A cisplatina (cis-diaminodicloroplatina II) é um efetivo agente anticâncer, porém seu uso clínico é altamente limitado, predominantemente devido à sua nefrotoxicidade. Muitos estudos têm demonstrado que a cisplatina causa disfunção mitocondrial em células epiteliais renais devido à ação de espécies reativas de oxigênio tais como ânions superóxido e radicais hidroxila. A proteção seletiva das mitocôndrias renais contra espécies reativas de oxigênio geradas pela cisplatina é fundamental na quimioterapia de pacientes com câncer. Vários estudos têm sugerido que o carvedilol é capaz de proteger contra a toxicidade mitocondrial cardíaca induzida pelo quimioterápico doxorubicina. Assim, no presente estudo investigou-se o potencial protetor deste fármaco contra a toxicidade mitocondrial renal induzida pela cisplatina, bem como os mecanismos moleculares envolvidos nesta proteção. Foram estudados 4 grupos (n=6, cada) de ratos Wistar machos tratados da seguinte forma: (i) Grupo controle: uma injeção intraperitoneal (i.p.) de DMSO (0,2mL/200g, i.p.) imediatamente antes da injeção de solução salina isotônica (2 ml/200g, i.p.) e posteriormente uma injeção diária de DMSO (0,2mL/200g, i.p.) em dois dias consecutivos; (ii) Grupo cisplatina (CISP): uma injeção de cisplatina (10 mg/kg, i.p.); (iii) Grupo carvedilol (CV): uma injeção de carvedilol (1 mg/kg, i.p.), seguida de uma injeção diária de carvedilol em dois dias consecutivos (1 mg/Kg, i.p) e (iv) Grupo carvedilol + cisplatina (CV+CISP): uma injeção de carvedilol (1mg/kg, i.p.), imediatamente antes da injeção de cisplatina (10 mg/Kg, i.p.) seguida de uma injeção diária de carvedilol nos dois dias seguintes (1 mg/Kg, i.p.). Os animais foram sacrificados 72 horas após o início do tratamento. O grupo CV+CISP apresentou uma significativa redução na lesão renal, marcada pela diminuição da concentração de uréia e creatinina plasmáticas, quando comparado ao grupo CISP. A avaliação da função mitocondrial comprovou o efeito protetor do carvedilol contra a toxicidade mitocondrial renal da cisplatina através da significativa melhora nos valores da (i) RCR, (ii) do consumo de oxigênio no estado 3 e (iii) da razão ADP/O. Além disso, no grupo CV+CISP o potencial de membrana mitocondrial e a captação de cálcio mitocondrial foram preservados. Adicionalmente, a redução da oxidação do NADPH, da cardiolipina, da glutationa e das proteínas sulfidrilas, bem como a redução na formação de MDA no grupo CV+CISP sugerem efeito protetor do carvedilol contra o estresse oxidativo mitocondrial. O grupo CV+CISP também apresentou menor ativação da caspase-3, o que sugere menor indução de apoptose. Os grupos CISP e CV+CISP apresentaram concentrações semelhantes de platina na suspensão mitocondrial renal, indicando que o mecanismo de proteção do carvedilol provavelmente não envolve a formação de complexos e a subseqüente inativação da cisplatina. Os resultados do presente estudo são promissores, pois o carvedilol é um fármaco de uso seguro e já estabelecido na clínica e a comprovação do seu efeito protetor contribuirá para o desenvolvimento de novas estratégias de prevenção dos danos nefrotóxicos da cisplatina. / Cisplatin (cis-diamminedichloridoplatinum II) is an affective anticancer agent; however its clinical use is highly limited, predominantly due to its nephrotoxicity. Many studies have shown that cisplatin cause mitochondrial dysfunction in renal epithelial cells due to the generation of reactive oxygen species, such as superoxide anions and hydroxyl radicals. The selective protection of the renal mitochondria against the reactive oxygen species generated by cisplatin is of critical importance in the chemotherapy of cancer patients. Some studies have suggested that carvedilol can protect against the cardiac mitochondrial toxicity induced by the chemotherapeutic agent doxorubicin. Therefore, in the present study we investigated the protective effect of carvedilol against renal mitochondrial toxicity, as well as the molecular mechanisms involved in this protection. We studied 4 groups (n=6, each) of male Wistar rats treated as follows: (i) Control group: one injection of DMSO (0,2 mL/200g body weight, i.p.), intraperitoneal injection (i.p.), immediately before the injection of isotonic saline solution (2mL/200g body weight) followed by one injection of DMSO (0,2 mL/200g body weight, i.p.) in the two following days; (ii) Cisplatin group (CISP): one injection of cisplatin (10 mg/kg body weight, i.p.); (iii) Carvedilol group (CV): one injection of carvedilol (CV) (1mg/kg body weight, i.p.) in three consecutive days and (iv) Carvedilol + Cisplatin group (CV+CISP): one injection of carvedilol (1mg/kg, body weight, i.p.) immediately before the injection of cisplatin (10mg/kg, body weight, i.p.), followed by one injection of carvedilol (1mg/kg, body weight, i.p.) in the two following days. Animals were killed 72h after the beginning of the treatment. CV+CISP group presented a significantly reduced renal injury, marked by the decrease of urea and creatinine plasmatic levels, as compared to the CISP group. The evaluation of the mitochondrial function showed the protective effect of carvedilol against the renal mitochondria toxicity induced by cisplatin, as demonstrated by the improvement in the values of (i) RCR; (ii) the oxygen consumption on state 3 respiration and ADP/O ratio. Besides that, in the CV+CISP group the mitochondrial membrane potential and the mitochondrial calcium uptake were preserved. Additionally, the lower oxidation of NADPH, cardiolipin, glutathione and sulfhydryl proteins, as well as the lower values of MDA in the CV+CISP group, suggests a protective effect of carvedilol against the mitochondrial oxidative stress. CV+CISP group also presented lower values of caspase 3, which suggests lower induction of apoptosis. The groups CISP and CV+CISP presented a similar platinum concentration in the mitochondrial suspension, which indicates that the protective mechanism of carvedilol probably does not involve complex formation with cisplatin and its ensuing inactivation. The present results are promising, since carvedilol is a safe drug, which is currently used in the clinical practice and the evidence of its protective effect will contribute to the development of new strategies to prevent the nephrotoxic damage of cisplatin.

A produção de espécies reativas de oxigênio (ROS) nos sistemas biológicos é contrabalanceada pelos sistemas antioxidantes enzimáticos e não-enzimáticos. Quando há um desequilíbrio entre a geração de ROS e esses sistemas, ocorre um aumento dessas espécies reativas causando estresse oxidativo, que pode levar a danos a macromoléculas, como lipídios, proteínas e o DNA. Os fármacos doxorrubicina (antineoplásico) e benzonidazol (antiparasitário) são conhecidos por induzir efeitos colaterais que podem estar relacionados ao aumento de ROS. Além disso, ensaios de mutagenicidade demonstram que esses fármacos apresentam atividade mutagênica por danos oxidativos. O Grupo NEQUIMED desenvolve substâncias com potencial atividade antineoplásica e antiparasitária, as quais ainda não foram avaliadas em relação às propriedades tóxicas e genotóxicas. Dessa forma, o objetivo deste trabalho foi analisar as propriedades mutagênicas e de estresse oxidativo dessas novas substâncias, comparando aos fármacos benzonidazol e doxorrubicina. Para detecção de ROS foi realizado o ensaio fluorimétrico utilizando o marcador 2,7-diacetato de diclorofluoresceína (DCFH-DA) em linhagens celulares de hepatocarcinoma humano (HepG2) e fibroblasto de camundongo (Balb/C 3T3 clone A31). O estado redox destas células foi avaliado através da quantificação da expressão gênica e do conteúdo proteico das enzimas antioxidantes através das técnicas de qRT-PCR e Western blot, respectivamente. A atividade mutagênica foi analisada com o ensaio Ames miniaturizado Salmonella/microssoma utilizando a linhagem TA102 de Salmonella typhimurium que detecta agentes mutagênicos que causam danos por oxidação. Os resultados mostraram que as substâncias estudadas pelo Grupo não induzem aumento na produção de ROS ou induzem em menores níveis do que doxorrubicina e benzonidazol, além de levar a alterações menos proeminentes que os fármacos para a expressão das proteínas antioxidantes. No ensaio mutagênico, o benzonidazol apresentou o pior perfil, doxorrubicina e Neq0438 somente foram mutagênicos com ativação enzimática, enquanto Neq0551 foi inativo. Assim, as novas substâncias (Neq0438 e Neq0551) apresentaram um perfil melhor do que os fármacos de referência, tornando-os candidatos promissores para estudos in vitro e in vivo subsequentes. / The production of reactive oxygen species (ROS) in biological systems is compensated by the enzymatic and non-enzymatic antioxidant systems. The excessive ROS production causes oxidative stress, which can damage important cellular macromolecules such as lipids, proteins and DNA. The drugs doxorubicin (antineoplastic) and benzonidazole (antiparasitic) are both known for their side effects, which can be related to the increase of ROS. Besides, mutagenicity assays show that these drugs have a mutagenic activity via oxidative damages. The research group NEQUIMED studies new substances with potential antineoplastic and antiparasitic activities, but their toxic and genotoxic properties have not been fully evaluated yet. Thus, the aim of this work is to assess the mutagenic potential and the oxidative stress generated by these substances, comparing them to benzonidazole and doxorubicin. The fluorimetric assay using the probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) was used for ROS detection in human hepatocarcinoma (HepG2) and mouse fibroblast (Balb/C 3T3 clone A31) cell lines. The redox state of these cells was evaluate by qRT-PCR and Western blot methods to quantifying gene expression and protein content of the antioxidant enzymes. The mutagenic potential was assessed by the miniaturized Ames text with the Salmonella/microssome mutagenicity assay, using the TA102 strain of Salmonella typhimurium, which detects oxidation damages to the DNA. The new substances did not induce an increase on ROS production, or did in lower levels when compared to doxorubicin and benzonidazole. Moreover, reference drugs also induced greater changes on the expression of the antioxidant enzymes. Benznidazole had a higher mutagenic activity, while Neq0438 and doxorubicin were mutagenic only when incubated with enzymatic activation. Neq0551 was inactive for Ames assay. Therefore, these new substances (Neq0438 and Neq0551) had a better overall profile than the reference drugs, turning out to be promising candidates for further in vitro and in vivo studies.

Associações entre fungos micorrízicos arbusculares e plantas hospedeiras podem transferir césio (137Cs) para o vegetal. A mobilidade do césio (Cs+) nos solos e sua similaridade química com o potássio (K+) constituem a principal ameaça à contaminação da vegetação pelo radionuclídeo. Foi realizado experimento com plantas de feijão (Phaseolus vulgaris L.) variedade Iapar Tangará, com o objetivo de avaliar o coeficiente de transferência direta do 137Cs do solo para a planta, a absorção, o transporte e o acúmulo do radionuclídeo 137Cs nas folhas, caule e raízes das plantas, a produção de massa seca do feijoeiro, o diâmetro do caule das plantas, a peroxidação lipídica, avaliar as enzimas do sistema antioxidativo (catalase, ascorbato peroxidase, glutationa redutase e superóxido dismutase), o teor de clorofila em unidade SPAD, em função dos efeitos da: (I) inoculação ou não com fungos micorrízicos arbusculares (FMA) e (II) do nível de potássio em quatro tipos de solos: Nitossolo, Chernossolo, Argissolo e Gleissolo. O delineamento experimental foi o de blocos completos ao acaso, utilizando esquema fatorial de quatro tipos de solo x dois níveis de potássio x presença e ausência de micorrizas com três repetições, totalizando 48 unidades experimentais. Foi calculada a atividade específica do 137Cs no solo e na planta e, por fim, a determinação do coeficiente de transferência direta do 137Cs do solo para o vegetal. Nas plantas cultivadas em Argissolo, Chernossolo e Gleissolo, foi observada maior atividade específica de 137Cs nas raízes do feijão em relação às folhas e ao caule, na condição de baixo nível de potássio e ausência de inoculação de FMA. O coeficiente de transferência solo-planta do 137Cs e a atividade na planta foram maiores no Argissolo e Nitossolo, com baixo teor de potássio e sem a presença de FMA. Constatou-se interação entre os tipos de solo, teor de K e presença de FMA quanto à peroxidação de lipídeos, sendo que houve maior dano nas folhas das plantas cultivadas no Chernossolo, principalmente no tratamento sem aplicação de FMA e com baixo teor de K. Não foi observada interação entre os solos e a inoculação com FMA para a quantificação de peróxido de hidrogênio (H2O2) nas folhas, todavia, constatou-se efeito para os teores de K no solo. Quanto ao sistema enzimático antioxidativo, a atividade da catalase, glutationa redutase, ascorbato peroxidase e superóxido dismutase houve interação entre os tipos de solo, níveis de K e presença ou ausência de FMA. Maior atividade da superóxido dismutase (SOD) foi demonstrada nas plantas cultivadas no Argissolo, sem a presença de FMA, também independente dos teores de K. O aumento nos teores de K nos solos não impediu a transferência direta de 137Cs do solo para as plantas, porém, reduziu a absorção do nuclídeo pelo feijoeiro. O tipo de solo influenciou na disponibilidade de 137Cs às plantas de feijão e por sua vez, nas atividades das enzimas do sistema de defesa da planta. A inoculação com os FMA não foi efetiva para evitar a transferência e a absorção de 137Cs pelas plantas / Associations between arbuscular mycorrhizal fungi (AMF) and host plants might transfer cesium (137Cs) for the plant. The mobility of cesium (Cs+) in soils and their chemical similarity to potassium (K+) are the main threat to contamination of vegetation by radionuclide. Experiment with common bean (Phaseolus vulgaris L.) variety Iapar Tangara was conducted with the objective of evaluating the coefficient of direct transfer of 137Cs from soil to the plant, absorption, transport and accumulation of the radionuclide 137Cs in the leaves, stem and plant roots, the dry mass of bean stem diameter of plants, lipid peroxidation, evaluate the antioxidant system enzymes (catalase, ascorbate peroxidase, glutathione reductase and superoxide dismutase), the content of chlorophyll SPAD unit, due to the effects of: (I) or without inoculation with mycorrhizal fungi (AMF) and (II) the level of potassium in four types of soil: Nitosol Chernosol, Alfisol and Ultisol. The experimental design was a randomized complete block, factorial design using four soil types x two levels of potassium x presence and absence of mycorrhizae with three replications, totaling 48 experimental units. The specific activity of 137Cs in soil and plant and finally, the coefficient of 137Cs direct transfer from soil to plant was calculated. In plants grown in Alfisol and Ultisol Chernosol, higher specific activity of 137Cs in bean roots compared to the leaves and the stem was observed under the condition of low potassium and absence of AMF inoculation. The coefficient of soil-plant transfer of 137Cs activity and yield were higher in the Ultisol and Alfisol with low potassium and without the presence of AMF. Found an interaction between soil types, K content and the presence of AMF as to lipid peroxidation, and there was more damage on the leaves of plants grown at Chernosol, primarily in the treatment without application of AMF and low in K. No interaction between soil and AMF inoculation for quantification of hydrogen peroxide (H2O2) in the leaves was observed, however, it was found to effect the K in the soil. As for the antioxidant enzyme system, the activity of catalase, glutathione reductase, ascorbate peroxidase and superoxide dismutase was no interaction between soil types, levels of K and the presence or absence of AMF. Increased activity of superoxide dismutase (SOD) has been demonstrated in plants grown in Ultisol without the presence of AMF, independent of the levels of K. The increase of K in the soil did not prevent the direct transfer of 137Cs from soil to plants, however, reduced the uptake of the nuclide by bean. Soil type influenced the availability of 137Cs to bean plants and in turn, the enzyme activities of the plant defense system. Inoculation with AMF was not effective to prevent the transfer and uptake of 137Cs by plants

Neste trabalho, estudou-se a degradação do bisfenol A (BPA) em solução aquosa através da fotólise direta sob radiação UV em 254 nm. Os experimentos foram realizados um reator fotoquímico tubular de vidro de 3,9 L, equipado com uma lâmpada de vapor de mercúrio de baixa pressão tubular concêntrica (36 W). Avaliaram-se os efeitos da taxa específica de emissão de fótons (0,87×10 18 ; 1,4×1018 e 3,6×1018 fótons L-1 s-1 ) e da concentração inicial de BPA (10 a 50 mg L-1 ), conforme um projeto experimental Doehlert. Os resultados dos experimentos indicaram que a degradação do BPA diminui com o aumento de sua concentração inicial, seguindo decaimento de pseudo primeira-ordem. A constante cinética de velocidade de degradação do BPA variou de 0,001 a 0,0066 min-1 , enquanto a porcentagem de degradação de BPA ao final de 120 minutos variou de 14 a 55%; já a remoção do carbono orgânico total variou entre 1,5 e 12,5%. Houve a formação de produtos persistentes e as espécies reativas de oxigênio (1 O2 oOH ) mostraram-se muito importantes durante a degradação do BPA. O rendimento quântico para fotólise de BPA foi de 0,0075 mol BPA mol fótons-1, determinado a partir dos dados experimentais através de um modelo matemático para degradação fotolítica do BPA em função do tempo. Para tanto, considerou-se um sistema de tratamento formado por um reator tubular e um tanque de mistura com recirculação entre eles, sendo o reator tubular tratado como uma série de três reatores contínuos de mistura perfeita (CSTR) associados entre si. Através da análise estatística dos dados experimentais para duas respostas (constante específica de degradação do BPA e porcentagem de degradação do BPA ao final de 120 minutos) avaliaram-se os efeitos das variáveis envolvidas (concentração inicial de BPA e taxa específica de emissão de fótons). / The aim of this study was to evaluate bisphenol A (BPA) degradation in aqueous solution by direct photolysis under UV radiation at 254 nm, considering the effects of the specific rate of photon emission (0.87×1018; 1.4×1018 and 3.6×1018 fótons L-1 s-1) and the initial BPA concentration (10-50 mg L-1), according to a Doehlert experimental design. The experiments were performed in a glass tubular photochemical reactor of 3.9 L equipped with a concentric low pressure mercury vapor lamp of 36 W. The experimental results indicated that BPA degradation decreases with increasing initial concentration, following pseudo first-order decay, and increases with increasing specific rate of photon emission. The values of the specific BPA degradation rate varied in the range 0.001-0.0066 min-1, while BPA percent degradation at 120 minutes of irradiation varied from 14 to 55%; TOC removal varied from 1.5 to 12.5%. Persistent degradation products were formed, and oxygen reactive species (1O2, oHO) showed to exhibit an important role during BPA degradation. The obtained quantum yield of BPA photolysis was 0.0075 mol BPA mol photons-1, determined from the experimental data through a mathematical model for BPA photolytic degradation versus time. With that aim a treatment system formed by a tubular reactor connected with a mixing tank with recirculation between them was considered; the tubular reactor was treated as a series of three associated continuous perfect mixing reactors (CSTR). Through statistical analysis of the experimental data for two responses (specific BPA degradation rate and BPA percent degradation at 120 minutes), the effects of the variables involved in BPA degradation (initial BPA concentration and specific rate of photon emission) were discussed.

This master thesis deals with the methods of measurement of singlet oxygen steady state concentrations in surface water and the factors affecting this concentration. The first part of this work shows theoretical background and an introduction to photochemistry of surface water with respect to the reactive oxygen species. The second part describes the methodology of conducted experiments and discuss its results with respect to current literature.

Dietas hiperlipídicas e a esteatose hepática são condições extremamente prevalentes. Trabalhos anteriores mostraram que a esteatose está associada a um aumento na geração de espécies reativas de oxigênio (ERO), e que isso pode mediar danos no fígado. Neste trabalho nós investigamos os possíveis mecanismos que desencadeiam os aumentos nas taxas de geração de ERO por meio da administração de dietas hiperlipídicas. Nós descobrimos que mitocôndrias de animais sujeitos a dietas hiperlipídicas não apresentaram diferenças significativas quanto a capacidade respiratória máxima e acoplamento, mas eram capazes de gerar mais ERO especificamente quando usados substratos do metabolismo de ácidos graxos. Além disso, foi observado que muitas isoformas de acil-CoA desidrogenases estavam mais expressas nos fígados de animais alimentados pela dieta hiperlipídica. No entanto, quando realizados ensaios de atividade enzimática apenas a acil CoA desidrogenase de cadeia longa (VLCAD) foi mais ativa. Estudos conduzidos com mitocôndrias permeabilizadas e expostas a grupos acil-CoA de diferentes tamanhos sugerem que a VLCAD pode ser uma fonte da produção aumentada de ERO em animais submetidos a dietas hiperlipídicas. Esta produção foi estimulada pela ausência de NAD+. Concluindo, nossos estudos descobriram uma nova fonte importante na geração de ERO estimulada por dietas hiperlipídicas, a VLCAD / High fat diets and accompanying hepatic steatosis are highly prevalent conditions. Previous work has shown that steatosis occurs concomitantly with enhanced reactive oxygen species (ROS) generation, which may mediate further liver damage. Here we investigated mechanisms leading to enhanced ROS generation following high fat diets (HFD). We found that mitochondria from HFD livers present no differences in maximal respiratory rates and coupling, but generate more ROS specifically when fatty acids are used as substrates. Indeed, many acyl-CoA dehydrogenase isoforms were found to be more highly expressed in HFD livers, although only the very long chain acyl-CoA dehydrogenase (VLCAD) was more functionally active. Studies conducted with permeabilized mitochondria and different chain length acyl-CoA derivatives suggest that VLCAD is a source of enhanced ROS production in mitochondria from HFD animals. This production is stimulated by the lack of NAD+. Overall, our studies uncover VLCAD as a novel, diet-sensitive, source of mitochondrial ROS

A Araucaria angustifólia é uma conífera nativa do Brasil que apresenta sementes recalcitrantes. Devido a sua importância econômica, foi intensamente explorada ao longo dos anos, encontrando-se atualmente classificada como espécie em perigo crítico de extinção pela International Union for Conservation of Nature. A embriogênese somática apresenta-se como uma ferramenta biotecnológica de grande valia na propagação de espécies recalcitrantes e de difícil propagação, com aplicação em programas de conservação, reflorestamento, e melhoramento genético vegetal. Estudos comparativos dos processos de embriogênese somática e zigótica têm permitido o conhecimento dos fatores bioquímicos, fisiológicos e genéticos que controlam o desenvolvimento do embrião, e o estabelecimento as condições artificiais para o correto desenvolvimento embrionário in vitro. O objetivo deste trabalho foi estudar a participação do ácido salicílico como sinalizador do processo de embriogênese zigótica e somática em A. angustifólia. Para tanto foi determinado o conteúdo de ácido salicílico livre e conjugado ao longo da embriogênese zigótica, e o efeito de sua suplementação em culturas embriogênicas com diferentes potenciais para a maturação. Para a embriogênese somática, a presença do ácido salicílico foi correlacionada com a geração de óxido nítrico e espécies reativas de oxigênio , e com a expressão do gene \"Somatic Embryogenesis Receptor Kinase\" (AaSERK). Os resultados obtidos demonstram que: a) ocorre um maior conteúdo de ácido salicílico na forma livre e conjugada nas etapas iniciais da embriogênese zigótica; b) a suplementação de ácido salicílico, nas concentrações de 0,5 a 2 mM, inibiram a indução de culturas embriogênicas; c) culturas embriogênicas incubadas em ácido salicílico apresentaram redução da síntese endógena de espécies reativas de oxigênio e aumento no conteúdo de óxido nítrico; d) a redução de espécies reativas de oxigênio indicou uma relação dose dependente com o ácido salicílico; e) a adição de um doador de óxido nítrico e um sequestrador inibiram a produção de espécies reativas de oxigênio; f) a expressão do gene AaSERK atingiu o maior nível no período de quatro horas de incubação em 0,1 mM de AS / Araucaria angustifolia is a conifer native of Brazil with recalcitrant seeds. Due to its economic importance, the exploration has been extensively over the years, currently this specie is classified as critically endangered by the International Union for Conservation of Nature. Somatic embryogenesis is presented as a biotechnological tool of great value in the propagation of recalcitrant species and difficult to spread, with applications in conservation, reforestation programs, and plant breeding. Comparative studies of the processes of zygotic and somatic embryogenesis has allowed the knowledge of biochemical, physiological and genetic factors that control embryo development, and the establishment artificial conditions for proper embryonic development in vitro. The objective of this work was to study the role of salicylic acid as a marker of zygotic embryogenesis and somatic process in A. angustifolia. Thus, we determined the content of free salicylic acid and conjugated along the zygotic embryogenesis, and the effect of their supplementation with different potential embryogenic cultures for maturation. For somatic embryogenesis, the presence of salicylic acid was correlated with the generation of nitric oxide, reactive oxygen species and in the gene expression \"Somatic embryogenesis Receptor Kinase\" (AaSERK). The results show that: a) there is an increased content of salicylic acid in free and conjugated form in the initial stages of zygotic embryogenesis; b) salicylic acid supplementation, in concentrations from 0.5 to 2 mM, inhibited the induction of embryogenic cultures; c) embryos incubated in salicylic acid decreased endogenous synthesis of reactive oxygen species and increase in content of nitric oxide; d) the reduction of reactive oxygen species indicated a dose-dependent relationship with salicylic acid; e) the addition of a nitric oxide donor and a kidnapper inhibited the production of reactive oxygen species; f) the expression of the gene AaSERK reached the highest level in four hours of incubation in 0.1 mM AS

by Yau Ming Hon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 131-154). / Abstracts in English and Chinese. / Contents --- p.i / Acknowledgements --- p.ix / Abstract --- p.x / 槪論 --- p.xi / List of abbreviations --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Reactive oxygen species (ROS) --- p.2 / Chapter 1.1.1 --- Hydrogen peroxide --- p.2 / Chapter 1.1.2 --- Superoxide anion --- p.3 / Chapter 1.1.3 --- Hydroxyl radical --- p.3 / Chapter 1.1.4 --- Carbon centered radical --- p.4 / Chapter 1.1.5 --- Nitric oxide --- p.4 / Chapter 1.2 --- Physiological roles of ROS --- p.5 / Chapter 1.2.1 --- Signal transduction --- p.5 / Chapter 1.2.2 --- Phagocytic activity --- p.6 / Chapter 1.3 --- Defense systems against ROS --- p.7 / Chapter 1.3.1 --- Endogenous antioxidant enzymes --- p.8 / Chapter 1.3.1.1 --- Catalase --- p.8 / Chapter 1.3.1.2 --- Superoxide dismutase --- p.8 / Chapter 1.3.1.3 --- Selenium-glutathione peroxidase --- p.9 / Chapter 1.3.1.4 --- Glutathione reductase --- p.10 / Chapter 1.3.1.5 --- Glutathione-S-transferases --- p.10 / Chapter 1.3.2 --- Non-enzymatic antioxidants --- p.12 / Chapter 1.3.2.1 --- Vitamin E (tocopherols and tocotrienols) --- p.12 / Chapter 1.3.2.2 --- Vitamin C (L-ascorbic acid) --- p.13 / Chapter 1.3.2.3 --- Glutathione --- p.14 / Chapter 1.3.2.4 --- Flavonoids and polyphenols --- p.15 / Chapter 1.3.2.5 --- Uric acid --- p.16 / Chapter 1.4 --- Roles of ROS in pathogenesis --- p.16 / Chapter 1.4.1 --- Liver diseases --- p.17 / Chapter 1.4.2 --- Genital malfunctioning --- p.19 / Chapter 1.5 --- "The traditional Chinese medicinal formula, Wu-zi-yan-zong-wan" --- p.19 / Chapter 1.5.1 --- Pharmacology of individual herbal components --- p.20 / Chapter 1.5.1.1 --- Semen Cuscuta --- p.20 / Chapter 1.5.1.2 --- Fructus Lycii --- p.21 / Chapter 1.5.1.3 --- Fructus Schisandrae --- p.21 / Chapter 1.5.1.4 --- Fructus Rubi --- p.22 / Chapter 1.5.1.5 --- Semen Plantaginis --- p.22 / Chapter 1.5.2 --- Effect of Wu-zi-yan-zong-wan on infertility --- p.23 / Chapter 1.5.3 --- Effect of Wu-zi-yan-zong-wan on liver disease --- p.23 / Chapter 1.6 --- Objectives of the present study --- p.24 / Chapter Chapter 2 --- Antioxidant Activity of Aqueous Extracts of the Herbal Components of Wu-zi-yan-zong-wan in in vitro Free Radical Generating Systems --- p.26 / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.1.1 --- Application of in vitro ROS generating systems --- p.27 / Chapter 2.1.1.1 --- Superoxide generation --- p.27 / Chapter 2.1.1.2 --- Hydroxyl radical generation system --- p.28 / Chapter 2.1.1.3 --- "2,2'-Azobis(2-amidinopropane) dihydrochloride- induced hemolysis" --- p.28 / Chapter 2.1.1.4 --- Bleomycin-iron-dependent DNA damage --- p.28 / Chapter 2.1.2 --- Objective --- p.29 / Chapter 2.2 --- Materials and methods --- p.30 / Chapter 2.2.1 --- Materials --- p.30 / Chapter 2.2.2 --- Preparation of aqueous herbal extracts --- p.30 / Chapter 2.2.3 --- Superoxide-scavenging assay --- p.30 / Chapter 2.2.4 --- Microsome lipid peroxidation inhibition assay --- p.31 / Chapter 2.2.5 --- "2,2'-Azobis(2-amidinopropane) dihydrochloride-induced hemolysis inhibition assay" --- p.32 / Chapter 2.2.6 --- Bleomycin-iron-dependent DNA damage inhibition assay --- p.32 / Chapter 2.2.7 --- Statistical analysis --- p.33 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- Extraction yield --- p.34 / Chapter 2.3.2 --- Free radical scavenging activity of Wu-zi-yan-zong-wan extract --- p.34 / Chapter 2.3.3 --- Free radical scavenging activity of FR extract --- p.37 / Chapter 2.3.3.1 --- Superoxide-scavenging activity --- p.37 / Chapter 2.3.3.2 --- Effect on hydroxyl radical-induced lipid peroxidation --- p.37 / Chapter 2.3.3.3 --- Effect on AAPH-induced hemolysis --- p.40 / Chapter 2.3.3.4 --- Effect on bleomycin-iron-dependent DNA damage --- p.40 / Chapter 2.3.4 --- Pro-oxidant activity of FR extract --- p.40 / Chapter 2.3.5 --- Free radical scavenging activity of the remaining herbal extracts --- p.44 / Chapter 2.4 --- Discussion --- p.46 / Chapter Chapter 3 --- Effect of Aqueous Extract of the Herbal Components of Wu- zi-yan-zong-wan on tert-Butyl Hydroperoxide-Induced Oxidative Damage in Primary Rat Hepatocyte --- p.51 / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.1.1 --- Primary rat hepatocyte as pharmacological model --- p.52 / Chapter 3.1.2 --- tert-Butyl hydroperoxide as an oxidative stress inducer --- p.53 / Chapter 3.1.3 --- Detection of ROS --- p.54 / Chapter 3.1.4 --- Objective --- p.55 / Chapter 3.2 --- Materials and methods --- p.56 / Chapter 3.2.1 --- Materials --- p.56 / Chapter 3.2.2 --- Primary rat hepatocyte isolation --- p.56 / Chapter 3.2.2.1 --- Liver perfusion --- p.56 / Chapter 3.2.2.2 --- Collagen pre-coated plates preparation --- p.57 / Chapter 3.2.2.3 --- Hepatocyte culture --- p.58 / Chapter 3.2.3 --- Drug treatment and oxidative stress induction --- p.58 / Chapter 3.2.4 --- Cytotoxicity assessment --- p.58 / Chapter 3.2.4.1 --- Lactate dehydrogenase leakage measurement --- p.59 / Chapter 3.2.4.2 --- MTT assay --- p.59 / Chapter 3.2.5 --- Cellular GSH content determination --- p.59 / Chapter 3.2.6 --- Protein determination by Lowry's method --- p.60 / Chapter 3.2.7 --- MDA measurement --- p.60 / Chapter 3.2.8 --- GSSG measurement --- p.61 / Chapter 3.2.9 --- ROS measurement with fluorescent dye --- p.61 / Chapter 3.2.10 --- "Vitamin C, vitamin E and butylated hydroxytoluene treatment" --- p.62 / Chapter 3.2.11 --- Antioxidant enzyme activity measurement --- p.62 / Chapter 3.2.11.1 --- Catalase activity measurement --- p.62 / Chapter 3.2.11.2 --- Superoxide dismutase activity measurement --- p.63 / Chapter 3.2.11.3 --- Glutathione peroxidase activity measurement --- p.63 / Chapter 3.2.11.4 --- Glutathione-S-transferases activity measurement --- p.63 / Chapter 3.2.11.5 --- Glutathione reductase activity measurement --- p.64 / Chapter 3.2.12 --- Statistical analysis --- p.64 / Chapter 3.3 --- Results --- p.65 / Chapter 3.3.1 --- Cytotoxicity of FR extract on rat hepatocyte --- p.65 / Chapter 3.3.2 --- Effect of tBHP and FR extract on hepatocyte viability --- p.65 / Chapter 3.3.3 --- Time-dependent effect of FR extract on tBHP-induced cytotoxicity --- p.69 / Chapter 3.3.4 --- Effect of tBHP and FR extract on hepatocyte GSH content --- p.69 / Chapter 3.3.5 --- Effect of tBHP and FR extract on GSSG formation in hepatocyte --- p.72 / Chapter 3.3.6 --- Effect of tBHP and FR extract on MDA formation in hepatocyte --- p.72 / Chapter 3.3.7 --- ROS-scavenging activity of FR extract in hepatocyte --- p.77 / Chapter 3.3.8 --- Effect of FR extract on antioxidant enzymes activities --- p.77 / Chapter 3.3.9 --- Comparison between typical antioxidants --- p.77 / Chapter 3.3.10 --- Effect of WZ and remaining herbal extracts on tBHP-induced oxidative damage in hepatocyte --- p.81 / Chapter 3.4 --- Discussion --- p.84 / Chapter Chapter 4 --- Effect of Aqueous Extract of Wu-zi-yan-zong-wan and Fructus Rubi on tert-Buty Hydroperoxide Induced Oxidative Damage in Mouse Model --- p.91 / Chapter 4.1 --- Introduction --- p.92 / Chapter 4.2 --- Materials and methods --- p.93 / Chapter 4.2.1 --- Materials --- p.93 / Chapter 4.2.2 --- Animal treatments --- p.93 / Chapter 4.2.3 --- Serum preparation --- p.94 / Chapter 4.2.4 --- Marker enzyme measurement --- p.94 / Chapter 4.2.5 --- Liver MDA and GSH determination --- p.95 / Chapter 4.2.6 --- Statistical analysis --- p.95 / Chapter 4.3 --- Results --- p.97 / Chapter 4.3.1 --- Effect of tBHP and FR extract on mouse serum ALT and AST activities --- p.97 / Chapter 4.3.2 --- Effect of tBHP and FR extract on mouse liver MDA and GSH content --- p.97 / Chapter 4.3.3 --- Effect of WZ extract on tBHP-induced increase in serum ALT and AST activities --- p.97 / Chapter 4.4 --- Discussion --- p.102 / Chapter Chapter 5 --- Characterization of the Active Antioxidant Principlein Aqueous Extract of FR --- p.105 / Chapter 5.1 --- Introduction --- p.106 / Chapter 5.2 --- Materials and methods --- p.107 / Chapter 5.2.1 --- Materials --- p.107 / Chapter 5.2.2 --- Chemical/physical treatments on FR extract --- p.107 / Chapter 5.2.3 --- Digestion with enzymes --- p.108 / Chapter 5.2.4 --- Antioxidant activity determination --- p.109 / Chapter 5.2.5 --- Chemical composition determination --- p.109 / Chapter 5.2.5.1 --- Uronic acid determination --- p.109 / Chapter 5.2.5.2 --- Hexose determination --- p.109 / Chapter 5.2.5.3 --- Tannin determination --- p.110 / Chapter 5.2.5.4 --- Protein determination --- p.110 / Chapter 5.2.6 --- Column chromatography --- p.110 / Chapter 5.2.6.1 --- Polyamide CC6 resin column chromatography --- p.111 / Chapter 5.2.6.2 --- Sephadex LH-20 gel column chromatography --- p.111 / Chapter 5.2.7 --- Antioxidant activity of commercially available tannin --- p.111 / Chapter 5.2.8 --- Bovine serum albumin precipitation --- p.112 / Chapter 5.2.9 --- Statistical analysis --- p.112 / Chapter 5.3 --- Results --- p.113 / Chapter 5.3.1 --- Effect of chemical/physical treatments on antioxidant activity of FR extract --- p.113 / Chapter 5.3.2 --- Effect of enzyme digestions on antioxidant activity of FR extract --- p.113 / Chapter 5.3.3 --- Chemical composition of FR extract --- p.118 / Chapter 5.3.4 --- Polyamide CC6 resin column chromatography --- p.118 / Chapter 5.3.5 --- Sephadex LH-20 gel column chromatography --- p.118 / Chapter 5.3.6 --- Antioxidant activity of commercially available tannin --- p.123 / Chapter 5.3.7 --- Effect of BSA precipitation on superoxide-scavenging activity --- p.123 / Chapter 5.4 --- Discussion --- p.127 / Conclusion --- p.131 / References --- p.132

Dietas hiperlipídicas e a esteatose hepática são condições extremamente prevalentes. Trabalhos anteriores mostraram que a esteatose está associada a um aumento na geração de espécies reativas de oxigênio (ERO), e que isso pode mediar danos no fígado. Neste trabalho nós investigamos os possíveis mecanismos que desencadeiam os aumentos nas taxas de geração de ERO por meio da administração de dietas hiperlipídicas. Nós descobrimos que mitocôndrias de animais sujeitos a dietas hiperlipídicas não apresentaram diferenças significativas quanto a capacidade respiratória máxima e acoplamento, mas eram capazes de gerar mais ERO especificamente quando usados substratos do metabolismo de ácidos graxos. Além disso, foi observado que muitas isoformas de acil-CoA desidrogenases estavam mais expressas nos fígados de animais alimentados pela dieta hiperlipídica. No entanto, quando realizados ensaios de atividade enzimática apenas a acil CoA desidrogenase de cadeia longa (VLCAD) foi mais ativa. Estudos conduzidos com mitocôndrias permeabilizadas e expostas a grupos acil-CoA de diferentes tamanhos sugerem que a VLCAD pode ser uma fonte da produção aumentada de ERO em animais submetidos a dietas hiperlipídicas. Esta produção foi estimulada pela ausência de NAD+. Concluindo, nossos estudos descobriram uma nova fonte importante na geração de ERO estimulada por dietas hiperlipídicas, a VLCAD / High fat diets and accompanying hepatic steatosis are highly prevalent conditions. Previous work has shown that steatosis occurs concomitantly with enhanced reactive oxygen species (ROS) generation, which may mediate further liver damage. Here we investigated mechanisms leading to enhanced ROS generation following high fat diets (HFD). We found that mitochondria from HFD livers present no differences in maximal respiratory rates and coupling, but generate more ROS specifically when fatty acids are used as substrates. Indeed, many acyl-CoA dehydrogenase isoforms were found to be more highly expressed in HFD livers, although only the very long chain acyl-CoA dehydrogenase (VLCAD) was more functionally active. Studies conducted with permeabilized mitochondria and different chain length acyl-CoA derivatives suggest that VLCAD is a source of enhanced ROS production in mitochondria from HFD animals. This production is stimulated by the lack of NAD+. Overall, our studies uncover VLCAD as a novel, diet-sensitive, source of mitochondrial ROS

Ce travail montre qu’une protéine non encore décrite dans les phagocytes, la PAT1 « Protein interacting with APP Tail 1 », interagit avec la partie cytosolique de la p22phox (composant du cytochrome b558 membranaire de la NADPH oxydase). Nous avons utilisé différentes approches pour montrer cette interaction : le système double hybride, la technique de GST-pull down, la microscopie confocale et la technique de co-immunoprécipitation. De plus, nous avons montré que la PAT1a recombinante augmente l’activité de la NADPH oxydase, in vitro dans un système acellulaire reconstitué, et dans les cellules intactes (monocytes et cellules COS-phox). Cette nouvelle interaction régule donc l’activation de la NADPH oxydase et la production des FRO. Par ailleurs, la liaison de PAT1 aux microtubules pourrait favoriser l’assemblage du complexe NADPH oxydase pendant son activation. Ceci pourrait conduire à l’identification de nouvelles cibles thérapeutiques qui préviennent la survenue des lésions tissulaires dans les maladies inflammatoires. / Reactive oxygen species (ROS) production by the phagocyte NADPH oxidase plays a crucial role in host defenses. NADPH oxidase is composed of the membrane flavocytochrome b558 components (p22phox and gp91phox/NOX2), and cytosolic components (p40phox, p47phox, p67phox and a small GTPase Rac1 or Rac2). In this work we identified PAT1 by double hybrid system as a potential partner of p22phox. The interaction between p22phox and PAT1a was further confirmed by in vitro GST pull-down assay, confocal microscopy and co-immunoprecipitation. Addition of recombinant PAT1a to the cell free-system enhanced NADPH oxidase activation and it’s over-expression in human monocytes and in COSphox cells increased ROS production in resting and fMLP-stimulated cells.These data clearly identify PAT1 as a novel regulator of NADPH oxidase activation in phagocytes.Inhibition of p22phox/PAT1 interaction could be used as new approach to limit ROS production by phagocytes at inflammatory sites.