Standardization of a protocol for shotgun proteomic analysis of saliva

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 MethodsSaliva 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

Do commercial whitening dentifrices increase enamel erosive tooth wear?

Objective: This in vitro study evaluated the effect of commercial whitening dentifrices on erosive tooth wear (ETW) of bovine enamel samples, in comparison with commercial regular dentifrices. Methodology: Sixty bovine crowns were embedded in acrylic resin, polished and then had their baseline profile determined. They were randomly assigned to 5 groups (n=12/group), according to the type of commercial dentifrice to be tested: GI – Crest Anti-cavity Regular; GII – Crest 3D White; GIII – Colgate Total 12 Clean Mint; GIV – Colgate Optic White; GV – Placebo (negative control, fluoride-free dentifrice). The samples were submitted to daily erosive and abrasive challenges for 3 days. The erosive challenges were performed 3 times a day by immersing the specimens in 0.1% citric acid solution (pH 2.5) for 90 s. Each day after the first and last erosive challenges, the specimens were subjected to the abrasive challenge for 15 s, using a toothbrushing machine (Biopdi, São Carlos, SP, Brazil), soft toothbrushes and slurry (1:3 g/ml) of the tested toothpastes (1.5 N). The specimens were kept in artificial saliva between the challenges. The final profile was obtained and the ETW (µm) was calculated. Data were analyzed by Kruskal-Wallis and Dunn’s tests (p<0.05). Results: All dentifrices tested significantly reduced the enamel wear in comparison with the Placebo, except GIII. The median (95% CI) ETW was 1.35 (1.25-1.46)bc for GI, 1.17 (1.01-1.34)cd for GII, 1.36 (1.28-1.45)ab for GIII, 1.08 (1.04-1.14)d for GIV and 2.28 (2.18-2.39)a for GV. Conclusion: When dentifrices from the same manufacturer were compared, the whitening dentifrices led to similar or less wear than the regular ones

Interplay between chronic exercise and genetic background on fluoride metabolism

O metabolismo do fluoreto (F) é influenciado por fatores genéticos e epigenéticos, como o exercício físico. Vários estudos têm procurado desvendar a influência de fatores genéticos na suscetibilidade à fluorose dentária, utilizando linhagens de camundongos com respostas diferentes aos efeitos de F. No entanto, não existem estudos avaliando os efeitos combinados da genética e do exercício no metabolismo do F. Este estudo avaliou o efeito do exercício crônico no metabolismo do F e parâmetros relacionados à homeostase da glicose, bem como perfil proteômico do fígado e músculo gastrocnêmio, em camundongos suscetíveis (A/J; S) ou resistentes (129P3/J; R) à fluorose, em resposta à exposição ao (F). Quarenta e cinco camundongos machos de cada linhagem foram divididos em 3 grupos, de acordo com os tratamentos que receberam por 56 dias: a) água deionizada e nenhum exercício (I); b) água contendo 50ppmF (como NaF) e nenhum exercício (II); c) água contendo 50ppmF e exercício (corridas diárias em esteira, 5 dias por semana, durante 60 minutos em alta intensidade; III). A capacidade física de todos os camundongos foi medida ao longo do experimento. As concentrações plasmáticas, ósseas e renais de F foram analisadas com um eletrodo específico. Glicose e insulina plasmáticas foram analisadas pelos métodos da glicose-oxidase e ELISA, respectivamente. Análises proteômicas quantitativas, livres de marcadores, foram realizadas no fígado e músculo gastrocnêmio (n-LC-MS/MS). Os dados foram analisados por ANOVA a 2 critérios e Bonferroni (p<0,05). As concentrações plasmáticas e ósseas de F foram significativamente maiores nos grupos II e III em relação ao I, independentemente da linhagem. Após o exercício, os camundongos RIII apresentaram concentrações de F no osso significativamente maiores quando comparadas aos RII. As concentrações de F no rim foram significativamente maiores nos camundongos RIII em comparação aos RII e SIII. A capacidade física final foi significativamente menor nos camundongos SII em comparação aos RII. Os camundongos S, independentemente do tratamento, apresentaram níveis plasmáticos de glicose mais altos que os R (significativo para o grupo II). Os níveis plasmáticos de insulina foram semelhantes entre os grupos. Nos grupos I e II, houve um aumento nas proteínas envolvidas no fluxo energético e nas enzimas antioxidantes nos camundongos S. No entanto, no grupo III, houve uma redução nas proteínas envolvidas na síntese proteica, metabolismo energético e desintoxicação, mas as enzimas antioxidantes ainda estavam aumentadas nos camundongos S. No músculo, os camundongos SI tiveram uma diminuição na expressão proteica em comparação com os RI. Entretanto, nas comparações SIIxRII e SIIIxRIII, houve um aumento de proteínas relacionadas ao fluxo energético e contração muscular nos camundongos S. Os resultados indicam uma complexa interação entre genética e exercício no metabolismo F. O exercício físico parece aumentar o acúmulo de F no osso de camundongos R. O estresse oxidativo em camundongos S pode ser exacerbado pelo tratamento com F. Os resultados analisados em conjunto sugerem que os indivíduos susceptíveis aos efeitos do F podem se beneficiar mais do efeito do exercício físico na homeostase da glicose do que os indivíduos resistentes, mediante exposição a este íon, o que dá respaldo à utilização do F em saúde pública.Fluoride (F) metabolism is influenced by genetic and epigenetic factors such as exercise. Several studies have attempted to unravel the influence of genetic factors on the susceptibility to dental fluorosis, using mice strains with different responses to the effects of F. However, there are no studies evaluating the combined effects of genetics and exercise on F metabolism. This study evaluated the effect of chronic exercise on F metabolism and parameters related to glucose homeostasis, as well as liver and gastrocnemius muscle proteome, in mice susceptible (A/J; S) or resistant (129P3/J; R) to fluorosis, in response to F exposure. Forty-five male mice from each strain were divided into 3 groups according to the treatments they received for 56 days: a) deionized water and no exercise (I); b) water containing 50ppmF (as NaF) and no exercise (II); c) water containing 50ppmF and exercise (daily runs on treadmill, 5 days a week for 60 minutes at high intensity; III). The physical capacity of all mice was measured throughout the experiment. Plasma, bone and renal F concentrations were analyzed with a specific electrode. Plasma glucose and insulin were analyzed by glucose oxidase and ELISA methods, respectively. Labe-free quantitative label proteomic analyses were performed on the liver and gastrocnemius muscle (n-LC-MS/MS). Data were analyzed using 2-way ANOVA and Bonferroni´s tests (p <0.05). Plasma and bone concentrations of F were significantly higher in groups II and III than I, regardless of strain. After exercise, RIII mice had significantly higher bone F concentrations compared to RII mice. Kidney F concentrations were significantly higher in RIII mice compared to RII and SIII. Final physical capacity was significantly lower in SII mice compared to RII ones. S mice, regardless of treatment, had higher plasma glucose levels than R mice (significant for group II). Plasma insulin levels were similar between the groups. In groups I and II, there was an increase in proteins involved in energy flux and antioxidant enzymes in S mice. However, in group III, there was a reduction in proteins involved in protein synthesis, energy metabolism and detoxification, but antioxidant enzymes were still increased in S mice. In muscle, SI mice had a decrease in protein expression compared to RI mice. However, in SIIxRII and SIIIxRIII comparisons, there was an increase in proteins related to energy flux and muscle contraction in S mice. The results indicate a complex interplay between genetics and exercise in F metabolism. Exercise seems to increase F accumulation in the bone of R mice. Oxidative stress may be exacerbated by F treatment in S mice. When analyzed in conjunction, our results suggest that the individuals susceptible to the effects of F might benefit more from the effects of physical exercise on glucose homeostasis that the resistant ones, upon exposure to this ion, which gives additional support to the use of F in public health

Dose-response effect of fluoride in parameters related to insulin resistance in mice strains with different genetic susceptibilities to fluorosis

O íon fluoreto (F) provém do elemento flúor. Sua absorção é inversamente relacionada ao pH e ocorre rapidamente no estômago e posteriormente no intestino delgado. Após sua absorção, o F é distribuído pelos tecidos através da corrente sanguínea e armazenado nos tecidos calcificados e moles. Sua excreção acontece por via renal. Trata-se de um elemento relevante em termos de Saúde Pública, devido às suas propriedades de prevenir ou reverter lesões cariosas em indivíduos de todas as idades. No entanto, sua ingestão excessiva é capaz de afetar o metabolismo ósseo e desenvolvimento do esmalte dentário. Estudos sugerem que o F pode interferir em vias metabólicas, inibindo a ação de diversas enzimas. Entretanto, a literatura é conflitante em relação aos seus efeitos na homeostasia da glicose, o que poderia, talvez, ser explicado pela diferença genética entre as linhagens utilizadas. Sabe-se que camundongos da linhagem A/J são extremamente sensíveis aos efeitos do F, enquanto que os camundongos da linhagem 129P3/J são altamente resistentes ao tratamento com esse íon. Por este motivo, foi investigado se esses animais que sabidamente apresentam uma expressão proteica diferencial em função do F devido ao seu background genético apresentam também respostas diferentes em parâmetros bioquímicos (glicemia jejum, insulinemia, índice de resistência à insulina [HOMA2-IR] e teste de tolerância à insulina) e imunológicos (TNF-&alpha;). Após aprovação da Comissão de ética, 156 animais (78 da linhagem A/J e 78 da linhagem 129P3/J) foram divididos em 3 grupos para cada linhagem, e tratados por um período de 42 dias com doses de 0, 15 ou 50 ppm de F na água e ração com baixo teor de F. Após o término do tratamento, os camundongos foram eutanasiados para a obtenção de amostras de sangue. Os dados foram analisados por ANOVA a 2 critérios e testes de Tukey e Sidak para comparações individuais (p<0,05). Para a glicemia, os animais A/J que receberam água sem F e com a dose de 15 ppm F tiveram glicemia significativamente mais alta que os animais 129P3/J que receberam o mesmo tratamento. Para as dosagens de insulina no plasma, houve diferença significativa apenas entre os camundongos A/J 0 ppm F e 50 ppm F, sendo mais baixa a insulinemia para os animais tratados. O índice HOMA2-IR mostrou diferença significativa somente entre os animais da linhagem A/J, sendo que o grupo que recebeu água contendo 50 ppm F apresentou valores menores quando comparado para os grupos 0 ppm F e 15 ppm F. Quanto ao TNF-&alpha;, não foi observada diferença significativa entre as linhagens e entre os tratamentos. Entretanto, houve uma tendência para seu aumento nos grupos tratados com água contendo 15 ppm F nas duas linhagens. Para o teste de tolerância à insulina, também não foram observadas diferenças significativas entre as linhagens, nem entre os tratamentos. Levando em consideração os resultados, percebe-se que as diferentes concentrações de F alteram os resultados para os parâmetros analisados, e, as linhagens respondem diferentemente a essas alterações. No entanto, é necessário que se analisem outras variáveis para que esse assunto seja melhor elucidado.Fluoride (F) comes from the element fluorine. Its absorption is inversely related to the pH and occurs quickly in the stomach and later in the small intestine. After absorption, F is distributed to the tissues through the bloodstream and stored in calcified and soft tissues. Excretion occurs via the kidneys. It is an important element in terms of public health, due to its properties to prevent or reverse caries in individuals of all ages. However, its excessive intake can affect bone metabolism and the development of tooth enamel. Studies suggest that F can interfere with metabolic pathways, by inhibiting the action of several enzymes. However, there is contradiction in the literature regarding its effects on glucose homeostasis, which could possibly be explained by genetic differences between the strains used. A/J mice are extremely sensitive to the effects of F, whereas 129P3/J mice are highly resistant to treatment with this ion. For this reason, it was investigated whether these animals which are known to exhibit differential protein expression upon exposure to F due to their genetic background also exhibit distinct responses in biochemical (fasting glucose, insulin, insulin resistance index [HOMA2-IR] and insulin tolerance test) and immune (TNF-&alpha;) parameters. After approval by the Ethics Committee, 156 animals (78 of A/J strain and 78 of 129P3/J strain) were obtained, divided into 3 groups for each strain and treated for a period of 42 days with 0, 15 or 50 ppm F in the drinking water. They received low-F diet. After treatment, the mice were euthanized and blood samples were obtained. Data were analyzed by 2-way ANOVA and Tukey and Sidak tests for individual comparisons (p<0.05). For blood glucose analysis, A/J mice treated with water containing no F containing 15 ppm F had significantly higher levels of glucose than 129P3/J animals receiving the same treatment. For plasma insulin, there was significant difference only between A/J mice treated with no F and 50 ppm F, with lower values for the treated animals. HOMA2-IR index showed a significant difference only between A/J animals, where the group received water containing 50 ppm F had lower values when compared to those receiving water containing no F or 15 ppm F. Regarding TNF-&alpha;, no significant differences were observed between the strains or among the treatments. However, there was a trend towards an increase in the groups treated with water containing 15 ppm F for both strains. For insulin tolerance test, also no significant differences between the strains or among treatments were observed. The results suggest that the different F concentrations alter the results of the parameters analyzed, and the strains respond differently to these changes. However, it is necessary to analyze other variables in order to better elucidate these findings

Standardization of a protocol for shotgun proteomic analysis of saliva

<div><p>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.</p></div

Liver proteome of mice with different genetic susceptibilities to the effects of fluoride

A/J and 129P3/J mice strains have been widely studied over the last few years because they respond quite differently to fluoride (F) exposure. 129P3/J mice are remarkably resistant to the development of dental fluorosis, despite excreting less F in urine and having higher circulating F levels. These two strains also present different characteristics regardless of F exposure. Objective In this study, we investigated the differential pattern of protein expression in the liver of these mice to provide insights on why they have different responses to F. Material and Methods Weanling male A/J and 129P3/J mice (n=10 from each strain) were pared and housed in metabolic cages with ad libitum access to low-F food and deionized water for 42 days. Liver proteome profiles were examined using nLC-MS/MS. Protein function was classified by GO biological process (Cluego v2.0.7 + Clupedia v1.0.8) and protein-protein interaction network was constructed (PSICQUIC, Cytoscape). Results Most proteins with fold change were increased in A/J mice. The functional category with the highest percentage of altered genes was oxidation-reduction process (20%). Subnetwork analysis revealed that proteins with fold change interacted with Disks large homolog 4 and Calcium-activated potassium channel subunit alpha-1. A/J mice had an increase in proteins related to energy flux and oxidative stress. Conclusion This could be a possible explanation for the high susceptibility of these mice to the effects of F, since the exposure also induces oxidative stress

Liver proteome of mice with different genetic susceptibilities to the effects of fluoride

ABSTRACT A/J and 129P3/J mice strains have been widely studied over the last few years because they respond quite differently to fluoride (F) exposure. 129P3/J mice are remarkably resistant to the development of dental fluorosis, despite excreting less F in urine and having higher circulating F levels. These two strains also present different characteristics regardless of F exposure. Objective In this study, we investigated the differential pattern of protein expression in the liver of these mice to provide insights on why they have different responses to F. Material and Methods Weanling male A/J and 129P3/J mice (n=10 from each strain) were pared and housed in metabolic cages with ad libitum access to low-F food and deionized water for 42 days. Liver proteome profiles were examined using nLC-MS/MS. Protein function was classified by GO biological process (Cluego v2.0.7 + Clupedia v1.0.8) and protein-protein interaction network was constructed (PSICQUIC, Cytoscape). Results Most proteins with fold change were increased in A/J mice. The functional category with the highest percentage of altered genes was oxidation-reduction process (20%). Subnetwork analysis revealed that proteins with fold change interacted with Disks large homolog 4 and Calcium-activated potassium channel subunit alpha-1. A/J mice had an increase in proteins related to energy flux and oxidative stress. Conclusion This could be a possible explanation for the high susceptibility of these mice to the effects of F, since the exposure also induces oxidative stress

Intestinal changes associated with fluoride exposure in rats: Integrative morphological, proteomic and microbiome analyses

Gastrointestinal signs and symptoms are the first signs of toxicity due to exposure to fluoride (F). This suggests the possibility that lower levels of subchronic F exposure may affect the gut. The aim of this study was to evaluate changes in the morphology, proteome and microbiome of the ileum of rats, after subchronic exposure to F. Male rats ingested water with 0, 10, or 50&nbsp;mgF/L for thirty days. Treatment with F, regardless of the dose, significantly decreased the density of HuC/D-IR neurons, whereas CGRP-IR and SP-IR varicosities were significantly increased compared to the control group. Increased VIP-IR varicosities were significantly increased only in the group treated with 50&nbsp;mgF/L. A significant increase in thickness of the tunica muscularis, as well as in the total thickness of the ileum wall was observed at both F doses when compared to controls. In proteomics analysis, myosin isoforms were increased, and Gastrotopin was decreased in F-exposed mice. In the microbiome metagenomics analysis, Class Clostridia was significantly reduced upon exposure to 10&nbsp;mgF/L. At the higher F dose of 50&nbsp;mg/L, genus Ureaplasma was significantly reduced in comparison with controls. Morphological and proteomics alterations induced by F were marked by changes associated with inflammation, and alterations in the gut microbiome. Further studies are needed to determine whether F exposure increases inflammation with secondary effects of the gut microbiome, and/or whether primary effects of F on the gut microbiome enhance changes associated with inflammation