In vitro effect of the herbicide glyphosate on human blood platelet aggregation and coagulation / Efeito in vitro do herbicida glifosato na agregação plaquetária e coagulação sanguínea em humanos

Glyphosate [N-(phosphonomethyl)-glycine] is a broad-spectrum, non-selective, post-emergence herbicide that is extensively used in agriculture. Published data referring to the effects of this product on human health are contradictory. We showed previously that long-term treatment of rats with low doses of Glyphosate-Biocarb® may induce hepatic histological changes and bleeding without decreasing platelet counts. The aim of the current study was to investigate, in vitro, the effect of glyphosate on human blood platelet aggregation and coagulation. Materials and methods: Platelet aggregation was determined in the platelet-rich plasma using the agents: 6µM-adenosine diphosphate, 6µM-epinephrine and 4µg/mL-collagen. Pretreatment with 500µg/mL glyphosate showed significant hypofunction of the three aggregating agents. The inhibitory effect was dose-dependent at concentrations from 50 to 500 µg/mL. The release of ATP was lower for glyphosate-treated platelets after stimulation by collagen. On the other hand, glyphosate did not promote any inhibitory effects on prothrombin time, thromboplastin time and thrombin time. In conclusion, the results demonstrate that glyphosate promotes changes in the platelet metabolism with an inhibitory effect on primary hemostasis.

O glifosato [N-(phosphonomethyl)-glycine] é um herbicida pós-emergente não seletivo de amplo espectro muito utilizado na agricultura. Dados da literatura referentes aos efeitos desse produto na saúde humana são contraditórios. Em estudos prévios demonstramos que ratos previamente tratados com glifosato apresentavam lesões hepáticas e sangramento sem alterações quantitativas de plaquetas. O objetivo do presente estudo é investigar os efeitos in vitro do glifosato (GP) na agregação plaquetária e coagulação sanguínea em humanos. A agregação plaquetária foi determinada em plasma rico em plaquetas (PRP) usando os agentes adenosina difosfato (ADP) 6µM, epinefrina 6µM e colágeno 4µg/mL. Pré-tratamento com GP 500µg/mL demonstrou significativa hipofunção dos três agentes agregantes. O efeito inibitório foi dose dependente em concentrações de 50-500 µg/mL. Utilizando-se a quantificação de ATP como um índice da capacidade de secreção plaquetária, foi observado diminuição da liberação das plaquetas tratadas com GP. Por outro lado, o GP não promoveu efeito inibidor no tempo de protrombina (TP), tempo de tromboplastina parcial ativada (ATTP) e tempo de trombina (TT). Em conclusão, os resultados demonstram que o GP promove mudanças no metabolismo plaquetário com efeito inibitório na hemostasia primária.

Increased protein nitration burden in the atherosclerotic lesions and plasma of apolipoprotein A-I deficient mice.

Apolipoprotein A-I (apoA-I), the major protein constituent within high-density lipoprotein (HDL), has been associated with antiatherogenic protection by mechanisms that include reverse cholesterol transport and antiinflammatory functions. To evaluate the proposed protective function of apoA-I, proteins modified by nitrating oxidants were evaluated in the aortic tissue and plasma of mice lacking the low-density lipoprotein receptor and apobec (LA) and LA mice with genetic deletion of apoA-I (LA-apoA-I(-/-)). The levels of nitrated proteins in aortic tissue quantified by liquid chromatography with online electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) were 6-fold higher in the LA-apoA-I(-/-) as compared with the LA mice. The quantitative analyses were corroborated by immunohistochemical and high-resolution immunoelectron microscopic evaluation of the lesions, which revealed abundant staining for nitrated proteins in the aortic root lesions of LA-apoA-I(-/-) as compared with the LA mice. Proteomic approaches based on affinity enrichment and site-specific adduct mapping identified unique specific protein targets for nitration in the plasma of LA-apoA-I(-/-) that were not present in the plasma of LA mice. In particular the nitration of fibrinogen was shown to accelerate fibrin clot formation. Another consequence of the augmented levels of nitrated proteins was the induction of humoral responses documented by the increased circulating immunoglobulins that recognize nitrotyrosine in LA-apoA-I(-/-) as compared with the LA mice. These data collectively support a protective function of apoA-I diminishing the burden of nitrative oxidants in these mice models of atherosclerosis.

Subcellular localization of tyrosine-nitrated proteins is dictated by reactive oxygen species generating enzymes and by proximity to nitric oxide synthase.

Using high-resolution immuno-electron microscopy the steady-state subcellular distribution of tyrosine-nitrated proteins in different cells and tissues was evaluated. In quiescent eosinophils and neutrophils in the bone marrow intracellular nitrated proteins were mainly restricted to the peroxidase-containing secretory granules. The inducible nitric oxide synthase (iNOS) was expressed in the same granules. Proteins nitrated on tyrosine residues were also abundant in the cytosol of circulating erythrocytes. In the vasculature, nitrated proteins were mainly located in mitochondria and endoplasmic reticulum of the endothelial cells, fibroblasts, and smooth muscle cells. Endogenous nitrated proteins were also found in chondrocytes in cartilage, where it was typically associated with the cytoplasmic interface of the endoplasmic reticulum membrane. Nitrated proteins were also prominent in the peroxisomes of liver hepatocytes and of secretory cells in the lacrimal gland. Challenge of mouse dendritic cells with lipopolysaccharide induced iNOS protein expression in cytosol and peroxisomes and was associated with an increased 3-nitrotyrosine formation in cytosol, mitochondria, and peroxisomes. These data indicate that nitric oxide-dependent protein tyrosine nitration is a physiologically relevant process localized within specific subcellular compartments in close proximity to iNOS and to enzymes capable of peroxidative chemistry and reactive oxygen species production.

Autologous apoptotic cell engulfment stimulates chemokine secretion by vascular smooth muscle cells.

Apoptosis of vascular smooth muscle cells (VSMCs) occurs in vivo under both physiological and pathological settings. The clearance of apoptotic cells may be accomplished in part by the surrounding normal VSMCs. However, the fate of internalized apoptotic cells, the rate of intracellular degradation, and the consequences of these processes to VSMC biology are unknown. Electron microscopy and confocal fluorescence imaging showed that rat VSMCs effectively bound and internalized autologous apoptotic VSMCs in vitro. Within 2 hours, the internalized apoptotic cells were delivered to lysosomes, and the majority of these internalized cells and their proteins were efficiently degraded by 24 hours. After degradation was completed, the phagocytic VSMCs remained viable with normal rates of proliferation. Clearance of apoptotic cells by VSMCs did not induce the release of vascular wall matrix proteases but was associated with a 1.6-fold increase in transforming growth factor-beta1 release. Interestingly, clearance of apoptotic cells stimulated VSMCs to secrete monocyte-chemoattractant protein-1 and cytokine-induced neutrophil chemoattractant. The coordinated release of transforming growth factor-beta1 and chemokines suggests that autologous apoptotic cell clearance stimulates VSMCs to release molecules that specifically recruit professional phagocytes while simultaneously dampening the inflammatory response and preventing vascular injury.

Expression of inducible nitric-oxide synthase and intracellular protein tyrosine nitration in vascular smooth muscle cells: role of reactive oxygen species.

A significant increase in the induction of inducible nitric-oxide synthase (iNOS) protein expression and in the levels of nitrite plus nitrate was observed in rat aortic smooth muscle cells (RASMCs) stably transfected with catalase (RASMC-2C2) as compared with empty vector-transfected RASMC-V4 cells after exposure to cytokines and lipopolysaccharide. The increased expression of iNOS protein in the RASMC-2C2 cells was associated with a significant activation of nuclear transcription factor kappaB, one of the transcriptional regulators of iNOS expression. The induction of iNOS was also accompanied by increased protein tyrosine nitration in both cell types as revealed by immunocytochemical staining and high pressure liquid chromatography with on-line electrospray ionization tandem mass spectrometry. Nitrotyrosine formation was inhibited by 1400W, an iNOS inhibitor, by 4-(2-aminoethyl) benzenesulfonyl fluoride, an inhibitor of NADPH oxidase, and by the superoxide dismutase mimetic M40403, but not by the peroxidase inhibitor 4-aminobenzoic hydrazide. Electron microscopy using affinity-purified anti-nitrotyrosine antibodies revealed labeling at the cytosolic side of the rough endoplasmic reticulum membranes, in the nucleus, occasionally in mitochondria, and consistently within the fibrillar layer underneath the plasma membrane. Collectively, the data in this model system indicate that hydrogen peroxide, by inhibiting the activation of nuclear transcription factor kappaB, prevents iNOS expression, whereas superoxide contributes in a precise pattern of intracellular protein tyrosine nitration.

Interacao de plaquetas e leucocitos humanos: modulacao da resposta de agregacao / Interaction of platelets and human leucocytes: modulation of the aggregation reply

A interacao de plaquetas e leucocitos humanos foi avaliada em presenca dos agonistas ADP e PMA. Os leucocitos inibiram a resposta de agregacao e liberacao de ATP. A utilizacao de SOD e azul de metileno nao influenciaram no efeito de inibicao leucocitaria, por outro lado, dipiridamol potenciou o efeito de compostos antiagregantes resultante da interacao de plaquetas e leucocitos. Os resultados obtidos sugerem que a inibicao da funcao plaquetaria por leucocitos humanos em presenca de ADP e PMA e independente da formacao de especies de oxigenio e nitrogenio. Os dados do presente trabalho demonstram que a interacao de plaquetas e leucocitos expostos a agonistas ADP e PMA envolve a formacao de compostos antiagregantes mediados preferencialmente pelo metabolismo de nucleotideos.