Rinsing with Statherin-Derived Peptide Alters the Proteome of the Acquired Enamel Pellicle.

Changes in the proteomic profile of the acquired enamel pellicle (AEP) formed for 3 min or 2 h after rinsing with a peptide containing the 15 N-terminal residues of statherin, with serines 2 and 3 phosphorylated (StatpSpS), were evaluated. Nine volunteers participated in 2 consecutive days. Each day, after professional tooth cleaning, they rinsed for 1 min with 10 mL of phosphate buffer containing 1.88 × 10-5 M StatpSpS or phosphate buffer only (control). The acquired pellicle formed on enamel after 3 min or 2 h was collected with electrode filter papers soaked in 3% citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. In the 3-min AEP, 19 and 131 proteins were uniquely identified in the StatpSpS and control groups, respectively. Proteins typically found in the AEP were only found in the latter. Only 2 proteins (neutrophil defensins) were increased upon treatment with StatpSpS, while 65 proteins (among which are several typical AEP proteins) were decreased. In the 2-h AEP, 50 and 108 proteins were uniquely found in StatpSpS and control groups, respectively. Hemoglobin subunits and isoforms of keratin were only found in the StatpSpS group, while cystatin-C, cathepsin D, and cathepsin G, isoforms of heat shock 70 and protocadherin were exclusively found in the control group. In addition, 23 proteins were increased upon treatment with StatpSpS, among which are histatin-1, serum albumin, and isoforms of neutrophil defensin and keratin, while 77 were decreased, most of them were typical AEP proteins. In both evaluated periods, rinsing with StatpSpS profoundly changed the proteomic profile of the AEP, which might impact the protective role of this integument against carious or erosive demineralization. This study provides important insights on the dynamics of the protein composition of the AEP along time, after rinsing with a solution containing StatpSpS.

Statherin-derived peptide protects against intrinsic erosion.

OBJECTIVES: In the present study, we used an in vitro initial intrinsic erosion model to evaluate: (experiment 1) the influence of the degree of serine (Ser) phosphorylation of peptides containing the 15 N-terminal residues of statherin and (experiment 2) the effect of different concentrations of the peptide with the best performance in experiment 1 on initial enamel erosion. DESIGN: Bovine enamel specimens were divided into 6 groups (n = 15/group) for each experiment. In experiment 1, the peptides evaluated (at 1.88 × 10-5 M) were: not phosphorylated (StatSS), phosphorylated in Ser2 (StatpSS), phosphorylated in Ser3 (StatSpS) phosphorylated in Ser2 and Ser3 (StatpSpS). Phosphate buffer and human recombinant statherin were used as negative and positive controls, respectively. In experiment 2, StatpSpS was evaluated at different concentrations: 0.94, 1.88, 3.76 and 7.52 × 10-5 M. Phosphate buffer and 0.1 mg/mL CaneCPI-5 were employed as negative and positive controls, respectively. In each experiment, the specimens were incubated with the solutions for 2 h, then the AEP was allowed to form (under human pooled saliva) for 2 h. The specimens were then challenged with 0.01 M HCl for 10 s. Demineralization was evaluated by percentage of surface hardness change (%SHC). Data were analyzed by ANOVA and Tukey's test (p < 0.05). RESULTS: In experiment 1, only StatpSpS significantly reduced the % SHC in comparison with control. In experiment 2, 1.88 × 10-5 M StatpSpS significantly reduced the %SHC in comparison with control. CONCLUSIONS: This is the first study showing that statherin-derived peptide might protect against intrinsic erosion.

Chronic Exposure to Sodium Fluoride Triggers Oxidative Biochemistry Misbalance in Mice: Effects on Peripheral Blood Circulation.

The excessive fluoride (F) exposure is associated with damage to cellular processes of different tissue types, due to changes in enzymatic metabolism and breakdown of redox balance. However, few studies evaluate doses of F compatible with human consumption. Thus, this study evaluated the effects of chronic exposure to sodium fluoride (NaF) on peripheral blood of mice from the evaluation of biochemical parameters. The animals were divided into three groups (n = 10) and received three concentrations of NaF in the drinking water for 60 days: 0 mg/L F, 10 mg/L F, and 50 mg/L F. The blood was then collected for trolox equivalent antioxidant capacity (TEAC), thiobarbituric acid reactive substances (TBARS), concentrations of nitric oxide (NO), superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH). The results showed that doses of 10 mg/L F and 50 mg/L F were able to increase TBARS concentration and decrease NO levels and CAT activity in the blood, but there was no statistical difference for SOD levels. The 50 mg/L F group showed an increase in TEAC levels and a decrease in the GSH content when compared to the control group. In this way, oxidative changes in blood from chronic exposure to F, especially at the highest dose, indicate that F may be a toxic agent and, therefore, the long-term exposure to excessive doses should be avoided.

Proposed mechanism for understanding the dose- and time-dependency of the effects of fluoride in the liver.

Fluoride (F) can induce changes in the expression of several liver proteins. It is suggested that these changes are dose- and time-dependent. The objective of this study was to analyze the effect of different F concentrations and exposure times to this ion on the pattern of protein expression in the liver of rats. Thirt-six 21-day-old male Wistar rats were divided into 2 groups (n = 18) according to the treatment duration (20 or 60 days). Each of these groups was then divided into 3 subgroups (n = 6) according to the concentration of F administered in drinking water, as follows: 0 mg/L (control), 15 mg/L or 50 mg/L. After the experiment periods, the animals were anesthetized and the liver and blood were collected. F was analyzed in plasma and liver. Part of the liver was fixed for histological analysis. Liver proteins were extracted and prepared for quantitative label-free mass spectrometry analysis. F concentrations in plasma and liver were significantly higher in the group treated with 50 mg /L in comparison with control, regardless the time of exposure. Histological alterations in the liver were more evident in the subgroups treated for 20 days. The proteomic analysis revealed changes in proteins related to endoplasmic reticulum and mitochondrial oxidative stress, mitochondrial alteration, apoptosis and cellular respiration upon exposure to F. The results reinforce previous findings showing that the effects of F in the liver are dose- and time-dependent and provide the molecular basis for understanding the evolution of these effects.

Standardization of a protocol for shotgun proteomic analysis of saliva.

INTRODUCTION: Saliva contains numerous proteins and peptides, each of them carries a number of biological functions that are very important in maintaining the oral cavity health and also yields information about both local and systemic diseases. Currently, proteomic analysis is the basis for large-scale identification of these proteins and discovery of new biomarkers for distinct diseases. OBJECTIVE: This study compared methodologies to extract salivary proteins for proteomic analysis. MATERIAL AND METHODS: Saliva samples were collected from 10 healthy volunteers. In the first test, the necessity for using an albumin and IgG depletion column was evaluated, employing pooled samples from the 10 volunteers. In the second test, the analysis of the pooled samples was compared with individual analysis of one sample. Salivary proteins were extracted and processed for analysis by LC-ESI-MS/MS. RESULTS: In the first test, we identified only 35 proteins using the albumin and IgG depletion column, while we identified 248 proteins without using the column. In the second test, the pooled sample identified 212 proteins, such as carbonic anhydrase 6, cystatin isoforms, histatins 1 and 3, lysozyme C, mucin 7, protein S100A8 and S100A9, and statherin, while individual analysis identified 239 proteins, among which are carbonic anhydrase 6, cystatin isoforms, histatin 1 and 3, lactotransferrin, lyzozyme C, mucin 7, protein S100A8 and S100A9, serotransferrin, and statherin. CONCLUSIONS: The standardization of protocol for salivary proteomic analysis was satisfactory, since the identification detected typical salivary proteins, among others. The results indicate that using the column for depletion of albumin and IgG is not necessary and that performing individual analysis of saliva samples is possible.

Chronic treatment with fluoride affects the jejunum: insights from proteomics and enteric innervation analysis.

Gastrointestinal symptoms are the first signs of fluoride (F) toxicity. In the present study, the jejunum of rats chronically exposed to F was evaluated by proteomics, as well as by morphological analysis. Wistar rats received water containing 0, 10 or 50 mgF/L during 30 days. HuC/D, neuronal Nitric Oxide (nNOS), Vasoactive Intestinal Peptide (VIP), Calcitonin Gene Related Peptide (CGRP), and Substance P (SP) were detected in the myenteric plexus of the jejunum by immunofluorescence. The density of nNOS-IR neurons was significantly decreased (compared to both control and 10 mgF/L groups), while the VIP-IR varicosities were significantly increased (compared to control) in the group treated with the highest F concentration. Significant morphological changes were seen observed in the density of HUC/D-IR neurons and in the area of SP-IR varicosities for F-treated groups compared to control. Changes in the abundance of various proteins correlated with relevant biological processes, such as protein synthesis, glucose homeostasis and energy metabolism were revealed by proteomics.

Standardization of a protocol for shotgun proteomic analysis of saliva

Abstract Saliva contains numerous proteins and peptides, each of them carries a number of biological functions that are very important in maintaining the oral cavity health and also yields information about both local and systemic diseases. Currently, proteomic analysis is the basis for large-scale identification of these proteins and discovery of new biomarkers for distinct diseases. Objective This study compared methodologies to extract salivary proteins for proteomic analysis. Material and Methods Saliva samples were collected from 10 healthy volunteers. In the first test, the necessity for using an albumin and IgG depletion column was evaluated, employing pooled samples from the 10 volunteers. In the second test, the analysis of the pooled samples was compared with individual analysis of one sample. Salivary proteins were extracted and processed for analysis by LC-ESI-MS/MS. Results In the first test, we identified only 35 proteins using the albumin and IgG depletion column, while we identified 248 proteins without using the column. In the second test, the pooled sample identified 212 proteins, such as carbonic anhydrase 6, cystatin isoforms, histatins 1 and 3, lysozyme C, mucin 7, protein S100A8 and S100A9, and statherin, while individual analysis identified 239 proteins, among which are carbonic anhydrase 6, cystatin isoforms, histatin 1 and 3, lactotransferrin, lyzozyme C, mucin 7, protein S100A8 and S100A9, serotransferrin, and statherin. Conclusions The standardization of protocol for salivary proteomic analysis was satisfactory, since the identification detected typical salivary proteins, among others. The results indicate that using the column for depletion of albumin and IgG is not necessary and that performing individual analysis of saliva samples is possible.

Fluoride Intensifies Hypercaloric Diet-Induced ER Oxidative Stress and Alters Lipid Metabolism.

BACKGROUND: Here, we evaluated the relationship of diet and F-induced oxidative stress to lipid metabolism in the liver of rats eating normocaloric or hypercaloric diets for two time periods (20 or 60 days). METHODS: Seventy-two 21-day-old Wistar rats were divided into 2 groups (n = 36) based on the type of diet they were eating; each of these groups was then further divided into another two groups (n = 18) based on the time periods of either 20 or 60 days, for a total of four groups. Each of these was divided into 3 subgroups (n = 6 animals/subgroup), dependent on the dose of F administered in the drinking water (0 mg/L(control), 15 mg/L or 50 mg/L). After the experimental period, blood samples and the liver were collected. Plasma samples were analyzed for HDL, cholesterol and triglycerides. Western blots were performed to probe for GRP78, Erp29, SOD2, Apo-E and SREBP in hepatic tissues. RESULTS: As expected,the expression of target proteins involved in oxidative stress increased in the F-treated groups, especially in liver tissue obtained from animals eating a hypercaloric diet. Most changes in the lipid levels and pathological conditions were seen earlier in the time period, at day 20. The morphometric analyses showed a reduction in steatosis in groups on ahypercaloric diet and treated with 50 mg F/L compared to the control, while no changes were obtained in normocaloric-fed rats. Accordingly, plasma TG was reduced in the F-treated group. The reduced expression of Apo-E in a time- and diet-dependent pattern may account for the particular decrease in steatosis in hypercaloric-fed F-treated rats. CONCLUSIONS: These results suggest that F changes liver lipid homeostasis, possibly because of the induction of oxidative stress, which seems to be higher in animals fed hypercaloric diets.

Effect of time of chronic treatment with fluoride in mice with previously induced Non-Alcoholic Fatty Liver Disease (NAFLD) / Efeito do tempo de tratamento crônico com fluoreto em camundongos com Doença Hepática Gordurosa Não Alcoólica (DHGNA) induzida previamente

O primeiro estudo avaliou o efeito da duração do tratamento crônico com fluoreto (F) em camundongos com doença hepática gordurosa não alcoólica (DHGNA) previamente induzida por dieta hiperlipídica, sobre o perfil lipídico, gotículas lipídicas e triglicerídeos (TG) no fígado, em comparação com os animais alimentados com a dieta normocalórica. Camundongos Swiss machos receberam dieta hiperlipídica por 30 dias para a indução da DHGNA, enquanto os controles receberam dieta normocalórica. Em seguida, cada um destes grupos receberam água contendo 0 ou 50 mg/L F durante 10, 20 ou 30 dias, no primeiro estudo.Os animais foram sacrificados, o sangue foi coletado para análise de F e perfil lipídico e fígado foi coletado para análise de inclusão lipídica, TG e F. Quando os animais foram tratados com dieta normocalórica e F durante 10 e 20 dias, foi observada uma melhoria significativa em muitos parâmetros relacionados com o perfil lipídico, além de uma redução de inclusão lipídica em animais tratados por 20 dias. O tratamento durante 30 dias aumentou o colesterol total e lipoproteína de alta densidade (HDL). Quanto à dieta hiperlipídica, o tratamento com F durante 10 dias não causou quaisquer alterações, enquanto o tratamento durante 20 dias melhorou os parâmetros analisados (redução do colesterol total e lipoproteínas de baixa densidade (LDL)). No entanto, o tratamento por 30 dias apenas diminuiu o HDL. Além disso, a dieta hiperlipídica aumentou a acumulação de F no organismo. Após a realização do primeiro estudo conduzimos o segundo estudo, o qual foi empregado o método de ressonância magnética nuclear (RMN) para investigar a influência do F na de novo lipogênese. Camundongos Swiss machos receberam dieta hiperlipídica por 30 dias para a indução da DHGNA, enquanto os controles receberam dieta normocalórica. Em seguida, cada um destes grupos receberam água contendo 0 ou 50 mg/L F durante 20 dias. Três dias antes da eutanásia, os animais receberam óxido de deutério (2H2O) por sonda esofágica, para obter um enriquecimento da água corporal (cerca de 3%). Os animais foram eutanasiados e coletou-se o sangue para análise de F e o fígado foi coletado para análise de F e de novo lipogêneses. De novo lipogênese foi significativamente menor quando se utilizou a dieta hiperlipídica, em comparação com a dieta normocalórica. Além disso, a administração F tendeu a diminuir a de novo lipogênese quando utilizada a dieta normocalórica. Os nossos dados indicam que F interfere no metabolismo de lipídios e tem uma ação diferente, dependendo do tempo de exposição e do tipo de dieta administrada. Além disso, F não altera de novo lipogênese em animais com DHGNA instalada, mas tende a reduzir, quando os animais são tratados com dieta normocalórica.(AU)

The first study evaluated the effect of duration of chronic treatment with fluoride (F) in mice with nonalcoholic fat liver disease (NAFLD) previously induced by hyperlipidic diet on the lipid profile, lipid droplets and triglycerides (TG) in liver, in comparison with animals fed normocaloric diet. Male Swiss mice received hyperlipidic diet for 30 days for induction of NAFLD, while controls received normocaloric diet. Then each of these groups received water containing 0 or 50mg/L F for 10, 20 or 30 days. The animals were euthanized, blood was collected for analysis of F and lipid profile and liver was collected for lipid droplets, TG and F analysis. When the animals were treated with normocaloric diet and F for 10 and 20 days, a significant improvement in many parameters related to lipid metabolism was observed, in addition to a reduction in lipid droplets in animals treated for 20 days. Treatment for 30 days increased total cholesterol and high density lipoprotein (HDL). As for the hyperlipidic diet, treatment with F for 10 days did not cause any alterations, while treatment for 20 days improved the parameters analyzed (reduced total cholesterol and low density lipoprotein (LDL)). However, treatment for 30 days only decreased HDL. Moreover, hyperlipidic diet increased F accumulation in the body. After the first study we conducted the second study, which employed nuclear magnetic resonance (NMR) to investigate the influence of F on de novo lipogenesis. Male Swiss mice received hyperlipidic diet for 30 days for induction of NAFLD, while controls received normocaloric diet. Then each of these groups received water containing 0 or 50mg/L F for 20 days. Three days prior to euthanasia, the animals received deuterium oxide (2H2O) by gavage to obtain an enrichment of body water (around 3%). The animals were euthanized, blood was collected for F analysis and liver was collected for F analysis and de novo lipogenesis analysis. De novo lipogenesis was significantly lower when the hyperlipidic diet was used, in comparison with the normocaloric diet. In addition, F administration tended to decrease de novo lipogenesis when the normocaloric diet was used. Our data indicate that F interferes in lipid metabolism and has a different action depending on the exposure time and type of diet administered. Moreover, F does not alter de novo Lipogenesis in animals with installed NAFLD, but tends to reduce it when animals are treated with the normocaloric diet.(AU)