Protective effect of Ginkgo biloba extract against genotoxic stress

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Genotipagem RhD fetal não invasiva – impacto económico na profilaxia da isoimunização RhD na gravidez

Overview and Aims: The introduction of antenatal RhD isoimmunization prophylaxis was of utmost importance in decreasing the prevalence of perinatal haemolytic disease. Noninvasive genotyping of fetal RhD group from mothers' plasma allows administration of anti-D immunoglobulin only to pregnant women with RhD positive fetuses. Aim: To analyse the economic impact of this technique in a population of nonisoimmunized pregnant women in comparison to the current practice of systematic RhD isoimmunization prophylaxis. Study design: Retrospective, observational and analytic study. Population: 9272 pregnant women who performed obstetric ultrasound in our Fetal Medicine and Prenatal Diagnosis Department between January 2007 to December 2012. Methods: The costs of systematic RhD isoimmunization prophylaxis were compared to fetal RhD genotyping and further RhD isoimunization of RhD negative mothers whose fetuses were RhD positive. Results:In the studied population, 16.7% of pregnant women were RhD negative and 40.3% of their offspring were also RhD negative. Concerning the economic analysis, selective administration of anti-D immunoglobulin based on fetal genotyping would have been about 62 euros more expensive per pregnancy than the current practice of antenatal immunoprophylaxis. Conclusion: Although non-invasive genotyping of fetal RhD group is undoubtly associated with innumerous benefits, in a merely economic analysis, this method is more expensive than the systematic immunoprophylaxis.info:eu-repo/semantics/publishedVersio

Protection against nitric oxide genotoxicity by Ginkgo biloba extract

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DNA protective effect of Ginkgo biloba extract persists after simulation of the human digestion in vitro

This work is supported by: European Investment Funds by FEDER/COMPETE/POCI– Operational Competitiveness and Internationalization Programme, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2013.info:eu-repo/semantics/publishedVersio

Actividade protectora de extractos vegetais contra a genotoxicidade do óxido nítrico

Dissertação de mestrado em Genética MolecularA good reputation regarding the use of plant extracts has been growing consistently over the last years, the potential benefits of their use being heavily supported by the literature. However, as most studies aspire very little by investigating only their antioxidant activities, a lot of work remains to be done on other activities, such as antigenotoxicity. Excessive production of oxidative species, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), can reveal to be harmful to the cell. Amongst various targets, these molecules can affect DNA and may lead to the loss of its stability and integrity, imposing a very dangerous threat to survival. Base excision repair (BER) and homologous recombination (HR) are two major DNA repair mechanisms of the cell, and are responsible for the repair of several forms of DNA damage, including base modification and strand breakage. SNP is a NO-releasing agent and can be used to simulate an excessive increase of NO production inside the cell. NO may oxidize in many different compounds, depending on the molecules it encounters within the cell. The most damaging oxidation products include peroxynitrite and dinitrogen trioxide, a very powerful oxidant and a very strong nitrosating agent, respectively, both being able to directly interact and modify DNA. This project purposed to investigate the protective properties of extracts of Dittrichia viscosa (DVE) and Ginkgo biloba (GBE) against NO-mediated genotoxicity and in what ways protection occurs by using the Schizosaccharomyces pombe model. In the end, the chemical profiles of DVE and GBE agreed with many published others, as most of the detected molecules were also detected in those studies. In vitro assays revealed a relatively (to standard) low IC50 values of DVE in DPPH reduction and iron chelating activity and GBE in DPPH reduction and NO scavenging. DVE did not provide viability protection against NO-mediated damage. GBE, however, was able to protect the studied BER and HR Sch. pombe mutants. Additionally, treatment with GBE alone significantly induced a quicker progression of cell cycle and slightly attenuated the delay produced by SNP treatment. Both extracts appear to activate oxidative-stress response through Pap1, although the prevention of oxidative stress by SNP was not achieved in either cases. In conclusion, GBE protection appears to be independent of BER and it may result from a combination of effects including the scavenging of NO, the generated ROS and RNS by flavonoids and the activation of oxidativestress response proteins.Nos últimos anos, tem-se vindo a construir uma boa reputação no que toca ao uso de extractos vegetais, sendo os seus potenciais benefícios fortemente suportados pela literatura. No entanto, a maioria dos estudos aspira a muito pouco ao investigar apenas as suas actividades antioxidantes, pelo que há muito trabalho a ser feito em relação a outras actividades, como a antigenotoxicidade. A produção excessiva de espécies oxidativas, como as espécies reativas de oxigénio (ROS) e de nitrogénio (RNS), podem revelar-se prejudicial à célula. Entre os seus vários alvos, estas moléculas podem afectar o DNA, podendo levar à perda da sua estabilidade e integridade, impondo assim uma grave ameaça à sobrevivência. A reparação por excisão de bases (BER) e a recombinação homóloga (HR) são dois dos principais mecanismos de reparação de DNA da célula e são responáveis pela reparação de vários tipos de danos no DNA, incluindo modificações de bases e quebras de cadeia. O nitroprussiato de sódio é um agente que liberta NO e pode ser utilizado para simular um aumento excessivo da produção de NO por parte da célula. O NO é capaz de oxidar em muitos compostos diferentes, dependendo das moléculas que encontra dentro da célula. Os produtos de oxidação mais prejudiciais são o peroxinitrito e o trióxido de dinitrogénio, um poderoso oxidante e um forte agente nitrosante, respectivamente, sendo ambos capazes de interagir e modificar directamente o DNA. Este projecto pretendeu investigar as propriedades protectoras de extractos de Dittrichia viscosa (DVE) e Ginkgo biloba (GBE) contra a genotoxicidade do NO e de que forma essa protecção ocorreria, recorrendo ao modelo de Schizosaccharomyces pombe. No fim, os perfis químicos de DVE e GBE foram semelhantes a muitos outros publicados, pelo que a maioria das moléculas detectadas foram também detectadas nesses estudos. Os ensaios in vitro revelaram valores de IC50 relativamente (ao padrão) baixos para DVE na reducção do DPPH e na actividade quelante do ferro e para GBE na reducção do DPPH e na eliminação do NO. DVE não proporcionou protecção da viabilidade contra danos induzidos pelo NO. GBE, no entanto, foi capaz de proteger estirpes de Sch. pombe mutantes na BER e HR. Ainda, o tratamento apenas com GBE induziu uma progressão significativamente mais rápida do ciclo celular e atenuou ligeiramente o atraso produzido pelo tratamento com SNP. Ambos os extractos pareceram activar a resposta a stress oxidativo através da Pap1, embora a prevenção do stresse oxidativo induzido pelo SNP não tenha acontecido em nenhum dos casos. Concluindo, a protecção pelo GBE parece ser independente da BER e deve resultar de uma combinação de efeitos, incluindo a eliminação do NO, dos ROS e dos RNS gerados por parte dos flavonóides e a activação de proteínas de resposta a stress oxidativo

Ginkgo biloba extract-induced mild oxidation might adapt cells against nitric oxide cytotoxicity

Oxidative stress can induce serious damage in cellular components, such as lipids, proteins and DNA, which compromises cell viability and can contribute to health problems. This oxidative state can be induced by excessive levels of signalling molecules such as nitric oxide, which can be converted into other molecules that can affect DNA integrity. Antioxidant plant extracts such as G. biloba L. can protect cells from oxidative stress, and thus promote human health. In this work, we evaluated the protective effect of a G. biloba leaf extract (GBE) against oxidative stress generated by sodium nitroprusside (SNP), an in vitro generator of nitric oxide, in Schizosaccharomyces pombe. The chemical analysis of GBE by electrospray ionization-mass spectrometry showed that the extract is mostly composed of flavonoids recognized by their strong antioxidant properties. Wild-type cells incubated with SNP and GBE showed higher survival than cells only incubated with SNP, indicating a protective effect. Mutant strains affected in base excision repair (mag1, apn2, nth1) and homologous recombination repair (rhp55) pathways were also protected from SNP, suggesting that GBE protection occurs by a DNA-repair independent mechanism. Cell cycle progression analysis revealed that GBE slightly reduces the delay caused by exposure to SNP. Cells incubated with SNP or GBE, or both, induced the relocalization of Pap1 into the nucleus. Pap1 is a transcription factor that is recruited to the nucleus upon oxidative stress, contributing to the expression of genes involved in oxidative stress response. Therefore, these results suggest that GBE may induce mild stress in cells, which can activate the oxidative stress response and consequently provide protection against SNP.This work is supported by: FCT - Portuguese Foundation for Science and Technology (PD/BD/135329/2017), under the Doctoral Programme “Agricultural Production Chains – from fork to farm” (PD/00122/2012); and by European Investment Funds by FEDER/COMPETE/POCI– Operational Competitiveness and Internationalization Programme, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2013

Ginkgo biloba and Dittrichia viscosa extracts protect Schizosaccharomyces pombe cells exposed to camptothecin

Cells are continuously exposed to toxic agents that promote genotoxic stress, which affect genomic integrity and contribute to the development of human diseases. Therefore, there is an urgent need to find safe products that can protect cells from genotoxic damage. Medicinal plant extracts are associated to many health benefits, often linked to their antioxidant properties, and thus may contribute to the preservation of DNA integrity and consequently provide an anticancer effect. In this work, we evaluated the protective effect of two medicinal leaf extracts, G. biloba extract (GBE) and D. viscosa extract (DVE), on the survival of Schizosaccharomyces pombe cells exposed to genotoxic stress induced by camptothecin (CPT). This compound inhibits DNA topoisomerase I activity which leads to the generation of DNA double strand breaks, compromising cell survival. The chemical analysis by electrospray ionization-mass spectrometry revealed that GBE and DVE are mainly composed of polyphenols. In vitro antioxidant assays indicate that GBE and DVE have the capacity to scavenge the radical DPPH and to chelate iron, but low capacity to scavenge NO. Schizosaccharomyces pombe cells incubated with GBE or DVE and CPT exhibited higher survival than cells only incubated with CPT, indicating the protective effect of the extracts against CPT. This effect seems to result from the ability to stimulate DNA repair through homologous recombination, since the mutant rad51, affected in this repair pathway, failed to be protected by both extracts. To our knowledge, this is the first report indicating the potential antigenotoxicity of these extracts against CPT.This work is supported by: FCT - Portuguese Foundation for Science and Technology (PD/BD/135329/2017), under the Doctoral Programme “Agricultural Production Chains – from fork to farm” (PD/00122/2012); and by European Investment Funds by FEDER/COMPETE/POCI– Operational Competitiveness and Internationalization Programme, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2013

Antioxidant and antigenotoxic activities of post-digestion Ginkgo biloba extract

Ginkgo biloba has been used in Chinese traditional medicine for many years and its beneficial effects have been widely spread, making it one of the most sold medicinal plants. Accordingly, many studies have been devoted to this plant, showing its strong antioxidant activity, which is the basis for many of the claimed beneficial effects. In some western countries, G. biloba is prescribed by doctors for memory improvement and against dementia in elderly people. Due to the strong antioxidant activity, it is likely that G. biloba extract is able to protect cells from oxidative damage, including DNA damage, and therefore can act as a tumor preventive agent. By contributing to genomic stability of cells when taken orally, G. biloba extract might help to prevent one of the most prevalent cancers in western countries: colorectal cancer. Hence, G. biloba extract is a good candidate as functional ingredient of foods as it has the potential to increase shelf life and also contribute to a healthy colon. In this work, an ethanolic leaf extract of G. biloba (GBE) was prepared and subjected to in vitro simulated digestion to obtain a post-digestion sample (DGBE) to study. Chemical analysis of both samples showed that the digestion process affects the chemical composition, mainly by hydrolysis of flavonoid glycosides. In vitro analysis of antioxidant activity revealed that the in vitro digestion decreased the DPPH scavenging activity and increased the NO scavenging activity and iron chelating activity of the extract, and also its total phenolic content estimated by Folin-Ciocalteu method. With human colorectal adenocarcinoma cell line (HT-29), GBE and DGBE were not cytotoxic and displayed antigenotoxicity upon oxidative stress. All these results indicate that although chemical modification occur during the digestive process, the beneficial properties are retained. Therefore, G. biloba extract has potential for application as functional food ingredient.This work is supported by: European Investment Funds by FEDER/COMPETE/POCI– Operational Competitiveness and Internationalization Programme, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT - Portuguese Foundation for Science and Technology, under the project

Murine dopaminergic Müller cells restore motor function in a model of Parkinson's disease

Müller cells constitute the main glial cell type in the retina where it interacts with virtually all cells displaying relevant functions to retinal physiology. Under appropriate stimuli, Müller cells may undergo dedifferentiation, being able to generate other neural cell types. Here, we show that purified mouse Müller cells in culture express a group of proteins related to the dopaminergic phenotype, including the nuclear receptor-related 1 protein, required for dopaminergic differentiation, as well the enzyme tyrosine hydroxylase. These dopaminergic components are active, since Müller cells are able to synthesize and release dopamine to the extracellular medium. Moreover, Müller-derived tyrosine hydroxylase can be regulated, increasing its activity because of phosphorylation of serine residues in response to agents that increase intracellular cAMP levels. These observations were extended to glial cells obtained from adult monkey retinas with essentially the same results. To address the potential use of dopaminergic Müller cells as a source of dopamine in cell therapy procedures, we used a mouse model of Parkinson's disease, in which mouse Müller cells with the dopaminergic phenotype were transplanted into the striatum of hemi-parkinsonian mice generated by unilateral injection of 6-hydroxydopamine. These cells fully decreased the apomorphine-induced rotational behavior and restored motor functions in these animals, as measured by the rotarod and the forelimb-use asymmetry (cylinder) tests. The data indicate local restoration of dopaminergic signaling in hemi-parkinsonian mice confirmed by measurement of striatal dopamine after Müller cell grafting. Müller cells are the main glial cells in the retina. When these cells are cultured in the absence of neurons, they spontaneously express proteins of the dopaminergic phenotype, including the enzymes tyrosine hydroxylase (TH), L-DOPA-decarboxylase (DDC) and the dopamine transport system (DAT). In this study, we show this phenomenon is observed with Müller cells obtained from different species, including primates, and address the therapeutic potential of these cells, using a mouse model of Parkinson's disease (PD). ‘Dopaminergic Müller cells’ synthesize dopamine and release most of this neurotransmitter to the extracellular space, constituting a natural dopaminergic ‘pump’. When transplanted to the striatum of PD mice, Müller cells decreased their apomorphine-induced rotational behavior and restored their overall motor functions, measured by rotarod and forelimb use asymmetry tests. Local restoration of dopaminergic signaling was also observed in grafted PD mice, by measuring striatum dopamine and its metabolite (DOPAC) levels (SB: 20µm)