A disfun����o redox espinhal na dor neurop��tica

As esp��cies reativas de oxig��nio (ROS) est��o envolvidas no desenvolvimento de dor neurop��tica. No entanto, a aplica����o cl��nica de mol��culas antioxidantes no tratamento desta patologia tem demonstrado pouca efic��cia. A inibi����o da NADPH oxidase (NOX), uma das principais fontes de ROS, poder�� ser uma boa estrat��gia terap��utica. O nosso grupo verificou que a apocinina (inibidor da NOX) melhora parcialmente os sintomas de dor neurop��tica e a disfun����o redox espinhal no modelo SNI (spared nerve injury). De forma a melhorar este efeito terap��utico, o presente estudo insere-se num projeto maior, que visa identificar as isoformas da NOX envolvidas na fisiopatologia da doen��a e avaliar o efeito da administra����o de inibidores espec��ficos para essas isoformas. Assim, propusemo-nos a avaliar a disfun����o redox espinhal em fases precoces dador neurop��tica perif��rica induzida pelo modelo SNI no Rato, relacionando-a com os comportamentos de dor demonstrados pelos animais. Foram constitu��dos tr��s grupos experimentais: SNI, sham e na��ve, com subgrupos testados e sacrificados aos dias 1, 3, 7 e 14 ap��s a cirurgia. Avaliou-se a sensibilidade mec��nica (vonFrey e pinprick) e ao frio (acetona) dos animais, sacrificaram-se e recolheram-se as medulas espinhais para an��lise imunohistoqu��mica, com marcadores de dano oxidativo no DNA e de dano nitrosativo. Ao contr��rio dos animais sham, que demonstraram um comportamento muito pr��ximo dos na��ve, os animais SNI desenvolveram alod��nia mec��nica e ao frio e hiperalgesia mec��nica na pata ipsilateral. No entanto, o dano oxidativo no corno dorsal ipsilateral da medula espinhal apresentou-se id��ntico nos grupos SNI e sham ao longo dos 14 dias de estudo, n��o havendo tamb��m diferen��as entre os cornos ipsi e contralateral �� les��o nervosa. �� poss��vel que o desenvolvimento de dor neurop��tica nos animais SNI n��o se fa��a acompanhar de disfun����o redox espinhal, pelo menos at�� aos 14 dias p��s indu����o. O facto de a les��o nervosa no modelo SNI se localizar numa por����o distal do ci��tico, ao contr��rio de outros modelos em que o stresse oxidativo espinhal foi j�� descrito, poderia explicar essas diferen��as. Em todo o caso, considerando que os resultados comportamentais obtidos indicam que as cirurgias SNI e sham causam diferentes n��veis de sensibiliza����o nos animais, parece-nos fulcral prolongar os tempos de neuropatia, e executar uma avalia����o do estado redox com outros marcadores, de forma a elucidar se, de facto, existem ROS envolvidas nesta sensibiliza����o e, em caso positivo, poder identificar essas esp��cies, bem como as suas fontes.

Effect of oxidative stress upon absorption of glucose by the human placenta: in vitro studies with BeWo cells

Pregnancy is a dynamic state and the placenta is a temporary organ that, among other important functions, plays a crucial role in the transport of nutrients and metabolites between the mother and the fetus, which is essential for a successful pregnancy. Among these nutrients, glucose is considered a primary source of energy and, therefore, fundamental to insure proper fetus development. Several studies have shown that glucose uptake is dependent on several morphological and biochemical placental conditions. Oxidative stress results from the unbalance between reactive oxygen species (ROS) and antioxidants, in favor of the first. During pregnancy, ROS, and therefore oxidative stress, increase, due to increased tissue oxygenation. Moreover, the relation between ROS and some pathological conditions during pregnancy has been well established. For these reasons, it becomes essential to understand if oxidative stress can compromise the uptake of glucose by the placenta. To make this study possible, a trophoblastic cell line, the BeWo cell line, was used. Experiments regarding glucose uptake, either under normal or oxidative stress conditions, were conducted using tert-butylhydroperoxide (tBOOH) as an oxidative stress inducer, and 3H-2-deoxy-D-glucose (3H-DG) as a glucose analogue. Afterwards, studies regarding the involvement of glucose facilitative transporters (GLUT) and the phosphatidylinositol 3-kinases (PI3K) and protein kinase C (PKC) pathways were conducted, also under normal and oxidative stress conditions. A few antioxidants, endogenous and from diet, were also tested in order to study their possible reversible effect of the oxidative effect of tBOOH upon apical 3H-DG uptake. Finally, transepithelial studies gave interesting insights regarding the apical-to-basolateral transport of 3H-DG. Results showed that 3H-DG uptake, in BeWo cells, is roughly 50% GLUT-mediated and that tBOOH (100 ��M; 24h) decreases apical 3H-DG uptake in BeWo cells by about 33%, by reducing both GLUT- (by 28%) and non-GLUT-mediated (by 40%) 3H-DG uptake. Uptake of 3H-DG and the effect of tBOOH upon 3H-DG uptake are not dependent on PKC and PI3K. Moreover, the effect of tBOOH is not associated with a reduction in GLUT1 mRNA levels. Resveratrol, quercetin and epigallocatechin-3-gallate, at 50 ��M, reversed, by at least 45%, the effect of tBOOH upon 3H-DG uptake. Transwell studies show that the apical-to-basolateral transepithelial transport of 3H-DG is increased by tBOOH.In conclusion, our results show that tBOOH caused a marked decrease in both GLUT and non-GLUT-mediated apical uptake of 3H-DG by BeWo cells. Given the association of increased oxidative stress levels with several important pregnancy pathologies, and the important role of glucose for fetal development, the results of this study appear very interesting.

Electrochemical evaluation of total antioxidant capacity of beverages using a purine-biosensor

In this paper, it was evaluated the total antioxidant capacity (TAC) of beverages using an electrochemical biosensor. The biosensor consisted on the purine base (guanine or adenine) electro-immobilization on a glassy carbon electrode surface (GCE). Purine base damage was induced by the hydroxyl radical generated by Fenton-type reaction. Five antioxidants were applied to counteract the deleterious effects of the hydroxyl radical. The antioxidants used were ascorbic acid, gallic acid, caffeic acid, coumaric acid and resveratrol. These antioxidants have the ability to scavenger the hydroxyl radical and protect the guanine and adenine immobilized on the GCE surface. The interaction carried out between the purinebase immobilized and the free radical in the absence and presence of antioxidants was evaluated by means of changes in the guanine and adenine anodic peak obtained by square wave voltammetry (SWV). The results demonstrated that the purine-biosensors are suitable for rapid assessment of TAC in beverages.

Metal accumulation and oxidative stress biomarkers in octopus (Octopus vulgaris) from Northwest Atlantic

Metals are ubiquitous in the environment and accumulate in aquatic organisms and are known for their ability to enhance the production of reactive oxygen species (ROS). In aquatic species, oxidative stress mechanisms have been studied by measuring antioxidant enzyme activities and oxidative damages in tissues. The aim of this study was to apply and validate a set of oxidative stress biomarkers and correlate responses with metal contents in tissues of common octopus (Octopus vulgaris). Antioxidant enzyme activity (catalase ��� CAT, superoxide dismutase ��� SOD and glutathione S-transferases ��� GST), oxidative damages (lipid peroxidation ��� LPO and protein carbonyl content ��� PCO) andmetal content (Cu, Zn, Pb, Cd and As) in the digestive gland and armof octopus, collected in the NWPortuguese coast in different periods, were assessed after capture and after 14 days in captivity. CAT and SOD activitieswere highly responsive to fluctuations inmetal concentrations and able to reduce oxidative damage, LPO and PCO in the digestive gland. CAT activity was also positively correlated with SOD and GST activities, which emphasizes that the three enzymes respond in a coordinated way to metal induced oxidative stress. Our results validate the use of oxidative stress biomarkers to assess metal pollution effects in this ecological and commercial relevant species.Moreover, octopus seems to have the ability to control oxidative damage by triggering an antioxidant enzyme coordinated response in the digestive gland.

DNA-based biosensor for the electrocatalytic determination of antioxidant capacity in beverages

Reactive oxygen species (ROS) are produced as a consequence of normal aerobic metabolism and are able to induce DNA oxidative damage. At the cellular level, the evaluation of the protective effect of antioxidants can be achieved by examining the integrity of the DNA nucleobases using electrochemical techniques. Herein, the use of an adenine-rich oligonucleotide (dA21) adsorbed on carbon paste electrodes for the assessment of the antioxidant capacity is proposed. The method was based on the partial damage of a DNA layer adsorbed on the electrode surface by OH��� radicals generated by Fenton reaction and the subsequent electrochemical oxidation of the intact adenine bases to generate an oxidation product that was able to catalyze the oxidation of NADH. The presence of antioxidant compounds scavenged hydroxyl radicals leaving more adenines unoxidized, and thus, increasing the electrocatalytic current of NADHmeasured by differential pulse voltammetry (DPV). Using ascorbic acid (AA) as a model antioxidant species, the detection of as low as 50nMof AA in aqueous solution was possible. The protection efficiency was evaluated for several antioxidant compounds. The biosensor was applied to the determination of the total antioxidant capacity (TAC) in beverages.

Electrochemical DNA-sensor for evaluation of total antioxidant capacity of flavours and flavoured waters using superoxide radical damage

In this paper, a biosensor based on a glassy carbon electrode (GCE) was used for the evaluation of the total antioxidant capacity (TAC) of flavours and flavoured waters. This biosensor was constructed by immobilising purine bases, guanine and adenine, on a GCE. Square wave voltammetry (SWV) was selected for the development of this methodology. Damage caused by the reactive oxygen species (ROS), superoxide radical (O2�����), generated by the xanthine/xanthine oxidase (XOD) system on the DNA-biosensor was evaluated. DNA-biosensor encountered with oxidative lesion when it was in contact with the O2�����. There was less oxidative damage when reactive antioxidants were added. The antioxidants used in this work were ascorbic acid, gallic acid, caffeic acid, coumaric acid and resveratrol. These antioxidants are capable of scavenging the superoxide radical and therefore protect the purine bases immobilized on the GCE surface. The results demonstrated that the DNA-based biosensor is suitable for the rapid assess of TAC in beverages.

Electrocatalytic evaluation of DNA damage by superoxide radical for antioxidant capacity assessment

The integrity of DNA purine bases was herein used to evaluate the antioxidant capacity. Unlike other DNA-based antioxidant sensors reported so far, the damaging agent chosen was the O 2 radical enzymatically generated by the xanthine/xanthine oxidase system. An adenine-rich oligonucleotide was adsorbed on carbon paste electrodes and subjected to radical damage in the presence/absence of several antioxidant compounds. As a result, partial damage on DNA was observed. A minor product of the radical oxidation was identified by cyclic voltammetry as a diimine adenine derivative also formed during the electrochemical oxidation of adenine/guanine bases. The protective efficiency of several antioxidant compounds was evaluated after electrochemical oxidation of the remaining unoxidized adenine bases, by measuring the electrocatalytic current of NADH mediated by the adsorbed catalyst species generated. A comparison between O 2 and OH radicals as a source of DNA lesions and the scavenging efficiency of various antioxidant compounds against both of them is discussed. Finally, the antioxidant capacity of beverages was evaluated and compared with the results obtained with an optical method.

Mitochondria are the main source and one of the targets of Pb (lead)-induced oxidative stress in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model organism for studying lead (Pb) toxicity. Yeast cells of a laboratory S. cerevisiae strain (WT strain) were incubated with Pb concentrations up to 1,000 ��mol/l for 3 h. Cells exposed to Pb lost proliferation capacity without damage to the cell membrane, and they accumulated intracellular superoxide anion (O2 .���) and hydrogen peroxide (H2O2). The involvement of the mitochondrial electron transport chain (ETC) in the generation of reactive oxygen species (ROS) induced by Pb was evaluated. For this purpose, an isogenic derivative ��0 strain, lacking mitochondrial DNA, was used. The ��0 strain, without respiratory competence, displayed a lower intracellular ROS accumulation and a higher resistance to Pb compared to the WT strain. The kinetic study of ROS generation in yeast cells exposed to Pb showed that the production of O2 .��� precedes the accumulation of H2O2, which is compatible with the leakage of electrons from the mitochondrial ETC. Yeast cells exposed to Pb displayed mutations at the mitochondrial DNA level. This is most likely a consequence of oxidative stress. In conclusion, mitochondria are an important source of Pb-induced ROS and, simultaneously, one of the targets of its toxicity.

Oxidative stress in pregnancy and fertility pathologies

Oxidative stress designates the state of imbalance between reactive oxygen species (ROS) production and antioxidant levels. In a healthy placenta, there is an increase in ROS production, due to formation of new tissues and inherent metabolism, but this is balanced by higher levels of antioxidants. However, this balance is lost in some situations, with a consequent increase in oxidative stress levels. Oxidative stress has been implicated in several placental disorders and pregnancy pathologies. The present review intends to summarize what is known about the relationship between oxidative stress and well-known pregnancy disorders.

Avalia����o do papel da glutationa e do vac��olo como mecanismos de defesa contra a toxicidade induzida pelo chumbo na levedura Saccharomyces cerevisiae

A presen��a de metais pesados no meio ambiente deve-se, principalmente, a actividades antropog��nicas. Ao contr��rio do Cu e do Zn, que em baixas concentra����es s��o essenciais para o normal funcionamento celular, n��o se conhece para o chumbo nenhuma fun����o biol��gica. O chumbo apresenta efeitos t��xicos, e considerado poss��vel agente carcinog��neo, sendo classificado como poluente priorit��rio pela Agencia de Protec����o Ambiental dos EUA (US-EPA). O presente trabalho teve como objetivo avaliar o papel da glutationa e do vac��olo, como mecanismos de defesa, contra os efeitos t��xicos induzidos pelo chumbo, usando como modelo a levedura Saccharomyces cerevisiae. A levedura S. cerevisiae quando exposta a varias concentra����es de chumbo, durante 3h, perde a viabilidade e acumula esp��cies reativas de oxig��nio (ROS). O estudo comparativo da perda de viabilidade e acumula����o de ROS em c��lulas de uma estirpe selvagem (WT) e de estirpes mutantes, incapazes de produzir glutationa devido a uma defici��ncia no gene GSH1 (���gsh1) ou GSH2 (���gsh2) mostrou que as estirpes ���gsh1 ou(���gsh2 n��o apresentavam um aumento da sensibilidade ao efeito toxico do chumbo. No entanto, o tratamento de c��lulas da estirpe WT com iodoacetamida (um agente alquilante que induz a deple����o de glutationa) aumentou a sensibilidade das c��lulas a presen��a de chumbo. Pelo contr��rio, o enriquecimento em GSH, atrav��s da incuba����o de c��lulas WT com glucose e uma mistura de amino��cidos que constituem a GSH (acido L-glut��mico, L-ciste��na e glicina), reduziu o stress oxidativo e a perda de viabilidade induzida por chumbo. A import��ncia do vac��olo, como mecanismo de defesa, foi avaliada atrav��s da utiliza����o de um mutante sem qualquer estrutura vacuolar (���vps16) ou de mutantes deficientes na subunidade catal��tica A (���vma1) ou B (���vma2) ou no proteol��tico - subunidade C (���vma3) da V-ATPase. As c��lulas da estirpe ����vps16 apresentaram uma elevada suscetibilidade a presen��a de chumbo. As c��lulas das estirpes deficientes na subunidade A, B ou c da V-ATPase, apresentaram uma maior perda de viabilidade, quando expostas a chumbo, do que as c��lulas da estirpe WT, mas menor do que a da estirpe ���vps16 Em conclus��o, os resultados obtidos, no seu conjunto, sugerem que a glutationa esta envolvida na defesa contra a toxicidade provocada por chumbo; todavia, a glutationa, por si s��, parece n��o ser suficiente para suster o stress oxidativo e a perda de viabilidade induzida por chumbo. O vac��olo parece constituir um importante mecanismo de defesa contra a toxicidade provocada por chumbo. A V-ATPase parece estar envolvida na compartimenta����o de chumbo no vac��olo.

Estudo da Toxicidade do Chumbo na Levedura Saccharomyces cerevisiae

O chumbo �� um importante poluente ambiental. A levedura Saccharomyces cerevisiae constitui um modelo ��til para o estudo dos efeitos t��xicos do chumbo. O conhecimento dos mecanismos de defesa e resist��ncia �� presen��a de metais pesados poder�� ser ��til em tecnologias de prote����o ambiental, nomeadamente no desenvolvimento de novas metodologias para a biorremedia����o de metais pesados. O presente trabalho teve como objetivo avaliar o impacto do Pb na capacidade proliferativa, na integridade membranar e na produ����o intracelular de esp��cies reativas de oxig��nio (ROS), na estirpe laboratorial da levedura Saccharomyces cerevisiae BY4741 (estirpe selvagem, WT). Foi tamb��m estudado o papel das mitoc��ndrias, como fonte de ROS induzida por Pb, e o envolvimento da H+-ATPase vacuolar (V-ATPase) e de transportadores vacuolares pertencentes �� superfam��lia ABC (de ATP-binding cassette) na defesa contra a toxicidade do Pb. O estudo cin��tico do impacto de duas concentra����es de Pb na viabilidade das leveduras (avaliado atrav��s de um ensaio clonog��nico), na integridade da membrana celular (determinada com iodeto de prop��dio) e na produ����o intracelular de ROS (o ani��o super��xido foi detetado com dihidroet��dio e o per��xido de hidrog��nio com 2���,7���- diclorodihidrofluoresce��na), revelou uma perda progressiva da capacidade proliferativa (53 e 17% de c��lulas vi��veis, ap��s a exposi����o durante 3h a 250 ou 1000 ��mol/l de chumbo, respetivamente), coincidente com a acumula����o intracelular de ani��o super��xido e de per��xido de hidrog��nio, na aus��ncia de perda da integridade membranar. A import��ncia das mitoc��ndrias na produ����o de ROS, induzida por chumbo, foi levada a cabo usando um mutante deficiente respirat��rio desprovido de ADN mitocondrial (��0). Quando comparado com a respetiva estirpe parental, o mutante ��0 apresentou uma maior resist��ncia ao Pb e uma menor produ����o de ROS induzida por Pb. A exposi����o das c��lulas da estirpe BY4741 a 250 e 1000 ��mol/l de chumbo originou a forma����o de 49 e 58% de c��lulas deficientes respirat��rias, respetivamente. A fun����o da V-ATPase, na desintoxica����o de chumbo, foi avaliada utilizando mutantes com uma estrutura vacuolar normal mas defetivos em subunidades da VATPase (vma1��, vma2��, vma3�� e vph1��). Comparativamente ��s c��lulas da estirpe WT, todos os mutantes testados, sem V-ATPase funcional, apresentaram uma maior suscetibilidade ao Pb. O papel dos transportadores vacuolares pertencentes �� superfam��lia ABC, na defesa contra a toxicidade induzida por chumbo, foi levada a cabo utilizando mutantes sem os transportadores Ycf1p ou Vmr1p. Os resultados preliminares mostraram que quando comparadas com as c��lulas da estirpe WT, as c��lulas das estirpes ycf1�� ou vmr1�� n��o apresentavam uma maior perda da viabilidade. A modifica����o da morfologia vacuolar, em c��lulas expostas a chumbo, foi visualizada utilizando a estirpe Vma2p-GFP. O tratamento das c��lulas com Pb originou a fus��o dos vac��olos de tamanho m��dio num ��nico vac��olo de grande dimens��o. Em conclus��o, os estudos desenvolvidos no presente trabalho, utilizando a estirpe laboratorial BY4741, mostraram que a perda da capacidade proliferativa das leveduras, induzida pelo chumbo, pode ser atribu��da �� acumula����o intracelular do ani��o super��xido e de per��xido de hidrog��nio. As mitoc��ndrias parecem ser uma das principais fontes de ROS induzido por Pb e, simultaneamente, um dos principais alvos da sua toxicidade. Em S. cerevisiae, o vac��olo desempenha um papel importante na desintoxica����o do Pb. A modifica����o da morfologia vacuolar ap��s exposi����o ao chumbo poder�� ser a consequ��ncia da acumula����o de Pb no vac��olo. Enquanto os transportadores da superfam��lia ABC parecem n��o estar envolvidos na sequestra����o vacuolar de Pb, �� necess��ria a presen��a, num estado funcional, da V-ATPase para que ocorra a compartimenta����o do Pb. Muito provavelmente, a compartimenta����o do Pb no vac��olo previne a sua acumula����o no citosol e o desencadear dos respetivos efeitos t��xicos.