Introdução

This Symposium is aimed at bringing together specialists with different backgrounds to discuss recent information on various aspects of lead toxicity, lead exposure in Brazil, the presence of lead in the environment, and the pre- and analytical methods for lead determination. Lead is a toxic metal usually found in the human environments because of it is very useful. Here we describe some historical aspects regarding lead and the human health that will highlight why even low level lead exposures should cause concern. At the end we present data from studies in apparently non-exposed children from Ribeirão Preto, SP, Brazil.O objetivo deste Simpósio é reunir especialistas de diversas áreas para trazer e discutir informações recentes sobre vários aspectos da toxicidade, exposição a chumbo no Brasil, presença do chumbo no ambiente, além dos métodos pré-analíticos e analíticos da determinação de chumbo. O chumbo é um metal tóxico comumente encontrado nos ambientes habitados pelo ser humano por ser muito útil. Nesta introdução são descritos alguns pontos históricos no conhecimento atual sobre chumbo e a saúde humana, que irão esclarecer por que mesmo exposições a baixas doses devem motivar preocupação. Finalizamos a introdução com a apresentação dos dados sobre chumbo em crianças aparentemente não expostas de Ribeirão Preto, SP, Brasil

Matrix metalloproteinases: Targets for doxycycline to prevent the vascular alterations of hypertension

AbstractHypertension is associated with well known structural and functional alterations in both resistance and conduit arteries, which may be the result from long-lasting high blood pressure and may also be the cause of maintained hypertension and its complications. Therefore, in addition to lowering blood pressure, therapeutic strategies targeting the structural and functional modifications found in hypertensive patients may prevent the cardiovascular events and decrease the death rates associated with hypertension. Mounting evidence indicates that many vascular alterations associated with sustained hypertension are due to imbalanced matrix metalloproteinases (MMPs), a family of zinc-endopeptidases that degrade not only proteins of extracellular matrix (ECM) but several other substrates. Recent observations showed that abnormal MMP activity is a feature of the pathogenesis of hypertension and other diseases, thus justifying the development of drugs aiming at MMP downregulation. This review focuses on the extracellular actions of MMPs in hypertension-induced chronic vascular alterations. We then discuss the effects of MMP inhibitors, especially doxycycline, on the vascular changes associated with hypertension. There is now strong evidence that MMP inhibition with doxycycline (and maybe other MMP inhibitors) may attenuate the functional and structural alterations associated with hypertension, including increases in arterial stiffness. These beneficial effects may be, at least in part, independent of their antihypertensive effects

Summary of the IADR Cariology Research, Craniofacial Biology, and Mineralized Tissue Groups Symposium, Iguaçu Falls, Brazil, June 2012: Gene-environment Interactions and Epigenetics in Oral Diseases: Enamel Formation and its Clinical Impact on Tooth Defects, Caries, and Erosion.

Characteristics of enamel may influence or modulate individual susceptibility to caries and erosion. These characteristics are defined during development, which is under strict genetic control, but can easily be modified in many ways by environmental factors. In the symposium, translational aspects of embryology, biochemistry, and genetics of amelogenesis were presented. The symposium provided unique insight into how basic sciences integrate with clinically relevant problems. The need for improved understanding of risks at the individual level, taking into consideration both environmental exposures and genetic background, was presented. The symposium was divided into four stepwise and interconnected topics as follows: 1) The Many Faces of Enamel Development; 2) Enamel Pathogenesis: Biochemistry Lessons; 3) Environmental Factors on Enamel Formation; and, 4) Genetic Variation in Enamel Formation Genes

Peptide characterization of mature fluorotic and control human enamel

Exposure to high fluoride levels during amelogenesis causes enamel fluorosis. This study aimed to determine and compare the amino acid sequences in the enamel of fluorotic and control teeth. This investigation included enamel samples obtained from erupted and non-erupted third molars with either TF grade 4-6 (n=7) fluorosis or no sign of fluorosis (controls, n=7). The samples were kept frozen at -20 °C until protein extraction. Samples were etched and processed with a cocktail of proteinase inhibitors and immediately analyzed. Matrix Assisted Laser Desorption/Ionization-Time-Of-Flight/Time-of-Flight Mass Spectrometry (MALDI-TOF/TOF) followed by MASCOT search aided the peptides analysis. The more abundant peptides bore the N-terminal amelogenin sequences WYQSIRPPYP (which is specific for the X-encoded amelogenin) and MPLPPHPGHPGYINF (which does not show sexual dimorphism) were not different in control or fluorotic enamel. There was no missing proteolytic cleavage in the fluorotic samples, which suggested that the increased amount of protein described in fluorotic enamel did not stem from the decreased ability of proteinases to cleave the proteins in humans. This study showed how to successfully obtain peptide from superficial enamel. A relatively low number of teeth was sufficient to provide good data on the actual peptides found in mature enamel2716671CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPSem informaçãoSem informaçãoExposição a altos níveis de flúor durante a amelogênese causa fluorose no esmalte. Este estudo tem como objetivo determinar e comparar as sequências de aminoácidos presentes no esmalte de dentes controles e fluoróticos. A investigação incluiu amostras de esmalte obtidas de terceiros molares erupcionados e não erupcionados, ambas ou com grau de fluorose TF 4-6 (n=7) ou sem sinais de fluorose (controles, n=7), congelados a -20 oC até a extração das proteínas. As amostras sofreram ataque ácido e foram processadas utilizando um coquetel de inibidores de proteinases, sendo imediatamente analisadas. MALDI-TOF/TOF seguido pela pesquisa com MASCOT foram utilizados para a análise dos peptídeos. Os peptídeos mais abundantes foram das amelogeninas com sequências N-terminal WYQSIRPPYP (que é codificada especificamente pela amelogenina X) e MPLPPHPGHPGYINF (que não apresenta dimorfismo sexual algum), não havendo diferenças entre dentes fluoróticos e controles. Nenhuma alteração na proteólise ocorreu nas amostras fluoróticas, o que sugere que o aumento na quantidade de proteínas existentes nas amostras fluoróticas não está correlacionada a habilidade das proteinases em clivar as proteínas em humanos. Este estudo mostrou como extrair com sucesso peptídeos do esmalte superficial. Um número relativamente baixo de dentes foram suficientes para se obter ótimos dados a respeito de peptídeos encontrados no esmalte madur

Atorvastatin and sildenafil decrease vascular TGF-β levels and MMP-2 activity and ameliorate arterial remodeling in a model of renovascular hypertension

AbstractImbalanced matrix metalloproteinase (MMP)-2 activity and transforming growth factor expression (TGF-β) are involved in vascular remodeling of hypertension. Atorvastatin and sildenafil exert antioxidant and pleiotropic effects that may result in cardiovascular protection. We hypothesized that atorvastatin and sildenafil alone or in association exert antiproliferative effects by down-regulating MMP-2 and TGF-β, thus reducing the vascular hypertrophy induced by two kidney, one clip (2K1C) hypertension.Sham and 2K1C rats were treated with oral atorvastatin 50mg/kg, sildenafil 45mg/kg, or both, daily for 8 weeks. Blood pressure was monitored weekly. Morphologic changes in the aortas were studied. TGF-β levels were determined by immunofluorescence. MMP-2 activity and expression were determined by in situ zymography, gel zymography, Western blotting, and immunofluorescence. The effects of both drugs on proliferative responses of aortic smooth muscle cells to PDGF and on on MMP-2 activity in vitro were determined. Atorvastatin, sildenafil, or both drugs exerted antiproliferative effects in vitro. All treatments attenuated 2K1C-induced hypertension and prevented the increases in the aortic cross-sectional area and media/lumen ratio in 2K1C rats. Aortas from 2K1C rats showed higher collagen deposition, TGF-β levels and MMP-2 activity and expression when compared with Sham-operated animals. Treatment with atorvastatin and/or sildenafil was associated with attenuation of 2K1C hypertension-induced increases in these pro-fibrotic factors. However, these drugs had no in vitro effects on hr-MMP-2 activity.Atorvastatin and sildenafil was associated with decreased vascular TGF-β levels and MMP-2 activity in renovascular hypertensive rats, thus ameliorating the vascular remodeling. These novel pleiotropic effects of both drugs may translate into protective effects in patients

In Situ Zymography and Immunolabeling in Fixed and Decalcified Craniofacial Tissues

In situ zymography is a very important technique that shows the proteolytic activity in sections and allows researchers to observe the specific sites of proteolysis in tissues or cells. It is normally performed in non-fixed frozen sections and is not routinely performed in calcified tissues. In this study, we describe a technique that maintains proteolytic activity in fixed and decalcified sections obtained after routine paraffin sectioning in conventional microtome and cryostat sections. We used adult rat hemimandibles, which presented bone, enamel, and dentine matrices; the substrate used was dye-quenched-gelatin. Gelatinolytic activity was colocalized with MMP-2 using fluorescent antibodies. Specific proteolytic activity was observed in all sections, compatible with metalloproteinase activity, particularly in dentine and bone. Furthermore, matrix metalloproteinase-2 was colocalized to the sites of green fluorescence in dentine. In conclusion, the technique presented here will allow in situ zymography reactions in fixed, decalcified, and paraffin-embedded tissues, and we showed that paraformaldehyde-lysine-periodate–fixed cryostat sections are suitable for colocalization of gelatinolytic activity and protein labeling with antibodies. (J Histochem Cytochem 57:615–622, 2009

Inverse relationship between markers of nitric oxide formation and plasma matrix metalloproteinase-9 levels in healthy volunteers

Background: Nitric oxide (NO) is a major regulator of cardiovascular homeostasis and has anti-atherogenic properties. Reduced NO formation is associated with endothelial dysfunction and with cardiovascular risk factors. Although NO downregulates the expression and activity of the pro-atherogenic enzyme matrix metalloproteinase-9 (MMP-9), no previous clinical study has examined whether endogenous NO formation is inversely associated with the circulating levels of pro-MMP-9, which are associated with cardiovascular events. We examined this hypothesis in 175 healthy male subjects who were non-smokers. Methods: To assess NO bioavailability, the plasma concentrations of nitrite, nitrate, and cGMP were determined using an ozone-based chemiluminescence assay and an enzyme immunoassay. Pro-MMP-9 and pro-MMP-2 levels were measured in plasma samples by gelatin zymography. Results: We found significant negative correlations between pro-MMP-9 levels and plasma nitrite (P=0.035, rs=-0.159), nitrate (P=0.040, rs=-0.158), and cGMP (P=0.011, rs=-0.189) concentrations. However, no significant correlations were found between pro-MMP-2 levels and the plasma concentrations of markers of NO bioavailability (all P>0.05). Conclusions: There is an inverse relationship between markers of NO formation and plasma MMP-9 levels. This finding may shed some light on the possible mechanisms involved in the increased cardiovascular risk of apparently healthy subjects with low NO bioavailability or high circulating levels of pro-MMP-9. (C) 2008 Elsevier B.V. All rights reserved

Matrix Metalloproteinases and Arterial Hypertension: Role of Oxidative Stress and Nitric Oxide in Vascular Functional and Structural Alterations

Various pathophysiological mechanisms have been implicated in hypertension, but those resulting in vascular dysfunction and remodeling are critical and may help to identify critical pharmacological targets. This mini-review article focuses on central mechanisms contributing to the vascular dysfunction and remodeling of hypertension, increased oxidative stress and impaired nitric oxide (NO) bioavailability, which enhance vascular matrix metalloproteinase (MMP) activity. The relationship between NO, MMP and oxidative stress culminating in the vascular alterations of hypertension is examined. While the alterations of hypertension are not fully attributable to these pathophysiological mechanisms, there is strong evidence that such mechanisms play critical roles in increasing vascular MMP expression and activity, thus resulting in abnormal degradation of extracellular matrix components, receptors, peptides, and intracellular proteins involved in the regulation of vascular function and structure. Imbalanced vascular MMP activity promotes vasoconstriction and impairs vasodilation, stimulating vascular smooth muscle cells (VSMC) to switch from contractile to synthetic phenotypes, thus facilitating cell growth or migration, which is associated with the deposition of extracellular matrix components. Finally, the protective effects of MMP inhibitors, antioxidants and drugs that enhance vascular NO activity are briefly discussed. Newly emerging therapies that address these essential mechanisms may offer significant advantages to prevent vascular remodeling in hypertensive patients

Circulating matrix metalloproteinases and their inhibitors in hypertension

Growing experimental evidence indicates that matrix metalloproteinases (MMPs) are implicated in many cardiovascular diseases including hypertension and its chronic complications. It is now clear that MMPs have many more substrates other than components of the extracellular matrix. In fact, intracellular targets now include those associated with the cardiovascular system. Clinical studies have suggested that circulating MMPs may predict cardiovascular morbidity and mortality. It is highly probable that increased MMPs may predispose hypertensive patients to additional complications and clinical sequelae. In this article, we review the basic principles linking MMP activity with hypertension and summarize clinical studies examining two specific MMPs (MMP-2 and -9) in hypertension. We also discuss how antihypertensive drugs may affect MMPs and their endogenous inhibitors, i.e., tissue inhibitors of matrix metalloproteinases (TIMPs). Circulating MMPs may predict increased risk of developing cardiovascular complications associated with hypertension. As such, patients could benefit from early pharmacologic intervention including use of MMP inhibitors4137-8656662CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPES

Should we measure serum or plasma lead concentrations?

Serum samples may not be appropriate to assess lead (Pb) concentrations because they may contain artificially higher Pb concentrations compared with those measured in plasma samples. Here, we compared Pb concentrations in serum versus heparin plasma separated from blood collected with or without vacuum. We have also examined the effects of sample standing time on Pb concentrations measured in serum, heparin plasma, and EDTA plasma. We studied plasma and serum samples from twelve healthy subjects. Blood samples were collected via venous drainage phlebotomy with and without vacuum into trace metal free tubes containing no anticoagulants (serum), or lithium heparin, or EDTA (to obtain plasma). Variable sample standing times (0, 5, and 30 min) prior to centrifugation were allowed. Plasma and serum Pb and iron concentrations were determined by inductively coupled plasma mass spectrometry. Plasma and serum cell-free hemoglobin concentrations were measured. Pb concentrations in serum and in heparin plasma from blood samples collected with or without vacuum were similar and not associated with significant changes in iron or hemoglobin concentrations. The sample standing time (up to 30 min) did not affect Pb concentrations in serum or in heparin plasma, which were approximately 50% lower than those found in EDTA plasma. Serum or heparin plasma separated from blood samples collected via venous phlebotomy with or without vacuum are appropriate medium to assess Pb concentrations, independently of the sample standing time243147151CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPnão temnão te