No / Drinking water disinfection by-products (DBPs) are generated by the chemical disinfection of water and may pose hazards to public health. Two major classes of DBPs are found in finished drinking water: haloacetic acids (HAAs) and trihalomethanes (THMs). HAAs are formed following disinfection with chlorine, which reacts with iodide and bromide in the water. Previously the HAAs were shown to be cytotoxic, genotoxic, mutagenic, teratogenic and carcinogenic. OBJECTIVES: To determine the effect of HAAs in human somatic and germ cells and whether oxidative stress is involved in genotoxic action. In the present study both somatic and germ cells have been examined as peripheral blood lymphocytes and sperm. The effects of three HAA compounds: iodoacetic acid (IAA), bromoacetic acid (BAA) and chloroacetic acid (CAA) were investigated. After determining appropriate concentration responses, oxygen radical involvement with the antioxidants, butylated hydroxanisole (BHA) and the enzyme catalase, were investigated in the single cell gel electrophoresis (Comet) assay under alkaline conditions, >pH 13 and the micronucleus assay. In the Comet assay, BHA and catalase were able to reduce DNA damage in each cell type compared to HAA alone. In the micronucleus assay, micronuclei (MNi) were found in peripheral lymphocytes exposed to all three HAAs and catalase and BHA were in general, able to reduce MNi induction, suggesting oxygen radicals play a role in both assays. These observations are of concern to public health since both human somatic and germ cells show similar genotoxic responses.

Elevada disponibilidade de bário (Ba) no meio de cultivo pode resultar em severa toxidez nas plantas. A toxidez causada pelo metal pesado nas plantas pode ser amenizada pelo fornecimento de nutrientes, particularmente o enxofre (S). Objetivou-se avaliar os parâmetros produtivos, sintomas visuais, estado nutricional, índice SPAD, indicadores de estresse (malondialdeído, peróxido de hidrogênio e prolina) e a atividade de enzimas do sistema antioxidante (glutationa redutase, superóxido dismutase, catalase, ascorbato peroxidase e guaiacol peroxidase) do capim tanzânia (Panicum maximum Jacq. cv. Tanzânia) cultivado com combinações de doses de S e de Ba na solução nutritiva, em casa de vegetação, em dois ciclos de crescimento das plantas. As plantas foram submetidas às combinações de cinco doses de S (0,1; 1,0; 1,9; 2,8 e 3,7 mmol L-1) com cinco doses de Ba (0,0; 2,5; 5,0; 10,0 e 20,0 mmol L-1), utilizando esquema fatorial fracionado (52), em blocos ao acaso, com seis repetições e seis plantas por unidade experimental. Os parâmetros produtivos e o estado nutricional das plantas foram severamente afetados pelas combinações de S e Ba em ambos os ciclos de crescimento. O aumento na dose de Ba na solução nutritiva prejudicou acentuadamente o crescimento e o estado nutricional das plantas, principalmente na área foliar, e nos nutrientes potássio e magnésio, enquanto o aumento da dose de S amenizou o efeito tóxico do metal. Maiores concentrações de malondialdeído foram observadas nas plantas que apresentaram maior acúmulo do metal pesado. As atividades das enzimas glutationa redutase, catalase e ascorbato peroxidase foram mais altas nas plantas cultivadas com baixo fornecimento de S, enquanto a atividade da guaiacol peroxidase foi aumentada com incremento das doses de Ba e de S. / High barium (Ba) availability in growth medium can cause severe toxicity in plants. Nutrient supply may mitigate heavy metal phytotoxicity, especially the sulphur (S) supply. The study was carried out to evaluate the plant growth, visual symptoms, nutritional status, indicators of stress (malondialdehyde, hydrogen peroxide and proline) and activities of the antioxidant enzymes (glutathione reductase, superoxide dismutase, catalase, ascorbate peroxidase and guaiacol peroxidase) in Tanzania guineagrass (Panicum maximum Jacq. cv. Tanzania) grown with combinations of S rates and Ba rates in nutrient solution, in a greenhouse, for two growth periods. Plants received nutrient solutions where combinations of five S rates (0.1, 1.0, 1.9, 2.8 and 3.7 mmol L-1) with five Ba rates (0.0, 2.5, 5.0, 10.0 and 20.0 mmol L-1) were used in a fractionated factorial (52), in randomized block design, with six replications and six plants per experimental unit. Plant production and nutritional status were highly affected by combinations of S and Ba rates, in both growth periods. Increase on Ba rates impaired growth, mainly on leaf area production. Barium rates impaired nutritional status, mainly on potassium and calcium, while increasing on S rates alleviated Ba toxic effects. High concentrations of malondialdehyde was observed in plants with increase of Ba application. The activities of catalase and ascorbate peroxidase were high in plants grown with low S availability, while guaiacol peroxidase activity was enhanced by increasing Ba and S rates in the nutrient solution.

Elevada disponibilidade de bário (Ba) no meio de cultivo pode resultar em severa toxidez nas plantas. A toxidez causada pelo metal pesado nas plantas pode ser amenizada pelo fornecimento de nutrientes, particularmente o enxofre (S). Objetivou-se avaliar os parâmetros produtivos, sintomas visuais, estado nutricional, índice SPAD, indicadores de estresse (malondialdeído, peróxido de hidrogênio e prolina) e a atividade de enzimas do sistema antioxidante (glutationa redutase, superóxido dismutase, catalase, ascorbato peroxidase e guaiacol peroxidase) do capim tanzânia (Panicum maximum Jacq. cv. Tanzânia) cultivado com combinações de doses de S e de Ba na solução nutritiva, em casa de vegetação, em dois ciclos de crescimento das plantas. As plantas foram submetidas às combinações de cinco doses de S (0,1; 1,0; 1,9; 2,8 e 3,7 mmol L-1) com cinco doses de Ba (0,0; 2,5; 5,0; 10,0 e 20,0 mmol L-1), utilizando esquema fatorial fracionado (52), em blocos ao acaso, com seis repetições e seis plantas por unidade experimental. Os parâmetros produtivos e o estado nutricional das plantas foram severamente afetados pelas combinações de S e Ba em ambos os ciclos de crescimento. O aumento na dose de Ba na solução nutritiva prejudicou acentuadamente o crescimento e o estado nutricional das plantas, principalmente na área foliar, e nos nutrientes potássio e magnésio, enquanto o aumento da dose de S amenizou o efeito tóxico do metal. Maiores concentrações de malondialdeído foram observadas nas plantas que apresentaram maior acúmulo do metal pesado. As atividades das enzimas glutationa redutase, catalase e ascorbato peroxidase foram mais altas nas plantas cultivadas com baixo fornecimento de S, enquanto a atividade da guaiacol peroxidase foi aumentada com incremento das doses de Ba e de S. / High barium (Ba) availability in growth medium can cause severe toxicity in plants. Nutrient supply may mitigate heavy metal phytotoxicity, especially the sulphur (S) supply. The study was carried out to evaluate the plant growth, visual symptoms, nutritional status, indicators of stress (malondialdehyde, hydrogen peroxide and proline) and activities of the antioxidant enzymes (glutathione reductase, superoxide dismutase, catalase, ascorbate peroxidase and guaiacol peroxidase) in Tanzania guineagrass (Panicum maximum Jacq. cv. Tanzania) grown with combinations of S rates and Ba rates in nutrient solution, in a greenhouse, for two growth periods. Plants received nutrient solutions where combinations of five S rates (0.1, 1.0, 1.9, 2.8 and 3.7 mmol L-1) with five Ba rates (0.0, 2.5, 5.0, 10.0 and 20.0 mmol L-1) were used in a fractionated factorial (52), in randomized block design, with six replications and six plants per experimental unit. Plant production and nutritional status were highly affected by combinations of S and Ba rates, in both growth periods. Increase on Ba rates impaired growth, mainly on leaf area production. Barium rates impaired nutritional status, mainly on potassium and calcium, while increasing on S rates alleviated Ba toxic effects. High concentrations of malondialdehyde was observed in plants with increase of Ba application. The activities of catalase and ascorbate peroxidase were high in plants grown with low S availability, while guaiacol peroxidase activity was enhanced by increasing Ba and S rates in the nutrient solution.

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No. of bitstreams: 1 cristianoferraraderesende.pdf: 2182147 bytes, checksum: a1f62e02cb7c97296c57ea6461357835 (MD5) Previous issue date: 2012-03-05 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Bromeliaceae é uma família essencialmente neotropical, cujos representantes são muito utilizados para fins paisagísticos. Uma das principais fontes de abastecimento do mercado consumidor de bromélias é o extrativismo o que, associado à destruição do ambiente natural, tem levado a perdas irreparáveis na biodiversidade da família, especialmente na Mata Atlântica, onde cerca de 40% das espécies encontram-se sob alguma categoria de ameaça. Pitcairnia encholirioides L. B. Sm. é uma espécie “criticamente em perigo” de extinção, sendo conhecida somente uma população dessa espécie, com cerca de 900 indivíduos, encontrada em 2004 em um afloramento rochoso muito degradado e sujeito ao fogo e ao pisoteio de animais, no município de Santa Maria Madalena, RJ. A utilização das técnicas de micropropagação pode reverter os riscos de extinção garantindo taxas elevadas de multiplicação, fornecendo material necessário ao mercado consumidor, evitando, dessa forma, o extrativismo das plantas nos seus locais de origem. A instalação e a conservação in vitro de bancos de germoplasma possuem especial importância, garantindo a sobrevivência de espécies raras e/ou endêmicas e fornecendo plantas para iniciativas de reintrodução. A etapa final da micropropagação é a aclimatização ex vitro, realizada após os procedimentos de multiplicação e enraizamento in vitro. A aclimatização é um período crítico para as plantas devido à perda de água, o que causa estresse hídrico e, como consequência, estresse oxidativo, podendo provocar problemas metabólicos e perdas elevadas. No presente trabalho, Pitcairnia encholirioides foi cultivada in vitro por 150 dias em meio de cultura adicionado de dois reguladores de crescimento (GA3 ou ANA), além de duas concentrações de sacarose (15 ou 30 g L-1) e sob dois tipos de vedação dos tubos de ensaio (vedação hermética com tampas e filme de PVC ou vedação com tampas que permitiam trocas gasosas), totalizando 8 tratamentos, período após o qual as plantas foram transferidas para condições ex vitro, em casa de vegetação, permanecendo por mais 180 dias. Dois desses tratamentos foram selecionados para as análises de desidratação, quando suas plantas foram submetidas a 30, 42 ou 54 dias sem irrigação, além do controle irrigado periodicamente, sendo posteriormente reidratadas durante 90 dias. Após os períodos de cultivo in vitro e permanência das plantas em casa de vegetação, foram realizadas análises dos conteúdos de prolina, proteínas totais, atividades das enzimas do metabolismo antioxidativo SOD, CAT, POD e PPO, além 2 dos teores de pigmentos fotossintetizantes. Além desses parâmetros, após desidratação e reidratação foram também avaliados os teores de carboidratos. De maneira geral, o cultivo in vitro em meio adicionado da menor concentração de sacarose e em tubos vedados hermeticamente se mostrou prejudicial, o que foi evidenciado pelo menor acúmulo de prolina, aumentos nas atividades das enzimas antioxidativas e menor acúmulo de pigmentos fotossintéticos. Após o período de aclimatização, não foram encontradas diferenças entre os tratamentos para a maioria dos parâmetros analisados, ou os dados oscilaram de maneira a não permitir uma generalização quanto aos efeitos residuais da sacarose, do tipo de tampa e dos reguladores de crescimento utilizados durante o cultivo in vitro. No período em que as plantas foram submetidas à desidratação progressiva, houve maior acúmulo de prolina e proteínas solúveis totais, além de aumento nos teores de pigmentos fotossintéticos nos tecidos provenientes de plantas cultivadas in vitro em meio contendo 30 g L-1 de sacarose e GA3. Houve também, em geral, queda mais acentuada nas atividades das enzimas em meio suplementado com a menor concentração de sacarose e ANA, além de oscilações nos teores de pigmentos. As relações entre os pigmentos sofreram leve redução em ambos os tratamentos, e os teores de carboidratos solúveis totais, sacarose, açúcares redutores e amido aumentaram com o prolongamento do estresse. Após a reidratação, todas as plantas demonstraram elevada capacidade de recuperação, apresentando em todas as análises realizadas valores muito similares aos das plantas do controle, não submetidas à desidratação. Os resultados obtidos no trabalho permitem concluir que o cultivo in vitro em meio adicionado de 30 g L-1 de sacarose e em tubos de ensaio que permitem trocas gasosas é o mais apropriado para plantas de P. encholirioides. Ademais, a concentração de sacarose adicionada ao meio de cultura também influencia o desenvolvimento das plantas após aclimatização, sendo encontrados melhores resultados em plantas cultivadas nos meios de cultura suplementados com a maior concentração desse carboidrato. Acredita-se que as plantas cultivadas nessas condições apresentariam maior capacidade de sobrevivência ao transplantio e maior resistência a períodos prolongados de estresse hídrico, aos quais, frequentemente, estão submetidas no ambiente natural. / Bromeliaceae is essentially a neotropical family, whose representatives are widely used for landscaping. One major source of supply for the consumer market is the extraction, which coupled with the destruction of the natural environment, has led to irreparable losses in biodiversity of the family, especially in the Atlantic Forest, where about 40% of bromeliad species are under some category of threat. Pitcairnia encholirioides L. B. Sm. is a critically endangered species. In 2004, a unique population of this species was found, with about 900 individuals in a very degraded rocky outcrop, subject to fire and trampling by animals, located in Santa Maria Madalena, RJ. The use of micropropagation can guarantee high multiplication rates, providing necessary material for the consumer market, effectively obviating the extraction of plants in their places of origin. In addition, installation and in vitro conservation of germplasm banks have special importance, ensuring the survival of rare species and/or endemic and providing micropropagated plants for reintroduction initiatives. Following the procedures of in vitro multiplication and rooting, the final stage of micropropagation is the ex vitro acclimatization, a critical period for the plants due to water loss, which causes water stress and, consequently, oxidative stress, which can cause metabolic problems and high losses. In this study, Pitcairnia encholirioides was grown for 150 days in vitro in a culture medium supplemented with two growth regulators (GA3 or NAA), and two concentrations of sucrose (15 or 30 g L-1) in two types of sealing of test tubes (hermetic seal with lids and PVC film and seal with lids that allowed gas exchange), totaling eight treatments, after which the plants were transferred to ex vitro conditions in a greenhouse, staying for more than 180 days. Two of these treatments were selected for analysis of dehydration when their plants were subjected to 30, 42 or 54 days without irrigation, apart from control irrigated periodically, being subsequently rehydrated for 90 days. After periods of in vitro cultivation and maintenance of the plants in the greenhouse, they were analyzed for proline content, total protein, activities of antioxidant metabolism enzymes SOD, CAT, POD and PPO, besides the content of photosynthetic pigments. Apart from these parameters, after dehydration and rehydration, levels of carbohydrates were also assessed. In general, the in vitro culture in medium containing the lowest concentration of sucrose and hermetically sealed tubes proved to be harmful, which was evidenced by lower levels of proline, increased 4 activity of antioxidant enzymes and lower accumulation of photosynthetic pigments. After the acclimatization period, no differences were found between treatments for most parameters, or data varied so as to not allow a generalization about the residual effects of sucrose, the type of cover and of the growth regulators used in the in vitro cultivation. During the period in which the plants were subjected to water stress, higher accumulation of proline and total soluble proteins occurred, and also increased levels of photosynthetic pigments in tissues from plants grown in vitro in medium containing 30 g L-1 sucrose and GA3. There was also, in general, sharper decrease in enzyme activity in medium with the lowest concentration of sucrose and NAA, as well as fluctuations in pigment. Relations between pigments suffered slight reduction in both treatments, and total soluble carbohydrates, sucrose, reducing sugars and starch increased with increasing stress. After rehydration, all plants showed high resilience, presenting for all analyzes values very similar to those of control plants not subjected to dehydration. The results indicate that the in vitro culture in medium supplemented with 30 g L-1 sucrose and tubes that allow gas exchange are the most appropriate. Moreover, the concentration of sucrose added to the culture medium also influences the development of plants after acclimatization, with best results found in plants grown in media supplemented with higher amount of this carbohydrate. It is believed that plants grown under these conditions would have higher capacity of resistance to transplanting and could survive for prolonged periods of water stress, which are often subjected in the natural environment.

<p>Women with pregestational diabetes have a 2-5 fold increased risk of giving birth to malformed babies compared with non-diabetic women. Diabetes-induced oxidative stress in maternal and embryonic tissues has been implicated in the teratogenic process. The malformations are likely to be induced before the seventh week of pregnancy, when the yolk sac is partly responsible for the transfer of metabolites to the embryo, and the uterine blood flow to the implantation site determines the net amount of nutrients available to the conceptus. We aimed to evaluate the effect on embryogenesis caused by a diabetes-induced disturbance in yolk sac morphology, uterine blood flow or altered maternal antioxidative status in conjunction with a varied severity of the maternal diabetic state.</p><p>We investigated to which extent maternal diabetes with or without folic acid (FA) supplementation affects mRNA levels and protein distribution of ROS scavenging enzymes (SOD, CAT, GPX), vascular endothelial growth factor-A (Vegf-A), folate binding protein-1 (Folbp-1), and apoptosis associated proteins (Bax, Bcl-2, Caspase-3) in the yolk sacs of rat embryos on gestational days 10 and 11. We found that maternal diabetes impairs, and that FA supplementation restores, yolk sac vessel morphology, and that maternal diabetes is associated with increased apoptotic rate in embryos and yolk sacs, as well as impaired SOD gene expression. We assessed uterine blood flow with a laser-Doppler-flow-meter and found increased blood flow to implantation sites of diabetic rats compared with controls. Furthermore, resorbed and malformed offspring showed increased and decreased blood flow to their implantation sites, respectively. In mice with genetically altered CuZnSOD levels, maternal diabetes increased embryonic dysmorphogenesis irrespective of CuZnSOD expression. We thus found the maternal diabetic state to be a major determinant of diabetic embryopathy and that the CuZnSOD status exerts a partial protection for the embryo in diabetic pregnancy. </p>

L’expression ‘programmation périnatale’ est employée pour décrire les effets à long terme d’un environnement gestationel néfaste observés chez la progéniture. Ce concept est aujourd’hui bien reconnu. Notre laboratoire a déjà démontré l’impact de l’hyperglycémie maternelle sur le développement rénal des embryons à l’aide des souris HoxB7-GFP transgéniques (Tg) et qui se traduit par une augmentation des espèces réactives de l’oxygène (ROS) et une néphrogenèse perturbée. Les rejetons affectés présentent ainsi des reins plus petits et possédant un nombre inférieur de néphrons à la naissance, et développent une hypertension et des dommages rénaux à l’âge adulte (20 semaines). Dans la première étude, nous avons tenté de réduire la production excessive de ROS dans les reins en développement par la surexpression de la catalase (CAT). Pour ce faire, nous avons croisé les souris CAT-Tg qui surexpriment la CAT dans les cellules des tubules proximaux rénaux (RPTCs) aux souris HoxB7-GFP-Tg afin de générer les souris HoxB7/CAT-GFP-Tg. Nous espérons observer la normalisation du nombre de néphrons et la prévention de l’hypertension et des dommages rénaux observés chez la progéniture issue d’un environnement gestational hyperglycémique. Nous avons observé que la surexpression de CAT dans les RPTCs permet de normaliser la dysmorphogenèse rénale présente chez les embryons de mères diabétiques. À l’âge adulte, la surexpression de CAT dans les RPTCs permet également de réduire la génération des ROS et l’hypertension, tout en améliorant la morphologie et la fonction rénale. Afin de définir les mécanismes impliqués dans ce processus, nous avons étudié le rôle potentiel de Nrf2 (‘nuclear factor-erythroid 2p45 (NF-E2) related factor-2’; un facteur de transcription des gènes antioxidants) et HO-1 (hème oxygénase-1’; une enzyme antioxidante). À la fois Nrf2 et HO-1 sont de forts antioxidants et ont été rapportés comme protecteurs pour le rein. Nous avons observé une surexpression des gènes et protéines Nrf2 et HO-1, en plus d’une translocation nucléaire accrue de Nrf2, dans les RPTCs de la progéniture des mères diabétiques, indiquant que chez les souris surexprimant CAT, Nrf2 et HO-1 sont tous deux bien activés et fonctionnels. En conclusion, nos études suggèrent que la surexpression de CAT dans les RPTCs permet de prévenir la programmation de l’hypertension et les dommages rénaux observés à l’âge adulte chez la progéniture issue de mères diabétiques, en partie suite à l’activation du système de défense Nrf2-HO-1 dans leurs reins. Il a déjà été démontré que l’activation du système rénine-angiotensine (RAS) intrarénal induit l’hypertension en augmentant la constriction des artérioles et la réabsorption du sodium par les tubules rénaux. Une activation du récepteur AT1R et de ses voies de signalisation induit également les dommages rénaux observés dans plusieurs pathologies. Dans le cadre de mon second article, nous avons identifié un nouveau mécanisme par lequel l’angiotensine (Agt) intrarénale induit l’hypertension et des dommages rénaux en réduisant l’expression de l’aquaporine 1 (AQP1, le canal pour l’eau le plus important dans les RPTCs). Des souris transgéniques surexprimant l’Agt de rat (rAgt-Tg) dans leurs RPTCs et des clones stables de cellules immortalisées de tubule proximal de rein de rat (IRPTCs) surexprimant le rAgt (pRSV/rAgt-IRPTC) ont été étudiés. Lorsque comparés aux souris non-transgéniques, les souris rAgt-Tg développent de l’hypertension et des dommages rénaux. Ces changements sont atténués par le traitement avec une double inhibition du RAS (losartan et perindopril). L’expression des protéines AQP1 et HO-1 est réduite dans les RPTCs, tandis que Nrf2 et le transporteur sodique NHE3 sont augmentés, à la fois in vivo et in vitro. Ces changements sont renversés par la double inhibition du RAS chez les animaux expérimentaux. Même si les niveaux de Nrf2 sont élevés, une accumulation cytosolique causée par une augmentation de l’export nucléaire induit par GSK3β se produit et ne parvient donc pas à induire l’expression des gènes en aval comme HO-1, ni à réduire l’expression de l’AQP1. En conclusion, nos résultats suggèrent qu’une déficience en Nrf2 nucléaire mène à une diminution de l’expression de HO-1 et une régulation négative de l’AQP1, jouant un role dans l’hypertension et les dommages rénaux induits par l’Agt intrarénal. L’hypertension et les dommages rénaux sont des maladies très hétérogènes et multifactorielles qui impliquent l’interaction de diverses molécules et voies de signalisations, et sont influencées par plusieurs facteurs environnementaux tels la diète ou la programmation périnatale. Tous ces différents facteurs contribuent à la progression de l’hypertension et des dommages rénaux, rendant les stratégies de traitement d’autant plus complexes. Dans notre étude, nous avons évalué le développement de l’hypertension dans deux circonstances : l’hypertension de la progéniture programmée par le diabète maternel et l’hypertension induite par l’activation du RAS intrarénal. Nous avons démontré que la génération des ROS dans les reins constitue un facteur majeur commun dans nos deux modèles d’hypertension chez la souris. De plus, le gène/facteur de transcription antioxydant Nrf2, sensible aux ROS, joue un rôle important dans le processus. Grâce à une meilleure compréhension des diverses voies qui mènent à la progression de l’hypertension, nous espérons qu’il sera possible de développer de meilleurs traitements pour faire face à l’hypertension. / The term ‘perinatal programming’ is used to describe the phenomenon that maternal adverse environment during pregnancies which have profound influences to their offspring later in life. And this concept is well accepted. Previously, we successfully created an in vivo murine model and demonstrated that maternal diabetes constitutes an adverse in utero environment that may fundamentally impair nephrogenesis and subsequently program of the offspring to develop hypertension and kidney injury in adulthood. It appears that enhanced reactive oxygen species (ROS) generation, activation of the nuclear factor-kappa B (NF-kB), intrarenal renin- angiotensin system (RAS) and p53 pathways were involved in the underlying mechanisms. In our first study, we investigated whether overexpression of catalase (CAT) in renal proximal tubular cells (RPTCs) could prevent the perinatal programming of hypertension and kidney injury in male offspring of diabetic dams and examined the potential underlying mechanisms both in vivo and in vitro. Our data demonstrate that CAT overexpression in RPTCs exert a direct effect on nephrogenesis in utero and ameliorate maternal diabetes- induced dysnephrogenesis. And further consequently, CAT overexpression in RPTCs preventing maternal diabetes-induced perinatal programming, mediated at least in part, via the nuclear factor-erythroid 2p45 (NF-E2) related factor-2 (Nrf2)- heme oxygenase (HO)- 1 defense system. Intrarenal RAS activation has attracted more attention in recent years due to studies have been reported that activation of the intrarenal RAS can elicit hypertension and kidney injury independently from the systemic RAS. Previously, we established a murine model (Agt-Tg) that specifically overexpress rat angiotensinogen (Agt) in their RPTCs and develops hypertension and nephropathy. Aquaporin 1 (AQP1) is the major water channel within renal RPTCs, but whether it has a regulatory role in the development of hypertension and nephropathy remains elusive. Our second study aimed to examine the regulation of AQP1 expression in an intrarenal RAS-induced hypertension and kidney injury, focusing on underlying molecular mechanisms. We believe that both our in vivo and in vitro studies identified a novel mechanism(s) in which Agt overexpression in RPTCs enhances cytosolic accumulation of Nrf2 via the phosphorylation of pGSK3β Y216. Consequently, less intranuclear Nrf2 is available to trigger HO-1 expression as a defense mechanism and subsequently diminishes AQP1 expression in RPTCs. In conclusion, our data suggest that Agt mediated-downregulation of AQP1 and Nrf2 signaling may play an important role in intrarenal RAS-induced hypertension and kidney injury. Hypertension and kidney injury is a heterogeneous and multifactorial disease that involves the interaction of various molecules/pathways and the influence of environmental factors, for instance, diet and perinatal programming. Such diverse causes contribute to the progression of hypertension and kidney disease, making the strategy of treatment even more complex. In our present study, we evaluated the development of hypertension under two circumstances: maternal diabetes-programmed hypertension in offspring and intrarenal RAS activation-induced hypertension. We found that ROS generation in the kidneys is a major and common factor in both hypertensive mice model. Also, the ROS-sensitive antioxidant gene/transcription factor – Nrf2, plays an important role in the process. By understanding the pathways that lead to hypertension progression, we can hopefully develop more effective treatments to cope with the disease.

Women with pregestational diabetes have a 2-5 fold increased risk of giving birth to malformed babies compared with non-diabetic women. Diabetes-induced oxidative stress in maternal and embryonic tissues has been implicated in the teratogenic process. The malformations are likely to be induced before the seventh week of pregnancy, when the yolk sac is partly responsible for the transfer of metabolites to the embryo, and the uterine blood flow to the implantation site determines the net amount of nutrients available to the conceptus. We aimed to evaluate the effect on embryogenesis caused by a diabetes-induced disturbance in yolk sac morphology, uterine blood flow or altered maternal antioxidative status in conjunction with a varied severity of the maternal diabetic state. We investigated to which extent maternal diabetes with or without folic acid (FA) supplementation affects mRNA levels and protein distribution of ROS scavenging enzymes (SOD, CAT, GPX), vascular endothelial growth factor-A (Vegf-A), folate binding protein-1 (Folbp-1), and apoptosis associated proteins (Bax, Bcl-2, Caspase-3) in the yolk sacs of rat embryos on gestational days 10 and 11. We found that maternal diabetes impairs, and that FA supplementation restores, yolk sac vessel morphology, and that maternal diabetes is associated with increased apoptotic rate in embryos and yolk sacs, as well as impaired SOD gene expression. We assessed uterine blood flow with a laser-Doppler-flow-meter and found increased blood flow to implantation sites of diabetic rats compared with controls. Furthermore, resorbed and malformed offspring showed increased and decreased blood flow to their implantation sites, respectively. In mice with genetically altered CuZnSOD levels, maternal diabetes increased embryonic dysmorphogenesis irrespective of CuZnSOD expression. We thus found the maternal diabetic state to be a major determinant of diabetic embryopathy and that the CuZnSOD status exerts a partial protection for the embryo in diabetic pregnancy.

Στόχος της παρούσας μελέτης ήταν να μελετήσει αν το εκχύλισμα των στιγμάτων του Crocus sativus L. αναστέλλει την επαγόμενη από σεληνιώδες νάτριο ανάπτυξη καταρράκτη σε ένα in vivo πείραμα με νεογνά επίμυων του γένους Wistar. Μέθοδοι: Τα νεογνά των επίμυων κατατάχθηκαν τυχαία σε 3 ομάδες. Ομάδα Ι (ομάδα μαρτύρων) όπου χορηγήθηκε υποδόρια φυσιολογικός ορός τη 10η ημέρα της ζωής. Ομάδα ΙΙ (ομάδα σεληνιώδους νατρίου) στην οποία χορηγήθηκε υποδόρια σεληνιώδες νάτριο (20 µmol/kg σωματικού βάρους) τη 10η ημέρα της ζωής. Ομάδα ΙΙΙ (ομάδα σεληνιώδους νατρίου και εκχυλίσματος στιγμάτων Crocus sativus L.) στην οποία εκτός από το σεληνιώδες νάτριο τη 10η ημέρα της ζωής χορηγήθηκε και εκχύλισμα στιγμάτων του Crocus sativus L. (60 mg/kg σωματικού βάρους) την 9η και 12η ημέρα της ζωής. Την 21η ημέρα της ζωής οι επίμυες θυσιάστηκαν και οι κρυσταλλοειδείς φακοί απομονώθηκαν και εξετάστηκαν για την εμφάνιση καταρράκτη. Ακολούθησε προσδιορισμός στους κρυσταλλοειδείς φακούς της δραστικότητας των αντιοξειδωτικών ενζύμων δισμουτάση του σουπεροξειδίου (SOD), της υπεροξειδάσης της γλουταθειόνης (GPx) καθώς και της καταλάσης (CAT). Προσδιορίστηκαν επίσης τα επίπεδα της γλουταθειόνης στους φακούς. Επιπλέον, μετρήθηκαν τα επίπεδα της μηλονικής διαλδεΰδης (MDA), ως δείκτη υπεροξείδωσης των λιπιδίων, καθώς και η συγκέντρωση των ελεύθερων σουλφυδρυλομάδων, ως δείκτη οξειδωτικής βλάβης των πρωτεϊνών, στους κρυσταλλοειδείς φακούς των επίμυων. Η επίδραση των χορηγούμενων παραγόντων στο πρωτεϊνικό προφίλ των φακών εκτιμήθηκε μέσω προσδιορισμού του λόγου των υδατοδιαλυτών προς τις μη υδατοδιαλυτές πρωτεΐνες του φακού. Τέλος έγινε ανάλυση των υδατοδιαλυτών πρωτεϊνών με ηλεκτροφόρηση σε πήκτωμα πολυακρυλαμιδίου. Αποτελέσματα: Το εκχύλισμα αποξηραμένων στιγμάτων του Crocus sativus L. επέδειξε σημαντική προστασία έναντι στην επαγόμενη από σεληνιώδες νάτριο καταρρακτογένεση στο in vivo πειραματικό μοντέλο που χρησιμοποιήσαμε. Οι μέσες τιμές των δραστικοτήτων των αντιοξειδωτικών ενζύμων SOD, GPx, CAT καθώς και της συγκέντρωσης της γλουταθειόνης αυξήθηκαν σημαντικά στην ομάδα που έλαβε εκχύλισμα στιγμάτων του Crocus sativus L. σε σύγκριση με την ομάδα των επίμυων που δέχθηκε μόνο την τοξική δράση του σεληνιώδους νατρίου. Το εκχύλισμα των στιγμάτων του Crocus sativus L. απέτρεψε σε σημαντικό βαθμό την υπεροξείδωση των λιπιδίων καθώς και την οξειδωτική βλάβη στις πρωτεΐνες του φακού. Επίσης απέτρεψε την πρωτεόλυση των υδατοδιαλυτών πρωτεϊνών του φακού. Συμπεράσματα: Χορήγηση εκχυλίσματος αποξηραμένων στιγμάτων του Crocus sativus L. απέτρεψε την επαγόμενη από το σεληνιώδες νάτριο καταρρακτογένεση σε νεογνά επίμυων του γένους Wistar πιθανώς μέσω ενίσχυσης της αντιοξειδωτικής άμυνας του κρυσταλλοειδούς φακού, μέσω αναστολής του βαθμού της υπεροξείδωσης των λιπιδίων, μέσω προστασίας των σουλφυδρυλομάδων των πρωτεϊνών καθώς και μέσω αναστολής της πρωτεόλυσης των υδατοδιαλυτών πρωτεϊνών του φακού. Αυτά τα ευρήματα τονίζουν τις πιθανές αντικαταρρακτογενετικές δράσεις των αποξηραμένων στιγμάτων του Crocus sativus L. οι οποίες αποδίδονται στις αντιοξειδωτικές τους ιδιότητες. / The present study sought to investigate whether Crocus sativus L. stigmas extract prevents selenite-induced cataractogenesis in vivo and to study the possible protective mechanism. Methods: Wistar rat pups were randomized into 3 groups. Group I (control) received subcutaneous injection of normal saline on postnatal day 10. Groups II (selenite treated) and III (selenite and Crocus sativus L. treated) received subcutaneous injection of sodium selenite (20 µmol/kg body weight) on postnatal day 10. Group III received intraperitoneal injections of Crocus sativus L. stigmas extract (60 mg/kg body weight) on postnatal days 9 and 12. On postpartum day 21, rats were sacrificed and the lenses were isolated and examined for cataract formation. Activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) and glutathione levels, as markers of antioxidant defense, were measured in the isolated lenses. Levels of the indicator of lipid peroxidation, malondialdehyde (MDA), and protein oxidation (sulfhydryl content) in lens were also determined. Effect of the different treatments on lens’ protein profile was evaluated with the estimation of soluble to insoluble protein ratio and SDS-PAGE analysis of water-soluble fraction (WSF) of lens proteins. Results: Crocus sativus L. stigmas extract demonstrated significant protection against selenite-induced cataractogenesis in vivo. The mean activities of SOD, GPx, CAT and glutathione levels were significantly increased in group III compared to the selenite-treated group. Crocus sativus L. stigmas extract significantly prevented selenite-induced lipid peroxidation, protein oxidation, as well as proteolysis and insolubilization of the lens WSF. Conclusions: Crocus sativus L. stigmas extract prevented selenite-induced cataract formation in Wistar rats possibly by reinforcement of antioxidant status, reduction of the intensity of lipid peroxidation, protection of the sulfhydryl groups, and inhibition of proteolysis of the lens WSF. These findings highlight the anti-cataractogenic potential of Crocus sativus L. stigmas by virtue of their antioxidant properties.

No description available.

L'étude de l'activité de la catalase immobilisée nous a permis de trouver là un catalyseur qui permet une décomposition rapide de l'eau oxygénée. On peut facilement préparer l'enzyme fixée et en assurer une bonne conservation au froid : Son activité reste la même pendant un mois et ne diminue que très légèrement au bout de six mois. Nous avons montré que l'activité de la catalase fixée diminue à partir d'un certain seuil de température. Pour maintenir cette activité, nous avons intérêt à travailler dans un domaine de faibles concentrations en eau oxygénée afin de ne pas trop élever la température Ceci provient du fait que, la cinétique de la réaction étant approximativement d'ordre 1 dans ces conditions, la quantité de chaleur dégagée est proportionnelle à la concentration de l'eau oxygénée utilisée. L'analyse théorique de la diffusion-réaction a permis de voir que la température de la membrane enzymatique est proportionnelle à la concentration en eau oxygénée utilisée ; Elle atteint sa valeur maximale au milieu de la membrane. Ces conclusions ne sont valables que dans un domaine assez restreint de températures. Dans le cas où il n'est pas possible de travailler à faible concentration, il est nécessaire de maintenir une température faible au niveau de l'enzyme par un refroidissement tel que nous l'avons décrit dans la deuxième partie. Nous pouvons penser que, soit par une dilution progressive soit par une membrane protectrice à gradient de H₂0₂, nous pourrions concilier le stockage peu encombrant de l'eau oxygénée concentrée et son utilisation à l'état dilué nécessaire pour préserver l'activité enzymatique. Une application directe de l'utilisation de la catalase fixée s'est matérialisée dans le réacteur enzymatique que nous avons construit. Le caractère exothermique de la réaction a pu être maîtrisé grâce à la conception et à la réalisation d'un réacteur qui permet de bien fixer et protéger la couche d'enzyme et d'évacuer les calories. On empêche aussi une baisse d'activité trop rapide de l'enzyme. Le réacteur, de dimensions modestes, peut fournir, à la pression atmosphérique, de l'oxygène à raison de 40 litres/heure, à partir de 260 mg de catalase et d'une surface géométrique de mousse enzymatique égale à 300 cm². Le nettoyage du réacteur est aisé du fait de l'utilisation des ailettes démontables. Grâce à son pouvoir d'emmagasiner de l'oxygène sous un faible volume [jusqu'à 478 litres d'oxygène par litre d'eau oxygénée pure], l'eau oxygénée nous permet d'avoir rapidement à notre disposition de l'oxygène au moyen du réacteur à catalase fixée. L'utilisation, pour l'alimentation en oxygène d'une personne, de ce réacteur sous la forme décrite ne semble pas poser de problèmes importants ; Il pourrait être utile notamment pour des premiers secours. Sous pression, nous avons vu que l'enzyme fixée donne lieu à une réaction comparable à celle sous pression atmosphérique. A 50°C, l'utilisation de pressions de plus en plus élevées pour la réaction de décomposition de l'eau oxygénée à 0,5 mole par litre provoque une baisse de l'activité apparente de la catalase fixée. Des températures de travail trop élevées provoquent, sous pression, une dénaturation rapide de l'enzyme fixée par effet thermique. Les résultats de l'expérience de fonctionnement de la catalase immobilisée sous 2 atmosphères, à 50°C, en utilisant une concentration en eau oxygénée de 0,05 à 0,5 mole par litre, montrent qu'il n'y a pas dénaturation de l'enzyme dans ces conditions. Si la cinétique de la réaction a pu ainsi être étudiée jusqu'à 5 atmosphères, l'utilisation du réacteur enzymatique en vue des plongées sous-marines de courte durée demande, quant à elle, une mise au point ultérieure plus approfondie.