Changes in the Proteomic Profile of Acquired Enamel Pellicles as a Function of Their Time of Formation and Hydrochloric Acid Exposure.

OBJECTIVE: Changes in the protein profile of acquired enamel pellicles (AEP) formed in vivo over different time periods were evaluated after the application of hydrochloric acid (HCl). METHODS: Nine subjects were submitted to dental prophylaxis with pumice. After 3 or 120 min, the teeth were isolated with cotton rolls and 50 µL of 0.1 M HCl (pH 1.0), 0.01 M HCl (pH 2.0), or deionized water were applied on the buccal surface of the teeth for 10 s. The AEP was then collected using an electrode filter paper presoaked in 3% citric acid. After protein extraction, the samples were submitted to reverse-phase liquid chromatography coupled to mass spectrometry (nano LC-ESI-MS/MS). Label-free quantification was performed (Protein Lynx Global Service software). RESULTS: A total of 180 proteins were successfully identified in the AEP samples. The number of identified proteins increased with the time of pellicle formation. Only 4 proteins were present in all the groups (isoforms of IgA, serum albumin, and statherin). The greatest number of proteins identified uniquely in one of the groups was obtained for the groups treated with HCl after 2 h of pellicle formation (approx. 50 proteins). CONCLUSION: Proteins resistant to removal by HCl, such as serum albumin and statherin, were identified even in the short-term AEP. In addition, 120-min pellicles present many proteins that are resistant to removal by HCl. This suggests an increase in protection against intrinsic acids with the time of pellicle formation, which should be evaluated in future studies.

Protein Profile of the Acquired Enamel Pellicle after Rinsing with Whole Milk, Fat-Free Milk, and Water: An in vivo Study.

This study detected changes in the protein profile of the acquired enamel pellicle (AEP) formed in vivo after rinsing with whole milk, fat-free milk, or water. Nine subjects in good oral condition took part in the study. The acquired pellicle was formed in the morning, for 120 min, after prophylaxis with pumice. Following this, the volunteers rinsed with 10 mL of whole milk, fat-free milk, or deionized water for 30 s, following a blinded crossover protocol. After 60 min, the pellicle was collected with filter paper soaked in 3% citric acid and processed for analysis by liquid chromatography-electrospray ionization tandem mass spectrometry. The obtained tandem mass spectrometry spectra were searched against a human protein database (Swiss-Prot). The proteomic data related to protein quantification were analysed using the PLGS software. A total of 260 proteins were successfully identified in the AEP samples collected from all groups. Forty-nine were common to all 3 groups, while 72, 62, and 49 were specific to the groups rinsing with whole milk, fat-free milk, and water, respectively. Some were typical components of the AEP, such as cystatin-B, cystatin-SN, isoforms of α-amylase, IgA and IgG, lysozyme C, protein S100 A78, histatin-1, proline-rich protein 27, statherin, and lactotransferrin. Other proteins are not commonly described as part of the AEP but could act in defence of the organism against pathogens. Distinct proteomic profiles were found in the AEP after rinsing with whole or fat-free milk, which could have an impact on bacterial adhesion and tooth dissolution. The use of fat-free milk could favourably modulate the adhesion of bacteria to the AEP as well as biofilm formation when compared with whole milk.

The effect of fluoride on the structure, function, and proteome of intestinal epithelia.

Fluoride exposure is widespread, with drinking water commonly containing natural and artificially added sources of the ion. Ingested fluoride undergoes absorption across the gastric and intestinal epithelia. Previous studies have reported adverse gastrointestinal effects with high levels of fluoride exposure. Here, we examined the effects of fluoride on the transepithelial ion transport and resistance of three intestinal epithelia. We used the Caco-2 cell line as a model of human intestinal epithelium, and rat and mouse colonic epithelia for purposes of comparison. Fluoride caused a concentration-dependent decline in forskolin-induced Cl- secretion and transepithelial resistance of Caco-2 cell monolayers, with an IC50 for fluoride of about 3 mM for both parameters. In the presence of 5 mM fluoride, transepithelial resistance fell exponentially with time, with a t1/2 of about 7 hours. Subsequent imaging by immunofluorescence and scanning electron microscopy showed structural abnormalities in Caco-2 cell monolayers exposed to fluoride. The Young's modulus of the epithelium was not affected by fluoride, although proteomic analysis revealed changes in expression of a number of proteins, particularly those involved in cell-cell adhesion. In line with its effects on Caco-2 cell monolayers, fluoride, at 5 mM, also had profound effects on Cl- secretion and transepithelial resistance of both rat and mouse colonic epithelia. Our results show that treatment with fluoride has major effects on the structure, function, and proteome of intestinal epithelia, but only at concentrations considerably higher than those likely to be encountered in vivo, when much lower fluoride doses are normally ingested on a chronic basis.

The effect of fluoride on the structure, function, and proteome of a renal epithelial cell monolayer.

High concentrations of fluoride in the body may cause toxic effects. Here, we investigated the effects of fluoride on the structure, function, and proteome of a cortical collecting duct epithelium in vitro. Kidney tubule cells (M-1) were chosen because the concentration of fluoride in the kidney is 4-5-fold higher than that in plasma. Mouse M-1 cell monolayers were incubated in fluoride-containing media, and the amiloride-sensitive short-circuit current and transepithelial resistance were measured. The Young's modulus of the epithelium was determined using atomic force microscopy, and the effect of fluoride on epithelial structure was assessed using scanning and transmission electron microscopy, and immunofluorescence. Differences in the expression of membrane proteins were evaluated using proteomics and bioinformatics. Fluoride exposure reduced both transepithelial Na+ transport and resistance. The IC50 for fluoride was ∼300 µM for both effects, and the half-times for the decays of ion transport and resistance were 8.4 h and 3.6 days, respectively. Fluoride treatment did not affect the sensitivity of Na+ transport to amiloride. The Young's modulus of the epithelium was also unaffected by fluoride; however, the functional effects of fluoride were accompanied by marked structural effects. Proteomic analysis revealed changes in expression of a number of proteins, and particularly mitochondrial proteins. Treatment with fluoride had profound effects on the structure, function and proteome of a model cortical collecting duct epithelium. Significantly, however, these effects were produced only at concentrations considerably higher than those likely to be encountered in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1455-1467, 2017.

Determination of the Mercury Fraction Linked to Protein of Muscle and Liver Tissue of Tucunaré (Cichla spp.) from the Amazon Region of Brazil.

This study used metalloproteomic techniques to characterize mercury (Hg)-bound proteins in the muscle and liver tissue of Tucunaré (Cichla spp.) collected at the Jirau Hydroelectric Power Plant in Madeira River Basin, Brazil. The proteome of the muscle and liver tissue was obtained after two steps of fractional precipitation and separating the proteins by 2-D polyacrylamide gel electrophoresis. Hg was identified and quantified in the protein spots by graphite furnace atomic absorption spectrometry after acid mineralization in an ultrasound bath. Hg with a molecular weight <20 kDa and a concentration between 13.30 and 33.40 mg g(-1) was found in the protein spots. These protein spots were characterized by electrospray ionization tandem mass spectrometry after trypsin digestion. From a total of 12 analyzed spots, seven proteins showing Hg biomarker characteristics were identified: parvalbumin and its isoforms, ubiquitin-40S ribosomal protein S27a, zinc (Zn) finger and BTB domain-containing protein 24, and dual-specificity protein phosphatase 22-B.

Low intensity lasers differently induce primary human osteoblast proliferation and differentiation.

Among various compounds used in research and clinic for degenerative bone diseases, low level laser therapy (LLLT), comprising low level lasers (LLL) and light emitting diodes (LEDs), has been investigated regarding its effects on bone metabolism. They have specific wavelengths but in general act as a cellular biomodulator, and as a therapeutic agent, rebalancing and normalizing their activity. However, they are not standardized yet, since their parameters of use are relevant for the effects and mechanisms of action. Therefore, the aim of this study was to compare the influence of two spectrums of LLL and LED phototherapy, at the same energy densities (10 and 50J/cm(2)), on human osteoblasts proliferation and differentiation. The involvement of ERK signaling on proliferation was also investigated by evaluating its activation during proliferation under different phototherapies by western blotting and CFSE-based osteoblast proliferation was measured in a presence or absence of the ERK-specific inhibitor. Osteogenic differentiation was evaluated through in vitro mineralization and gene expression of type I collagen (COL1A1) and osteonectin (SPARC) by Real Time- PCR. Increases in viable cells and proliferation were obtained after irradiation, regardless of LLLT type. However, only red at 10J/cm(2) and infrared at both doses, but not LED, induced ERK1/2 activation. In the presence of ERK inhibitor, the LLL-induced proliferation was prevented. In addition, while COL1A1 gene expression was upregulated by red laser, SPARC does so by infrared stimulation. However, LED, at both doses, increased both COL1A1 and SPARC expression. All LLLT increased mineralization, dependent on the dose and time. Thus, LLL and LED differently modulated the metabolism of human osteoblasts, increasing proliferation by mechanism dependent or not of ERK signaling activation and osteogenic differentiation markers.

Aquaporin 5 Interacts with Fluoride and Possibly Protects against Caries.

Aquaporins (AQP) are water channel proteins and the genes coding for AQP2, AQP5, and AQP6 are clustered in 12q13. Since AQP5 is expressed in serous acinar cells of salivary glands, we investigated its involvement in caries. DNA samples from 1,383 individuals from six groups were studied. Genotypes of eight single nucleotide polymorphisms covering the aquaporin locus were tested for association with caries experience. Interaction with genes involved in enamel formation was tested. The association between enamel microhardness at baseline, after creation of artificial caries lesion, and after exposure to fluoride and the genetic markers in AQP5 was tested. Finally, AQP5 expression in human whole saliva, after exposure to fluoride in a mammary gland cell line, which is known to express AQP5, and in Wistar rats was also verified. Nominal associations were found between caries experience and markers in the AQP5 locus. Since these associations suggested that AQP5 may be inhibited by levels of fluoride in the drinking water that cause fluorosis, we showed that fluoride levels above optimal levels change AQP5 expression in humans, cell lines, and rats. We have shown that AQP5 is involved in the pathogenesis of caries and likely interacts with fluoride.

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.

Proteomic analysis of kidney in rats chronically exposed to fluoride.

Two-dimensional gel electrophoresis (2-DE) was used to better understand alterations in renal metabolism induced by fluoride (F). Three groups of weanling male Wistar rats were treated with drinking water containing 0 (control), 5, or 50 ppm F for 60 days (n=6/group). Kidneys were collected for proteomic and histological (HE) analysis. After protein isolation, renal proteome profiles were examined using 2-DE and Colloidal Coomassie Blue staining. Protein spots with a 2-fold significant difference as detected by quantitative intensity analysis (Image Master Platinum software) and t-test (p<0.05) were excised and analyzed by MALDI-TOF MS (matrix assisted laser desorption ionization-time-of-flight mass spectrometry). The histological analysis revealed no damage in kidneys induced by F, except for a vascular congestion in the 50 ppm F group. Between control vs 50 ppm F, and control vs 5 ppm F groups, 12 and 6 differentially expressed proteins were detected, respectively. Six proteins, mainly related with metabolism, detoxification and housekeeping, were successfully identified. At the high F group, pyruvate carboxylase, a protein involved in the formation of oxaloacetate was found to be downregulated, while enoyl coenzyme A hydratase, involved in fatty acids oxidation, was found to be upregulated. Thus, proteomic analysis can provide new insights into the alterations in renal metabolism after F exposure, even in low doses.