Efeito do sildenafil na genotoxicidade induzida pelo estresse oxidativo em camundongos ateroscleróticos

Detalhes bibliográficos
Autor(a) principal: Rodrigues, Bianca de Paula e
Data de Publicação: 2013
Tipo de documento: Dissertação
Idioma: por
Título da fonte: Repositório Institucional da Universidade Federal do Espírito Santo (riUfes)
Texto Completo: http://repositorio.ufes.br/handle/10/7983
Resumo: It is well known that enhanced production of reactive oxygen species (ROS) leads to oxidative stress observed in atherosclerosis and that ROS can also cause damage in cellular macromolecules, including DNA. Considering previous report that sildenafil, an inhibitor of phosphodiesterase 5 (PDE5), have antioxidant effects, in the present study we evaluated the effect of this drug on genotoxicity of blood mononuclear cells (MNC) and liver cells from atherosclerotic apolipoprotein E knockout mice (apoE-/- ). To accelerate and aggravate the spontaneous hyperlipidemia and atherosclerosis in apoE−/− mice, 8-week-old animals were fed a Western-type diet. Animals were randomly distributed into three different groups: (a) apoE−/− mice administered with the PDE5 inhibitor sildenafil (apoE−/− sildenafil, Viagra® , 40 mg/kg/day, for 3 weeks, by oral gavage, n=18), (b) apoE-/- mice administered with vehicle (apoE-/- vehicle, n=14) and (c) Wild-type C57Black/6 mice (WT, n=16). Then, animals (18-week-old) were euthanized and MNC isolated by density gradient and liver cells extracted by enzymatic action. Oxidative stress was determined by superoxide anion (•O2 - ) production using DHE probe. 106 cells were diluted with 1mL PBS and incubated with 160 μM DHE for 30 min at 37ºC in the dark, then washed and resuspended in 0.5ml of PBS-iFBS. A FACSCanto II cytometer was used for the flow cytometric analysis. Measurements were performed in triplicate and 10,000 events were used for each measurement. The genotoxicity analysis was performed using the alkaline comet assay that consists to obtain from individual cells, an array with a fluorescent halo in the format of a comet corresponding to the quantity of the fragmented DNA. The DNA helices ruptures extensions were evaluated by the image intensification method, where 100 cells were randomly selected (50 in each slide) using the analysis program CASP. The measurements of the comets were obtained by the parameters of the tail moment and percentage tail DNA. In addition, comets with more than 25% DNA in the tail (damage level moderate-high) were quantified between groups. Data are expressed as mean±SEM. Statistical analysis were performed by one-way ANOVA followed by Bonferroni post hoc test (p<0.05). Flow cytometric analysis showed a significant increase (78%) in superoxide anion (•O2 - ) production in apoE-/- vehicle mice MNC compared to WT and treatment with sildenafil decreases the levels of •O2 - in animals apoE-/- . In the DNA damage analysis all parameters used (%tail DNA, tail moment and number of comets with damage level moderate-high) showed an increase in the DNA fragmentation in both MNC and liver cells of animals apoE-/- vehicle when compared to WT control, and sildenafiladministered apoE-/- mice exhibited minimal DNA damage in those cells similar to WT mice. Our data shows that atherosclerosis increases ROS production leading to oxidative stress. Apparently, increased ROS interact with the DNA leading to fragmentation and, interestingly, the treatment with sildenafil was able to reduce ROS production inhibiting DNA damage. The novelty of this study is that sildenafil may offer a new perspective to the use of PDE5 inhibitors to protect against DNA damage, in cells involved in the inflammatory and dyslipidemic processes that accompany atherosclerosis.
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spelling Vasquez, Elisardo CorralRodrigues, Bianca de Paula eCampagnaro, Bianca PrandiSantos, Leonardo dos2018-08-01T22:58:44Z2018-08-012018-08-01T22:58:44Z2013-05-24It is well known that enhanced production of reactive oxygen species (ROS) leads to oxidative stress observed in atherosclerosis and that ROS can also cause damage in cellular macromolecules, including DNA. Considering previous report that sildenafil, an inhibitor of phosphodiesterase 5 (PDE5), have antioxidant effects, in the present study we evaluated the effect of this drug on genotoxicity of blood mononuclear cells (MNC) and liver cells from atherosclerotic apolipoprotein E knockout mice (apoE-/- ). To accelerate and aggravate the spontaneous hyperlipidemia and atherosclerosis in apoE−/− mice, 8-week-old animals were fed a Western-type diet. Animals were randomly distributed into three different groups: (a) apoE−/− mice administered with the PDE5 inhibitor sildenafil (apoE−/− sildenafil, Viagra® , 40 mg/kg/day, for 3 weeks, by oral gavage, n=18), (b) apoE-/- mice administered with vehicle (apoE-/- vehicle, n=14) and (c) Wild-type C57Black/6 mice (WT, n=16). Then, animals (18-week-old) were euthanized and MNC isolated by density gradient and liver cells extracted by enzymatic action. Oxidative stress was determined by superoxide anion (•O2 - ) production using DHE probe. 106 cells were diluted with 1mL PBS and incubated with 160 μM DHE for 30 min at 37ºC in the dark, then washed and resuspended in 0.5ml of PBS-iFBS. A FACSCanto II cytometer was used for the flow cytometric analysis. Measurements were performed in triplicate and 10,000 events were used for each measurement. The genotoxicity analysis was performed using the alkaline comet assay that consists to obtain from individual cells, an array with a fluorescent halo in the format of a comet corresponding to the quantity of the fragmented DNA. The DNA helices ruptures extensions were evaluated by the image intensification method, where 100 cells were randomly selected (50 in each slide) using the analysis program CASP. The measurements of the comets were obtained by the parameters of the tail moment and percentage tail DNA. In addition, comets with more than 25% DNA in the tail (damage level moderate-high) were quantified between groups. Data are expressed as mean±SEM. Statistical analysis were performed by one-way ANOVA followed by Bonferroni post hoc test (p<0.05). Flow cytometric analysis showed a significant increase (78%) in superoxide anion (•O2 - ) production in apoE-/- vehicle mice MNC compared to WT and treatment with sildenafil decreases the levels of •O2 - in animals apoE-/- . In the DNA damage analysis all parameters used (%tail DNA, tail moment and number of comets with damage level moderate-high) showed an increase in the DNA fragmentation in both MNC and liver cells of animals apoE-/- vehicle when compared to WT control, and sildenafiladministered apoE-/- mice exhibited minimal DNA damage in those cells similar to WT mice. Our data shows that atherosclerosis increases ROS production leading to oxidative stress. Apparently, increased ROS interact with the DNA leading to fragmentation and, interestingly, the treatment with sildenafil was able to reduce ROS production inhibiting DNA damage. The novelty of this study is that sildenafil may offer a new perspective to the use of PDE5 inhibitors to protect against DNA damage, in cells involved in the inflammatory and dyslipidemic processes that accompany atherosclerosis.Na aterosclerose ocorre aumento de agentes genotóxicos endógenos, como espécies reativas de oxigênio (ROS), que podem causar danos oxidativos ao DNA. Sabe-se que o sildenafil, inibidor da fosfodiesterase 5 (PDE5), apresenta efeito antioxidante, diminuindo o estresse oxidativo e a peroxidação lipídica. O objetivo do presente estudo foi analisar o efeito do tratamento crônico com sildenafil sobre a genotoxicidade causada pelo estresse oxidativo em células mononucleares (MNC) do sangue e células hepáticas de camundongos ateroscleróticos apoE knockout (apoE-/-). Foram utilizados camundongos machos apoE-/- com 8 semanas de idade que receberam dieta aterogênica até a 18° semana de vida. Com 15 semanas de idade os animais foram divididos em dois grupos: tratado com veículo (apoE-/- veículo, n=14) ou sildenafil (Viagra®; 40 mg/Kg/dia; apoE-/- sildenafil, n=18), por gavagem. Animais da linhagem C57Black/6 wild-type (WT, n=16) foram submetidos ao mesmo protocolo que os animais do grupo apoE-/- veículo, exceto pela administração da dieta. Ao final do tratamento, os animais foram eutanasiados, as MNC isoladas utilizando gradiente de densidade e células hepáticas extraídas através de ação enzimática. Para análise do estresse oxidativo 106 MNC foram diluídas em PBS e incubadas com 160 μM de DHE por 30 minutos a 37ºC no escuro e, depois lavadas e ressuspendidas em 0,5 ml de PBS-iSFB. As amostras foram mantidas em gelo até o momento da aquisição dos dados pelo citômetro de fluxo FACSCanto II. Em cada experimento foram avaliadas 30.000 células. A análise de genotoxicidade foi realizada utilizando o ensaio do cometa alcalino que consiste em obter, a partir de células individuais, uma matriz com um halo fluorescente no formato de um cometa correspondendo à quantidade de DNA fragmentado. Foi avaliada a extensão das rupturas das hélices do DNA com método de intensificação de imagem, em 100 células escolhidas aleatoriamente de cada animal. Os parâmetros analisados foram a porcentagem de DNA na cauda (% tail DNA) e o momento da cauda (tail moment). Além disso, foi realizada a quantificação de cometas com mais de 25% de DNA na cauda (nível moderado-elevado de fragmentação) entre os grupos. Os dados estão expressos como média±EPM. A análise estatística foi realizada por ANOVA de uma via, seguida de post hoc de Bonferroni, adotando o nível de significância de 5%. A análise por citometria de fluxo mostrou um aumento de 78% na produção de ânion superóxido (O2-) nas MNC de animais apoE-/- veículo quando comparados aos animais WT, e o tratamento com sildenafil diminuiu os níveis de O2- nos animais apoE-/- próximo aos níveis do controle. Todos os parâmetros utilizados para investigar o dano ao DNA (%tail DNA, tail moment e quantidade de cometas com nível moderado-elevado de dano) demonstraram um aumento da fragmentação do DNA tanto em MNC quanto em células hepáticas dos animais apoE-/- veículo quando comparados aos animais WT, sendo que o tratamento com sildenafil foi capaz de diminuir esses valores para os níveis observados no controle. Nossos resultados sugerem que a aterosclerose aumenta a produção de ROS levando ao estresse oxidativo. Assim, as ROS interagem com o DNA das células, fragmentando-o. Sendo que o tratamento com o sildenafil foi capaz de reduzir os níveis de produção de ROS e, consequentemente, diminuir o dano ao DNA. Estes resultados são inéditos na literatura e devem contribuir de maneira relevante na busca de novas terapias de prevenção e tratamento da aterosclerose e suas consequências.Texthttp://repositorio.ufes.br/handle/10/7983porUniversidade Federal do Espírito SantoMestrado em Ciências FisiológicasPrograma de Pós-Graduação em Ciências FisiológicasUFESBRCentro de Ciências da SaúdeAtherosclerosisOxidative stressDNA damageComet assayApoE-/- miceSildenafilCamundongos apoE-/-AteroscleroseEstresse oxidativoDano ao DNAEnsaio do cometaFisiologia612Efeito do sildenafil na genotoxicidade induzida pelo estresse oxidativo em camundongos ateroscleróticosinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da Universidade Federal do Espírito Santo (riUfes)instname:Universidade Federal do Espírito Santo (UFES)instacron:UFESORIGINALtese_6583_Dissertação Bianca de Paula e Rodrigues.pdfapplication/pdf1945250http://repositorio.ufes.br/bitstreams/2fe6a377-f183-497b-8763-b99b9e164c31/downloadb40ae223c37d2768fd970e0e1d00bd98MD5110/79832024-07-16 17:10:00.507oai:repositorio.ufes.br:10/7983http://repositorio.ufes.brRepositório InstitucionalPUBhttp://repositorio.ufes.br/oai/requestopendoar:21082024-10-15T17:59:41.681200Repositório Institucional da Universidade Federal do Espírito Santo (riUfes) - Universidade Federal do Espírito Santo (UFES)false
dc.title.none.fl_str_mv Efeito do sildenafil na genotoxicidade induzida pelo estresse oxidativo em camundongos ateroscleróticos
title Efeito do sildenafil na genotoxicidade induzida pelo estresse oxidativo em camundongos ateroscleróticos
spellingShingle Efeito do sildenafil na genotoxicidade induzida pelo estresse oxidativo em camundongos ateroscleróticos
Rodrigues, Bianca de Paula e
Atherosclerosis
Oxidative stress
DNA damage
Comet assay
ApoE-/- mice
Sildenafil
Camundongos apoE-/-
Aterosclerose
Estresse oxidativo
Dano ao DNA
Ensaio do cometa
Fisiologia
612
title_short Efeito do sildenafil na genotoxicidade induzida pelo estresse oxidativo em camundongos ateroscleróticos
title_full Efeito do sildenafil na genotoxicidade induzida pelo estresse oxidativo em camundongos ateroscleróticos
title_fullStr Efeito do sildenafil na genotoxicidade induzida pelo estresse oxidativo em camundongos ateroscleróticos
title_full_unstemmed Efeito do sildenafil na genotoxicidade induzida pelo estresse oxidativo em camundongos ateroscleróticos
title_sort Efeito do sildenafil na genotoxicidade induzida pelo estresse oxidativo em camundongos ateroscleróticos
author Rodrigues, Bianca de Paula e
author_facet Rodrigues, Bianca de Paula e
author_role author
dc.contributor.advisor1.fl_str_mv Vasquez, Elisardo Corral
dc.contributor.author.fl_str_mv Rodrigues, Bianca de Paula e
dc.contributor.referee1.fl_str_mv Campagnaro, Bianca Prandi
dc.contributor.referee2.fl_str_mv Santos, Leonardo dos
contributor_str_mv Vasquez, Elisardo Corral
Campagnaro, Bianca Prandi
Santos, Leonardo dos
dc.subject.eng.fl_str_mv Atherosclerosis
Oxidative stress
DNA damage
Comet assay
ApoE-/- mice
topic Atherosclerosis
Oxidative stress
DNA damage
Comet assay
ApoE-/- mice
Sildenafil
Camundongos apoE-/-
Aterosclerose
Estresse oxidativo
Dano ao DNA
Ensaio do cometa
Fisiologia
612
dc.subject.por.fl_str_mv Sildenafil
Camundongos apoE-/-
Aterosclerose
Estresse oxidativo
Dano ao DNA
Ensaio do cometa
dc.subject.cnpq.fl_str_mv Fisiologia
dc.subject.udc.none.fl_str_mv 612
description It is well known that enhanced production of reactive oxygen species (ROS) leads to oxidative stress observed in atherosclerosis and that ROS can also cause damage in cellular macromolecules, including DNA. Considering previous report that sildenafil, an inhibitor of phosphodiesterase 5 (PDE5), have antioxidant effects, in the present study we evaluated the effect of this drug on genotoxicity of blood mononuclear cells (MNC) and liver cells from atherosclerotic apolipoprotein E knockout mice (apoE-/- ). To accelerate and aggravate the spontaneous hyperlipidemia and atherosclerosis in apoE−/− mice, 8-week-old animals were fed a Western-type diet. Animals were randomly distributed into three different groups: (a) apoE−/− mice administered with the PDE5 inhibitor sildenafil (apoE−/− sildenafil, Viagra® , 40 mg/kg/day, for 3 weeks, by oral gavage, n=18), (b) apoE-/- mice administered with vehicle (apoE-/- vehicle, n=14) and (c) Wild-type C57Black/6 mice (WT, n=16). Then, animals (18-week-old) were euthanized and MNC isolated by density gradient and liver cells extracted by enzymatic action. Oxidative stress was determined by superoxide anion (•O2 - ) production using DHE probe. 106 cells were diluted with 1mL PBS and incubated with 160 μM DHE for 30 min at 37ºC in the dark, then washed and resuspended in 0.5ml of PBS-iFBS. A FACSCanto II cytometer was used for the flow cytometric analysis. Measurements were performed in triplicate and 10,000 events were used for each measurement. The genotoxicity analysis was performed using the alkaline comet assay that consists to obtain from individual cells, an array with a fluorescent halo in the format of a comet corresponding to the quantity of the fragmented DNA. The DNA helices ruptures extensions were evaluated by the image intensification method, where 100 cells were randomly selected (50 in each slide) using the analysis program CASP. The measurements of the comets were obtained by the parameters of the tail moment and percentage tail DNA. In addition, comets with more than 25% DNA in the tail (damage level moderate-high) were quantified between groups. Data are expressed as mean±SEM. Statistical analysis were performed by one-way ANOVA followed by Bonferroni post hoc test (p<0.05). Flow cytometric analysis showed a significant increase (78%) in superoxide anion (•O2 - ) production in apoE-/- vehicle mice MNC compared to WT and treatment with sildenafil decreases the levels of •O2 - in animals apoE-/- . In the DNA damage analysis all parameters used (%tail DNA, tail moment and number of comets with damage level moderate-high) showed an increase in the DNA fragmentation in both MNC and liver cells of animals apoE-/- vehicle when compared to WT control, and sildenafiladministered apoE-/- mice exhibited minimal DNA damage in those cells similar to WT mice. Our data shows that atherosclerosis increases ROS production leading to oxidative stress. Apparently, increased ROS interact with the DNA leading to fragmentation and, interestingly, the treatment with sildenafil was able to reduce ROS production inhibiting DNA damage. The novelty of this study is that sildenafil may offer a new perspective to the use of PDE5 inhibitors to protect against DNA damage, in cells involved in the inflammatory and dyslipidemic processes that accompany atherosclerosis.
publishDate 2013
dc.date.issued.fl_str_mv 2013-05-24
dc.date.accessioned.fl_str_mv 2018-08-01T22:58:44Z
dc.date.available.fl_str_mv 2018-08-01
2018-08-01T22:58:44Z
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dc.publisher.none.fl_str_mv Universidade Federal do Espírito Santo
Mestrado em Ciências Fisiológicas
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dc.publisher.department.fl_str_mv Centro de Ciências da Saúde
publisher.none.fl_str_mv Universidade Federal do Espírito Santo
Mestrado em Ciências Fisiológicas
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