O experimento foi conduzido na FZEA/USP com objetivo de comparar a utilização de fontes orgânicas de selênio (Se) com o selenito de sódio na dieta de cordeiros, pela análise da concentração de Se nos tecidos, da atividade da enzima glutationa peroxidase no fígado, do balanço metabólico e do cálculo da biodisponibilidade. Foram utilizados 40 cordeiros Suffolk, os quais foram submetidos a três fontes e três níveis de Se suplementar por 84 dias. Os tratamentos foram: tratamento 1: sem suplementação; tratamentos 2, 3 e 4: 0,2; 0,8 e 1,4 mg/kg de Se suplementar na forma de selenito de sódio; tratamentos 5, 6 e 7: 0,2; 0,8 e 1,4 mg/kg de Se suplementar na forma de Se-levedura; tratamentos 8, 9 e 10: 0,2; 0,8 e 1,4 mg/kg de Se suplementar na forma de Se-metionina. Foram colhidas amostras de sangue para dosagem sérica de Se e ao final do experimento, os animais foram abatidos para colheita de amostras de fígado, músculo e rim, para determinação dos teores de Se e da atividade da glutationa peroxidase (fígado). Nos últimos cinco dias de experimento foi realizado um balanço metabólico de Se. A biodisponibilidade foi calculada através da técnica “slope ratio", utilizando como parâmetros a concentração de selênio no fígado, músculo, rim e a atividade da glutationa peroxidase. Não houve efeito da fonte de Se utilizada na ingestão, absorção aparente e retenção de Se, atividade da glutationa peroxidase e nas concentrações de selênio no fígado, rim e soro; porém, as concentrações de selênio no músculo foram maiores nos animais suplementados com fontes orgânicas do que nos outros animais (P<0,0001). A biodisponibilidade de Se no músculo foi maior quando foram utilizadas fontes orgânicas de selênio. O uso de fontes orgânicas de Se promove maior acúmulo de Se no músculo de cordeiros e promoveria maior ingestão de Se por consumidores de carne ovina. / This research was carried out at FZEA/USP to compare the utilization of organic selenium (Se) sources to sodium selenite in lambs’ diet, through analyses of tissues Se concentrations, liver glutathione peroxidase activity, metabolic balance of Se and bioavailability assay. Forty Suffolk lambs were used and submitted to three sources and three levels of supplementary Se for 84 days. Treatments were: treatment 1: no supplement; treatments 2, 3 and 4: 0.2, 0.8 and 1.4 mg/kg of supplementary sodium selenite-selenium; treatments 5, 6 and 7: 0.2, 0.8 and 1.4 mg/kg of supplementary selenoyeast-selenium; treatments 8, 9 and 10: 0.2, 0.8 and 1.4 mg/kg of supplementary selenomethionine-selenium. Blood samples were taken to Se serum dosage and in the end of the experiment the animals were killed and samples of liver, muscle and kidney were taken to Se concentrations dosage and glutathione peroxidase activity (liver). In the last five days of the experiment, a Se metabolic balance was realized. Bioavailability was calculated by “Slope Ration Assay", using liver, muscle and kidney Se concentrations and glutathione peroxidase activity as parameters. Se sources did not affect Se intake, apparent absorption and retention, glutathione peroxidase activity and Se concentrations in liver, kidney and serum; however, selenium concentrations in muscle of animals supplied with organic sources were higher than in other animals (P<0.0001). Se bioavailability in muscle was higher when organic Se sources were used. Using organic Se sources provides higher accumulation of Se in lambs’ muscle which would provide higher Se intake to consumers of sheep meat.

O objetivo deste estudo foi avaliar o estado nutricional relativo ao selênio em indivíduos adultos relacionando-o com polimorfismos no gene da enzima GPx e sua influência sobre a enzima e o balanço redox. Foram selecionados 343 estudantes da Universidade Federal do Piauí entre 20 e 50 anos, de ambos os sexos, selecionados de acordo com critérios de inclusão adotados, como ausência de doenças crônicas não transmissíveis (DCNT) dentre outros. Sangue venoso foi coletado para análise de Se, genotipagem dos SNP da GPx1 (Pro198Leu, -602A/G e Arg5Pro), da atividade das enzimas antioxidantes (GSH-Px e SOD) eritrocitárias, malondialdeído (MDA) e capacidade de absorção de radicais de oxigênio (ORAC) plasmáticas. A análise de Se foi realizada por meio de espectrometria de absorção atômica por geração de hidretos, a genotipagem por PCR em tempo real em Step One Plus, as enzimas em analisador bioquímico automático utilizando kits comerciais, o MDA em cromatografia líquida de alta eficiência e ORAC em um leitor de microplaca. A análise estatística foi realizada por meio do software R 3.0.2. Foram realizados testes de comparação de duas e de mais que três médias entre as variáveis genéticas e os parâmetros de avaliação do Se e do balanço redox. Análises de regressão linear e linear generalizada foram realizadas para identificar a influência das variáveis genéticas, antropométricas, do perfil lipídico e estilo de vida sobre o Se sanguíneo e as variáveis do balanço redox. Os dados foram considerados significativos com p menor que 5%. A idade média dos participantes foi de 24,4±5,0 anos sendo 57,7% do sexo feminino. Entre os participantes, 95,7% eram carreadores do alelo Leu do SNP Pro198Leu e G do -602A/G e não possuíam quantidades mínimas de Se plasmático para otimizar a atividade da GPx. A atividade da GPx foi significativamente mais baixa e de ORAC mais alta nos indivíduos com o genótipo Leu/Leu em relação ao Pro/Pro do SNP Pro198Leu. Os indivíduos com o genótipo Arg/Pro apresentaram atividade da GPx significativamente maior que aqueles com o genótipo Arg/Arg do SNP Arg5Pro. Não houve diferença significativa entre as médias de MDA e SOD e os genótipos dos três SNP. As variáveis genéticas, de avaliação antropométrica, do perfil lipídico e estilo de vida mostraram influência sobre os marcadores do balanço redox, alterando o perfil antioxidante dos participantes. Os indivíduos são deficientes em Se e aqueles com o alelo Leu do SNP Pro198Leu apresentam em seu organismo maior concentração de ORAC, provavelmente para proteção contra radicais livres. O alelo variante do SNP Arg5Pro mostrou-se benéfico para o estado nutricional relativo ao Se. / The aim of this study was to evaluate the nutritional status relative to Se of adult individuals relating it to polymorphisms in the GPx enzyme gene and its influence on the enzyme and the redox balance. We selected 343 students of the Federal University of Piauí between 20 and 50 years, of both genders, selected according to inclusion criteria, such as the absence of chronic noncommunicable diseases (NCD) among others. Venous blood was collected for analysis of Se, genotyping of SNP of GPx1 (Pro198Leu, -602A / G and Arg5Pro), the activity of antioxidant enzymes (GSH-Px and SOD) erythrocyte, malondialdehyde (MDA) and absorption capacity of plasma oxygen radicals (ORAC). The analysis of Se was performed by atomic absorption spectrometry through hydride generation, genotyping by real time PCR in Step One Plus, enzymes in automatic biochemical analyzer by the use of commercial kits, MDA analysis was conducted in high-performance liquid chromatography and ORAC in a microplate reader. Statistical analysis was obtained using the software R version 3.0.2. Comparison tests were performed with two and more than three averages between the genetic variables and the evaluation parameters from Se and redox balance. Linear regression analysis and generalized linear were carried out to identify the influence of genetic variables, anthropometric, lipid profile and lifestyle on the sanguine Se and the variables of the redox balance. Data were considered significant for p less than 5%. The average age of participants was 24.4±5.0 years and 57.7% were female. Among the participants, 95.7% were allele carriers Leu of SNP Pro198Leu and G of -602A/G, and they did not have minimal amounts of Se plasma to optimize the activity of GPx. The GPx activity was significantly lower and that of ORAC was higher in subjects with the Leu/Leu genotype compared to Pro/Pro of SNP Pro198Leu. Individuals with the Arg/Pro genotype had GPx activity significantly higher than those with genotype Arg/Arg of SNP Arg5Pro. There was no significant difference between the means of MDA and SOD of the genotypes of the three SNP. The genetic variables, anthropometric measurements, lipid profile and lifestyle showed influence on markers of redox balance by changing the antioxidant profile of the participants. Individuals are deficient in Se, and those with the Leu allele of SNP Pro198Leu present in their bodies highest concentration of ORAC, probably to protect against free radicals. The variant allele of the SNP Arg5Pro proved to be beneficial to the nutritional status of the Se.

Foi demonstrado o envolvimento das espécies reativas de oxigênio (ERO) na patogênese de algumas retroviroses, levando muitas vezes à progressão e/ou persistência das mesmas. No entanto, o papel das espécies reativas de oxigênio resultante da modulação do sistema antioxidante, e seu envolvimento na infecção de bovinos naturalmente infectados pelo VLEB, até o presente trabalho, não tinha sido ainda investigado. Desta forma, o presente estudo objetivou-se avaliar o envolvimento do estresse oxidativo na infecção pelo VLEB em bovinos naturalmente infectados, associado ou não ao estabelecimento da linfocitose persistente (LP). Assim, a avaliação do estresse oxidativo se deu pelo teor de malondialdeído, concentração de glutationa reduzida, atividade da glutationa peroxidase e da superóxido dismutase, e pela atividade antioxidante total; e a resposta imune pela quantificação das subpopulações de linfócitos, função fagocítica e produção intracelular de peróxido de hidrogênio de leuccitos polimorfonucleares, morte celular e proliferação linfocitária, além da avaliação hematológica e sorodiagnóstico da leucose enzoótica bovina (LEB). Os resultados do presente estudo apontaram para o envolvimento do estresse oxidativo na infecção pelo vírus da leucose enzoótica bovina, dado pela menor concentração de glutationa peroxidase e a tendência a menor atividade da superxido dismutase eritrocitárias nos animais infectados pelo VLEB, não podendo, contudo, ser associado à fase da infecção. Ademais, os marcadores de estresse oxidativo não apresentaram alterados nem sequer a atividade antioxidante total destes animais. Além disso, o presente trabalho apontou para menor proliferação de linfócitos e redução da apoptose de células CD5+ nos animais infectados pelo VLEB manifestando LP, corroborados pelo significante aumento da população de linfcitos B (CD21+), e dos principais co-marcadores das células infectadas, no caso células CD5+ e CD11b+, nestes animais. No entanto, não observou diferenças significativas na população de linfócitos T destes animais, nem sequer alteração da capacidade fagocítica e produção intracelular de peróxido de hidrogênio pela população de leucócitos polimorfonucleares nos animais infectados. / Abundant observations of multiple pathogenic interactions between reactive oxygen species (ROS) and the pathogenesis of some retroviruses have drawn attention to role of ROS on disease progression and/or persistence of infection. Therefore, the role of the oxidative stress resulted from the modulation of the antioxidant system in dairy cows naturally infected with BLV has not been studied yet. So, the present study tries to investigate the involvement of oxidative stress in dairy cows naturally infected with BLV associated or not with the persistent lymphocytosis (PL). Thus, the oxidative stress was evaluated by the malondialdehyde, reduced glutathione content, and by the activity of glutathione peroxidase and superoxide dismutase, and also total antioxidant activity. The immune response was also evaluated by the quantification of lymphocyte subsets, lymphocyte proliferation, polymorfonuclear leukocytes phagocytosis and intracellular hydrogen peroxide production, and cell death. The results of the present work point out to an involvement of oxidative stress in dairy cows naturally infected with BLV, given by a lower glutathione peroxidase activity and also a tendency towards lower superoxide dismutase activity, but it can be not associated with PL. Futhermore, the oxidative stress markers and also the total antioxidant status was not altered in infected animals. The present study also showed a lower lymphocyte proliferarion and apoptosis rates of CD5+ cells, strengthen by a higher B cells (CD21+) counts and also by a significant augment of CD5+ and CD11b+ positive cells that are often co-expressed by the infected cells. Although, no significant difference was observed in T cells counts. Moreover, the phagocytosis rates and the intracellular hydrogen peroxide production by the polymorphonuclear leukocytes are not altered in BLV infected dairy cows.

Introdução: A polipose nasal (PN) é considerada uma condição inflamatória crônica da mucosa da cavidade nasal e seios paranasais de etiologia não muito clara. Há poucos dados sobre alterações epiteliais em PN e sua relação com a ação dos radicais livres. Muitas doenças estão ligadas a danos causados por espécies reativas de oxigênio (ROS) e de nitrogênio (RNSS) e ocorrem de um desequilíbrio entre eles e antioxidantes, o que for maior atividade de espécies reativas, o que chamamos de estresse oxidativo. Objetivo: O objetivo principal deste estudo é avaliar o estresse oxidativo em pólipos removidos cirurgicamente em 3 grupos de pacientes com polipose nasal (com PN unicamente, PN associado à asma e PN associado à asma e intolerância ao ácido acetilsalicílico) a fim de elucidar possíveis diferenças no perfil redox nestes grupos. Material e Métodos: Cinquenta e nove pacientes com diagnóstico de polipose nasal foram divididos em três grupos clínicos: um grupo controle PN unicamente, um grupo asma (PN associado à asma) e um grupo Widal (PN associado à asma e intolerância ao ácido acetilsalicílico). Medição e Resultados Principais: Neste trabalho defesas enzimáticas (superóxido dismutase, consumo de peróxido de hidrogênio, glutationa peroxidase e glutationa S-transferase) e defesas não enzimáticas (glutationa total, nitritos e nitratos, vitamina C e E) foram analisados. Também foi realizada a medição de danos em lipídios (malondialdeído) e proteínas (carbonila). No grupo asma, o consumo de peróxido de hidrogênio, atividade da glutationa peroxidase, níveis de malondialdeído e vitamina E foram significativamente menores do que no grupo de controle. Também foi realizada a medição de danos em lipídios (malondialdeído) e proteínas (carbonila). No grupo Widal foram encontrados níveis significativamente maiores de glutationa e nitritos e nitratos em relação ao grupo controle. Não foram encontradas diferenças entre os grupos em relação ao nível de carbonila e glutationa, tamanho dos pólipos, atividades da superóxido dismutase e S-transferase. Conclusões: A classificação redox dos grupos de PN foi parcialmente alcançada. Os pólipos do grupo asma possuem alterações nas defesas enzimáticas relacionadas com o peróxido de hidrogênio e a peroxidação lipídica, enquanto pólipos do grupo Widal apresentaram alterações nos níveis de óxido nítrico e glutationa. / Introduction: Nasal polyposis (NP) is considered a chronic inflammatory condition of the mucosa of the nasal cavity and paranasal sinuses of etiology is not very clear. There are few data on epithelial changes in nasal polyposis and its relation to the action of free radicals. Many diseases are linked to damage caused by reactive oxygen species (ROS) and nitrogen (RNSs) and occur from an imbalance between them and antioxidants, whichever is greater activity of reactive species, what we call oxidative stress. Objective: The primary objective of this study is to evaluate oxidative stress in polyps surgically removed in 3 groups of patients with nasal polyposis, in order to elucidate possible differences in redox profile in these groups. Methods: Fifty nine patients diagnosed with nasal polyposis were divided into three groups: a control group, an asthma group (NP with asthma) and a Widal group (NP with asthma and aspirin intolerance) in which patients had an association of NP, asthma and aspirin intolerance. Measurement and main results: In this work enzymatic defenses (superoxide dismutase, hydrogen peroxide consumption, glutathione peroxidase and glutathione S-transferase) and non-enzymatic defenses (total glutathione, measurement of nitrites and nitrates, vitamin C and E) were analyzed. Also the measurement of damage in lipids (malondialdehyde) and proteins (carbonyl) was conducted. In the asthma group, hydrogen peroxide consumption, glutathione peroxidase activity, malondialdehyde, and vitamin E levels were significantly lower than in the control group. The Widal group showed significant higher glutathione levels, nitrite and nitrate levels than found in the control group. No differences were found among the groups regarding carbonyl level, polyp size, superoxide dismutase, and glutathione S-transferase activities. Conclusions: The redox classification of the groups of NP was partly achieved. Polyps of patients with asthma have changes in enzymatic defense pathways related to hydrogen peroxide and lipid peroxidation while polyps of patients with Widal triad present changes in nitric oxide and glutathione.

O objetivo deste estudo foi avaliar o estado nutricional relativo ao selênio em indivíduos adultos relacionando-o com polimorfismos no gene da enzima GPx e sua influência sobre a enzima e o balanço redox. Foram selecionados 343 estudantes da Universidade Federal do Piauí entre 20 e 50 anos, de ambos os sexos, selecionados de acordo com critérios de inclusão adotados, como ausência de doenças crônicas não transmissíveis (DCNT) dentre outros. Sangue venoso foi coletado para análise de Se, genotipagem dos SNP da GPx1 (Pro198Leu, -602A/G e Arg5Pro), da atividade das enzimas antioxidantes (GSH-Px e SOD) eritrocitárias, malondialdeído (MDA) e capacidade de absorção de radicais de oxigênio (ORAC) plasmáticas. A análise de Se foi realizada por meio de espectrometria de absorção atômica por geração de hidretos, a genotipagem por PCR em tempo real em Step One Plus, as enzimas em analisador bioquímico automático utilizando kits comerciais, o MDA em cromatografia líquida de alta eficiência e ORAC em um leitor de microplaca. A análise estatística foi realizada por meio do software R 3.0.2. Foram realizados testes de comparação de duas e de mais que três médias entre as variáveis genéticas e os parâmetros de avaliação do Se e do balanço redox. Análises de regressão linear e linear generalizada foram realizadas para identificar a influência das variáveis genéticas, antropométricas, do perfil lipídico e estilo de vida sobre o Se sanguíneo e as variáveis do balanço redox. Os dados foram considerados significativos com p menor que 5%. A idade média dos participantes foi de 24,4±5,0 anos sendo 57,7% do sexo feminino. Entre os participantes, 95,7% eram carreadores do alelo Leu do SNP Pro198Leu e G do -602A/G e não possuíam quantidades mínimas de Se plasmático para otimizar a atividade da GPx. A atividade da GPx foi significativamente mais baixa e de ORAC mais alta nos indivíduos com o genótipo Leu/Leu em relação ao Pro/Pro do SNP Pro198Leu. Os indivíduos com o genótipo Arg/Pro apresentaram atividade da GPx significativamente maior que aqueles com o genótipo Arg/Arg do SNP Arg5Pro. Não houve diferença significativa entre as médias de MDA e SOD e os genótipos dos três SNP. As variáveis genéticas, de avaliação antropométrica, do perfil lipídico e estilo de vida mostraram influência sobre os marcadores do balanço redox, alterando o perfil antioxidante dos participantes. Os indivíduos são deficientes em Se e aqueles com o alelo Leu do SNP Pro198Leu apresentam em seu organismo maior concentração de ORAC, provavelmente para proteção contra radicais livres. O alelo variante do SNP Arg5Pro mostrou-se benéfico para o estado nutricional relativo ao Se. / The aim of this study was to evaluate the nutritional status relative to Se of adult individuals relating it to polymorphisms in the GPx enzyme gene and its influence on the enzyme and the redox balance. We selected 343 students of the Federal University of Piauí between 20 and 50 years, of both genders, selected according to inclusion criteria, such as the absence of chronic noncommunicable diseases (NCD) among others. Venous blood was collected for analysis of Se, genotyping of SNP of GPx1 (Pro198Leu, -602A / G and Arg5Pro), the activity of antioxidant enzymes (GSH-Px and SOD) erythrocyte, malondialdehyde (MDA) and absorption capacity of plasma oxygen radicals (ORAC). The analysis of Se was performed by atomic absorption spectrometry through hydride generation, genotyping by real time PCR in Step One Plus, enzymes in automatic biochemical analyzer by the use of commercial kits, MDA analysis was conducted in high-performance liquid chromatography and ORAC in a microplate reader. Statistical analysis was obtained using the software R version 3.0.2. Comparison tests were performed with two and more than three averages between the genetic variables and the evaluation parameters from Se and redox balance. Linear regression analysis and generalized linear were carried out to identify the influence of genetic variables, anthropometric, lipid profile and lifestyle on the sanguine Se and the variables of the redox balance. Data were considered significant for p less than 5%. The average age of participants was 24.4±5.0 years and 57.7% were female. Among the participants, 95.7% were allele carriers Leu of SNP Pro198Leu and G of -602A/G, and they did not have minimal amounts of Se plasma to optimize the activity of GPx. The GPx activity was significantly lower and that of ORAC was higher in subjects with the Leu/Leu genotype compared to Pro/Pro of SNP Pro198Leu. Individuals with the Arg/Pro genotype had GPx activity significantly higher than those with genotype Arg/Arg of SNP Arg5Pro. There was no significant difference between the means of MDA and SOD of the genotypes of the three SNP. The genetic variables, anthropometric measurements, lipid profile and lifestyle showed influence on markers of redox balance by changing the antioxidant profile of the participants. Individuals are deficient in Se, and those with the Leu allele of SNP Pro198Leu present in their bodies highest concentration of ORAC, probably to protect against free radicals. The variant allele of the SNP Arg5Pro proved to be beneficial to the nutritional status of the Se.

O experimento foi conduzido na FZEA/USP com objetivo de comparar a utilização de fontes orgânicas de selênio (Se) com o selenito de sódio na dieta de cordeiros, pela análise da concentração de Se nos tecidos, da atividade da enzima glutationa peroxidase no fígado, do balanço metabólico e do cálculo da biodisponibilidade. Foram utilizados 40 cordeiros Suffolk, os quais foram submetidos a três fontes e três níveis de Se suplementar por 84 dias. Os tratamentos foram: tratamento 1: sem suplementação; tratamentos 2, 3 e 4: 0,2; 0,8 e 1,4 mg/kg de Se suplementar na forma de selenito de sódio; tratamentos 5, 6 e 7: 0,2; 0,8 e 1,4 mg/kg de Se suplementar na forma de Se-levedura; tratamentos 8, 9 e 10: 0,2; 0,8 e 1,4 mg/kg de Se suplementar na forma de Se-metionina. Foram colhidas amostras de sangue para dosagem sérica de Se e ao final do experimento, os animais foram abatidos para colheita de amostras de fígado, músculo e rim, para determinação dos teores de Se e da atividade da glutationa peroxidase (fígado). Nos últimos cinco dias de experimento foi realizado um balanço metabólico de Se. A biodisponibilidade foi calculada através da técnica “slope ratio”, utilizando como parâmetros a concentração de selênio no fígado, músculo, rim e a atividade da glutationa peroxidase. Não houve efeito da fonte de Se utilizada na ingestão, absorção aparente e retenção de Se, atividade da glutationa peroxidase e nas concentrações de selênio no fígado, rim e soro; porém, as concentrações de selênio no músculo foram maiores nos animais suplementados com fontes orgânicas do que nos outros animais (P<0,0001). A biodisponibilidade de Se no músculo foi maior quando foram utilizadas fontes orgânicas de selênio. O uso de fontes orgânicas de Se promove maior acúmulo de Se no músculo de cordeiros e promoveria maior ingestão de Se por consumidores de carne ovina. / This research was carried out at FZEA/USP to compare the utilization of organic selenium (Se) sources to sodium selenite in lambs’ diet, through analyses of tissues Se concentrations, liver glutathione peroxidase activity, metabolic balance of Se and bioavailability assay. Forty Suffolk lambs were used and submitted to three sources and three levels of supplementary Se for 84 days. Treatments were: treatment 1: no supplement; treatments 2, 3 and 4: 0.2, 0.8 and 1.4 mg/kg of supplementary sodium selenite-selenium; treatments 5, 6 and 7: 0.2, 0.8 and 1.4 mg/kg of supplementary selenoyeast-selenium; treatments 8, 9 and 10: 0.2, 0.8 and 1.4 mg/kg of supplementary selenomethionine-selenium. Blood samples were taken to Se serum dosage and in the end of the experiment the animals were killed and samples of liver, muscle and kidney were taken to Se concentrations dosage and glutathione peroxidase activity (liver). In the last five days of the experiment, a Se metabolic balance was realized. Bioavailability was calculated by “Slope Ration Assay”, using liver, muscle and kidney Se concentrations and glutathione peroxidase activity as parameters. Se sources did not affect Se intake, apparent absorption and retention, glutathione peroxidase activity and Se concentrations in liver, kidney and serum; however, selenium concentrations in muscle of animals supplied with organic sources were higher than in other animals (P<0.0001). Se bioavailability in muscle was higher when organic Se sources were used. Using organic Se sources provides higher accumulation of Se in lambs’ muscle which would provide higher Se intake to consumers of sheep meat.

Die Induktion antioxidativer Enzyme gilt als eine Möglichkeit, die antioxidative Kapazität von Zellen zu steigern und dadurch mit oxidativem Stress assoziierten Erkrankungen (z. B. Herz-Kreislauf-Erkrankungen, Neurodegeneration, Atherosklerose) vorzubeugen. Ausgehend davon wurde in der vorliegenden Arbeit der Dual-Luciferase-Reportergen-(DLR)-Assay zum Nachweis der Induktion der antioxidativen Enzyme Katalase (CAT), zytosolische Glutathion-Peroxidase (GPX1) und Kupfer-Zink-Superoxid-Dismutase (SOD1) entwickelt. Im Zuge dessen wurden drei Säugetierzelllinien (CaCo2, IEC-18, V79) auf ihre Eignung zur Modellzelllinie untersucht. Aufgrund der Transfektionseffizienz wurde die Fibroblastenzelllinie V79 ausgewählt. Zur Gewährleistung eines hohen Substanzdurchsatzes des DLR-Assays wurden bei der Etablierung Parameter wie Kulturplattenformat, DNA-Menge, Luciferasen-Kinetik berücksichtigt. Nach erfolgreicher Etablierung des Versuchs im 96-Well-Format wurden L-Carnitin, Catechin, Epigallocatechingallat, Genistein, Wasserstoffperoxid (H2O2), Natrium-Ascorbat, Paraquat, Quercetin, 12-O-Tetradecanoylphorbol-13-Acetat (TPA) und Trolox in nicht-zytotoxischen Konzentrationen hinsichtlich der Aktivierung des Ratten-CAT-, des humanen GPX1- und des humanen SOD1-Promotors untersucht. Die Bestimmung der maximal tolerierbaren Behandlungskonzentration erfolgte im Vorfeld mittels Resazurintest. Von den zehn Verbindungen zeichneten sich drei Substanzen als potente Induktoren für die SOD1 und die GPX1 aus. Die 24-stündige Behandlung von mit Reportergenkonstrukten transient transfizierten V79-Zellen mit 100 µM Paraquat resultierte in einer Verdopplung der relativen SOD1-Promotor-Aktivität und einer Erhöhung der relativen GPX1-Promotor-Aktivität auf 1,6 bzw. 1,7. Die Stimulation mit 20 µM Genistein oder 10 µM Quercetin führte wiederum zu einer Verdopplung bis Verdreifachung der relativen SOD1- und GPX1-Promotor-Aktivität. Der Promotor der Rattenkatalase konnte demgegenüber nur durch 50 µM H2O2 aktiviert werden (1,5fach). Für diesen DLR-Assays bieten sich folglich Genistein, Quercetin wie auch H2O2 als Referenzsubstanzen an. Um aber eine qualitative Charakterisierung der einzelnen Verbindungen hinsichtlich ihres Induktionspotentials zu gewährleisten, sollten von allen getesteten Substanzen Dosis-Wirkungskurven aufgenommen werden. Zudem wird für den routinemäßigen Einsatz die Verwendung stabil transfizierter Zellen zur Vermeidung von mit der Transfektion verbundenen experimentellen Schwankungen empfohlen. / The induction of antioxidative enzymes might be an opportunity to elevate the cellular antioxidative capacity and, thus, to prevent oxidative stress associated diseases (e. g. cardio-vascular disease, neurodegenerative disease, atherosclerosis). Based on this idea the dual luciferase reporter gene (DLR) assay was developed to demonstrate the induction of three antioxidative enzymes: catalase (CAT), cytosolic glutathione peroxidase (GPX1), and copper-zinc superoxide dismutase (SOD1). In the course of the development three mammalian cell lines (CaCo2, IEC-18, V79) were tested for their ability to serve as a model cell line. The line V79 was chosen due to the transfection efficiency. To give consideration to a high-throughput several parameters were studied (e. g. format of the cultural plates, amount of DNA, kinetics of the luciferases) and the DLR assay was successfully established in 96 well plates. Subsequently, L-carnitine, catechin, epigallocatechin gallate, genistein, hydrogen peroxide (H2O2), sodium ascorbate, paraquat, quercetin, 12-O-tetradecanoylphorbol-13-acetate (TPA) and trolox were tested in non-cytotoxic concentrations for the activation of the rat CAT, human GPX1 and human SOD1 promoter. The maximally tolerable concentrations were determined by resazurin test in advance. Three out of these ten compounds were identified as potent inducers of GPX1 and SOD1. Stimulation of reporter gene construct transient transfected V79 cells for 24 hours with 100 µM paraquat caused a duplication of the relative GPX1 promoter activity and a 1.6-/1.7-fold increase of the relative SOD1 promoter activity. The incubation with 20 µM gen-istein or 10 µM quercetin resulted in duplication to triplication of both, the relative GPX1 and SOD1 promoter activity. In contrast, the rat CAT promoter was activated by 50 µM H2O2 (1.5-fold). Consequently, genistein, quercetin, and H2O2 are considered to be suitable reference substances for this DLR assay. To further characterize the inducing potential of the tested compounds all of them should be tested in different concentrations. Furthermore, for the routinely performed DLR assay it is recommended to use stably transfected cells to eliminate transfection caused variations.

Bei der Entdeckung der Glutathionperoxidase-2 (GPx2) wurde zunächst davon ausgegangen, dass die Funktion dieses Enzyms im Kryptengrund des Colons einzig in der Reduktion von H2O2 besteht. Im Laufe der weiteren Erforschung zeigte sich, dass GPx2 auch in verschiedenen Tumorgeweben vermehrt exprimiert wird. Dabei wird diskutiert, ob die Wirkung von GPx2 im Tumor eher als pro- oder als antikarzinogen einzustufen ist. Mehrere Experimente in vitro und in vivo zeigten antiinflammatorische Eigenschaften der GPx2. Aufgrund dieser Befunde wird derzeit über weitere Funktionen der GPx2 spekuliert. In dieser Arbeit wurde die physiologische Funktion von GPx2 näher erforscht, dazu wurden Wildtyp- und GPx2-Knockout-Mäuse in Hinblick auf Veränderungen der Enzymexpression und der Colonmorphologie untersucht. Es wurden drei verschiedene Selendiäten verfüttert: selenarmes, selenadäquates und selensupplementiertes Futter. Unter physiologischen Bedingungen ist am Kryptengrund des Colons, innerhalb der proliferierenden Zone, die Mitoserate am höchsten. Der Großteil der apoptotischen Zellen ist hingegen an der Kryptenspitze vorzufinden. Durch den Knockout von GPx2 kam es zu einer signifikanten Erhöhung der Apoptoserate am Kryptengrund. Dabei war der größte Effekt auf selenarmem Futter zu verzeichnen. Hierbei wurde sogar eine Veränderung der Colonmorphologie dokumentiert, da die Verschiebung der Proliferationszone in Richtung Kryptenspitze eine Verlängerung der Krypten nach sich zog. Im Wildtyp wurden keine Apoptosen im Kryptengrund detektiert. GPx1 wird unter physiologischen Bedingungen im Gegensatz zur GPx2 in der Kryptenspitze exprimiert und ist im Selenmangel nicht mehr detektierbar. Der Knockout von GPx2 erhöhte die GPx1-Expression im Kryptengrund auf allen drei Selendiäten. Diese Überexpression von GPx1 am Kryptengrund soll vermutlich den Verlust von GPx2 an dieser Stelle kompensieren. Da jedoch dort die massive Apoptoserate detektiert wurde, kann die GPx1 nicht die komplette Funktion von GPx2 kompensieren. Diese Ergebnisse deuten darauf hin, dass die Funktion von GPx2 nicht nur in der Reduktion von H2O2 liegt. Vielmehr kann eine Rolle bei der Aufrechterhaltung der Homöostase von Zellen postuliert werden. Ein weiterer Bestandteil dieser Arbeit war die Klärung der Frage, welchen Einfluss GPx2 auf die entzündungsassoziierte Colonkarzinogenese ausübt. In dem hierfür verwendeten AOM/DSS-Model wird der karzinogene Prozess durch Entzündung vorangetrieben. Es erfolgte sowohl im Wildtyp als auch im GPx2-Knockout zum einen die Bewertung des Entzündungsstatus des Colons und zum anderen wurde die Anzahl von ACF und Tumoren verglichen. Das Colon im GPx2-Knockout war wesentlich stärker entzündet als im Wildtyp. Diese Ergebnisse bestätigen die für die GPx2 postulierte antiinflammatorische Funktion. Normalerweise führt eine Erhöhung der Mitoseanzahl zur Regeneration des entzündeten Gewebes. Jedoch beeinflusst der Verlust von GPx2 vermutlich den Ablauf der Entzündung, indem beispielsweise die Regeneration des Gewebes durch die enorm hohe Apoptoserate am Kryptengrund verlangsamt wird. Des Weiteren hatten sich im GPx2-Knockout tendenziell mehr Tumore entwickelt. Somit korrelierte die Entzündung des Colons mit der Entwicklung von Tumoren. Der Verlust von GPx2 begünstigte vermutlich sowohl die Tumorinitiation als auch die Tumorprogression. Allerdings stimulierte die Expression von GPx2 ebenfalls das Tumorwachstum. Es kann geschlussfolgert werden, dass eine adäquate GPx2-Expression vor Entzündung schützt und somit das Risiko für Colonkrebs senkt. Ob GPx2 aber insgesamt pro- oder antikarzinogen wirkt, hängt vermutlich vom Stadium des Colonkarzinogenese ab. / Since the detection of glutathione peroxidase-2 (GPx2) it was assumed that reducing hydroperoxides is the only function of this enzyme in the crypt ground of the colon. But further studies showed that GPx2 is also highly expressed in tumor tissue. However, it is not known whether it acts a pro- or anticarcinogenic manner at this site. In vitro and in vivo experiments elucidate antiinflammatory features of GPx2, based on these findings additional functions of GPx2 are discussed. In this dissertation the physiological function of GPx2 was investigated. For this purpose in wild type and GPx2-knockout mice, changes of enzyme expression and colon morphology were analyzed. The mice were fed three diets containing different selenium concentrations: selenium deficient, selenium adequate and selenium supplemented. Under physiological conditions the mitosis rate is highest in the proliferating zone in the crypt ground of the colon. The majority of apoptotic cells are located at the tip of the crypt. The knockout of GPx2 significantly increased the rate of apoptosis in the crypt ground. The greatest effect was documented on the selenium deficient diet. Here, changes of the colonic morphology were detectable, because the shift of the proliferating zone towards the tip of the crypt lead to an extension of the crypts. In the wild type mice no apoptotic cells were detected on the crypt ground. Under physiological conditions GPx1, in contrast to GPx2, is mainly expressed on the top of the crypt, and this enzyme is no longer detectable under selenium deficiency. The knockout of GPx2 increased the expression of GPx1 in the crypt ground of the colon on all three selenium diets. It is likely that this over expression of GPx1 compensates for the loss of GPx2. However the massive apoptotic rate in the crypt ground shows that GPx1 can not compensate the complete function of GPx2. These results elucidate that GPx2 not only functions as a hydroperoxide reducer, but that it is also important for the maintenance of the stem cell character and the homeostasis of cells. The question if GPx2 influences the inflammation triggered by the coloncarcinogenic process was next assessed in this dissertation. Therefore the AOM/DSS model was used to trigger the carcinogenic process through inflammation. The amount of aberrant crypt foci (ACF) and tumors in the colon were analyzed in both wild type and GPx2-knockout mice. However initially the inflammation status was compared between the two genotypes. The inflammation of the colon was stronger in the GPx2-knockout mice than in wild type. These results support the postulated antiinflammatory features of GPx2. The loss of GPx2 may influence the inflammation process by decelerating the regeneration of the tissue caused by the increased apoptotic rate in the proliferating zone. Additionally, the GPx2-knockout mice developed more tumors in the colon. Therefore the inflammation of the colon correlated with the development of tumors. The loss of GPx2 may have enhanced both tumor initiation and progression. But the expression of GPx2 also stimulated the growth of tumors. These results indicate that an adequate GPx2-expression can protect from colonic inflammation, and therefore decrease the risk of developing colon cancer. Whether GPx2 acts in a pro- or anticarcinogenic manner appears to depend on the state of the carcinogenic process.

The study aimed to investigate the following: 1. Investigate a putative glutathione peroxidase gene (Glyma17g34110) within Glycine max by an in silico analysis and spatial expression. 2. Determine the effects of exogenously applied nitric oxide on the expression of Glyma17g34110. 3. Investigate the antioxidant mechanism with attention to Glyma17g34110,reactive oxygen species and cell death in the response to salt stress. 4. Establish whether Glyma17g34110 is a glutathione peroxidase or thioredoxindependent peroxidase gene. / Magister Scientiae - MSc

The production of reactive oxygen species (ROS) is prominent in all aerobic metabolisms including plants. For this reason, the redox homeostasis of the production and scavenging of these intermediates is imperative for growth, development and survival during unfavourable conditions. In this study, a putative glutathione peroxidase gene (Glyma17g34110) from Glycine max (soybean) was identified and analyzed. The successful characterisation of Glyma17g34110 provided evidence of it being a glutathione peroxidase using glutathione as its preferred electron donor and substrate. Furthermore, it is known that antioxidant enzymes such as GPX exist in various tissues, performing a diverse set of functions. By a bioinformatic analysis of Glyma17g34110 and its promoter region, it was indicated that Glyma17g34110 could be a putative chloroplast protein that could play an important role in photosynthesis.One of the major factors affecting plant growth and development worldwide is abiotic stresses such as salinity. In the presence of salinity the production of harmful ROS is increased, resulting in detrimental reactions with important biological features (DNA, protein and lipid membranes), leading to cell death. The analysis of Glyma17g34110 under salt stress revealed that it is a salt sensitive gene and thus, the down-regulation of Glyma17g34110 could be due to the lack of known defence and response cis-acting elements present in the promoter region. Furthermore, it was proven in previous studies that the application of exogenous nitric oxide (NO) increases the activity of antioxidant enzymes. In this thesis it was observed that the presence of exogenously applied NO increased the expression of Glyma17g34110 tremendously in all soybean tissues (leaves, roots and nodules) investigated.Studies have found numerous cis-acting elements to be NO responsive, however, none of these elements were found in the promoter region upstream of glyma17g34110. This suggests that novel cis-acting elements could be present in the promoter region of Glyma17g34110.Thus, increasing the expression of Glyma17g34110 during salinity in the presence of NO, as well as the identification of these novel cis-acting elements, could lead to the enhancement of the defence mechanisms against ROS, which could lead to increasing plant tolerance to stress. / >Magister Scientiae - MSc