineralized organic matrix stabilization, which acts as a barrier against the diffusion of the acids from erosion.

Metalloproteomic and differential expression in plasma in a rat model of type 1 diabetes.

Type 1 diabetes is characterized by hyperglycemia, which in the chronic stage is associated with abnormalities in lipids, protein and, carbohydrate metabolism, as well as oxidative stress. New strategies for prevention and treatment are needed, as type 1 diabetes affects life quality and survival, and involves high-cost treatment. Proteomic and metalloproteomic studies can elucidate the functional and physiological aspects of biomolecules. In the present study, differential proteomics was used to identify potential biomarkers of diabetes in rat plasma associated with copper, selenium, zinc, and magnesium fractionation in control and diabetic rats, as well as diabetic rats treated with insulin. 2D-PAGE was used in the plasma protein fractionation; graphite furnace atomic absorption spectrometry (GFAAS) and flame atomic absorption spectrometry (FAAS) were used for quantitative determination of copper, magnesium, selenium, and zinc in the spots that showed different expression; and protein spots were characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) after tryptic digestion. ESI-MS/MS analysis characterized 35 different proteins, indicating alpha-1-macroglobulin and haptoglobulin as potential candidates as biomarkers for diabetes treated with insulin; also, 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, transmembrane protein 11, serum amyloid P component, vitamin D-binding protein, and biliverdin reductase were identified as potential candidates as biomarkers for uncontrolled diabetes.

Lead contents in the surface enamel of deciduous teeth sampled in vivo from children in uncontaminated and in lead-contaminated areas.

This study aimed to: (1) measure lead contents in the surface enamel of two populations consisting of 4-6-year-old children, one from an apparently uncontaminated area (Ribeirão Preto, São Paulo State, SP, Brazil, n=247) and the other from an area notoriously contaminated with lead (Bauru, São Paulo State, Brazil, n=26); (2) compare biopsy depths between the two populations; (3) correlate biopsy depth with lead content; (4) stratify samples according to biopsy depth to compare lead contents in samples from similar biopsy depths. A surface enamel acid-etch microbiopsy was performed in vivo on a single upper deciduous incisor for each sample. Lead was measured by graphite furnace atomic absorption spectrometry (GFAAS) while phosphorus was measured colorimetrically to establish biopsy depth. Samples from both populations were classified into categories of similar biopsy depths based on biopsy depth quartiles. Median lead contents were statistically different between the Ribeirão Preto population (206 microg/g, range: 5-1399 microg/g) and the Bauru population (786 microg/g, range: 320-4711 microg/g) (p<0.001); however, biopsy depth did not differ between the Ribeirão Preto (3.9 microm, Standard Deviation, SD=0.9) and Bauru (3.8 microm, SD=0.9) populations (p=0.7940). Pearson's correlation coefficient for biopsy depths versus log10 lead values was -0.29 for Ribeirão Preto and -0.18 for Bauru. Lead contents were statistically different between the two populations for all quartiles of biopsy depth. These findings suggest that lead accumulated in the surface enamel of deciduous teeth is linked to the environment in which people reside, indicating that this tissue should be further explored as an accessible biomarker of lead exposure.

Fingernails and toenails as biomarkers of subchronic exposure to fluoride from dentifrice in 2- to 3-year-old children.

This work evaluated the use of fingernails and toenails as biomarkers of subchronic exposure to fluoride (F) from fluoride dentifrice (FD) in 2- to 3-year-old children. Ten 2- to 3-year-old children used a placebo dentifrice (without F) for 28 days, FD (1,570 ppm F as monofluorophosphate) for the following 28 days, and then placebo dentifrice for an additional 28 days, then returned to their usual dentifrices. Fingernails and toenails were clipped every 2 weeks, during the experimental period and for an additional 22 weeks. Nail F was analyzed by electrode following hexamethyldisiloxane-facilitated diffusion. There were no significant differences between fingernail and toenail F concentrations. Mean peak F concentrations occurred 16 weeks after starting the use of FD. Results suggest that fingernails and toenails may be suitable biomarkers of subchronic exposure to F from FD in small children.