Methylmercury (MeHg) is a pervasive environmental contaminant with potent neurotoxic, teratogenic and likely carcinogenic activity, for which the underlying molecular mechanisms remain largely unclear. Base excision repair (BER) is important in mitigating the pathogenic effects of oxidative stress, which has also been implicated in the mechanism of MeHg toxicity, however the importance of BER in MeHg toxicity is currently unknown. Accordingly, we addressed this question using: (1) spontaneously- and Simian virus 40 (SV40) large T antigen-immortalized oxoguanine glycosylase 1-null (Ogg1-/-) murine embryonic fibroblasts (MEFs); and, (2) human Ogg1 (hOgg1)- or formamidopyrimidine glycosylase (Fpg)-expressing human embryonic kidney (HEK) cells; reciprocal in vitro cellular models with deficient and enhanced ability to repair oxidatively damaged DNA respectively. When spontaneously-immortalized wild-type and Ogg1-/- MEFs were exposed to environmentally relevant, low micromolar concentrations of MeHg, both underwent cell cycle arrest but Ogg1-/- cells exhibited a greater sensitivity to MeHg than wild-type controls with reduced clonogenic survival and increased apoptosis, DNA damage and DNA damage response activation. Antioxidative catalase alleviated the MeHg-initiated DNA damage in both wild-type and Ogg1-/- cells, but failed to block MeHg-mediated apoptosis at micromolar concentrations. As in spontaneously immortalized MEFs, MeHg induced cell cycle arrest in SV40 large T antigen-immortalized MEFs, with increased sensitivity to MeHg persisting in the Ogg1-/- MEFs. Importantly, cells seeded at a higher density exhibited compromised proliferation, which protected against MeHg-mediated cell cycle arrest and DNA damage. In the reciprocal model of enhanced DNA repair, hOgg1- and Fpg-expressing cells appeared paradoxically more sensitive than wild-type controls to acute MeHg exposure for all cellular and biochemical parameters, potentially due to the accumulation of toxic intermediary abasic sites. Accordingly, our results provide the first evidence that Ogg1 status represents a critical determinant of risk for MeHg toxicity independent of cellular immortalization method, with variations in cellular proliferation and interindividual variability in antioxidative and DNA repair capacities constituting important determinants of risk for environmentally-initiated oxidatively damaged DNA and its pathological consequences.

Methylmercury (MeHg) is a pervasive environmental contaminant with potent neurotoxic, teratogenic and likely carcinogenic activity, for which the underlying molecular mechanisms remain largely unclear. Base excision repair (BER) is important in mitigating the pathogenic effects of oxidative stress, which has also been implicated in the mechanism of MeHg toxicity, however the importance of BER in MeHg toxicity is currently unknown. Accordingly, we addressed this question using: (1) spontaneously- and Simian virus 40 (SV40) large T antigen-immortalized oxoguanine glycosylase 1-null (Ogg1-/-) murine embryonic fibroblasts (MEFs); and, (2) human Ogg1 (hOgg1)- or formamidopyrimidine glycosylase (Fpg)-expressing human embryonic kidney (HEK) cells; reciprocal in vitro cellular models with deficient and enhanced ability to repair oxidatively damaged DNA respectively. When spontaneously-immortalized wild-type and Ogg1-/- MEFs were exposed to environmentally relevant, low micromolar concentrations of MeHg, both underwent cell cycle arrest but Ogg1-/- cells exhibited a greater sensitivity to MeHg than wild-type controls with reduced clonogenic survival and increased apoptosis, DNA damage and DNA damage response activation. Antioxidative catalase alleviated the MeHg-initiated DNA damage in both wild-type and Ogg1-/- cells, but failed to block MeHg-mediated apoptosis at micromolar concentrations. As in spontaneously immortalized MEFs, MeHg induced cell cycle arrest in SV40 large T antigen-immortalized MEFs, with increased sensitivity to MeHg persisting in the Ogg1-/- MEFs. Importantly, cells seeded at a higher density exhibited compromised proliferation, which protected against MeHg-mediated cell cycle arrest and DNA damage. In the reciprocal model of enhanced DNA repair, hOgg1- and Fpg-expressing cells appeared paradoxically more sensitive than wild-type controls to acute MeHg exposure for all cellular and biochemical parameters, potentially due to the accumulation of toxic intermediary abasic sites. Accordingly, our results provide the first evidence that Ogg1 status represents a critical determinant of risk for MeHg toxicity independent of cellular immortalization method, with variations in cellular proliferation and interindividual variability in antioxidative and DNA repair capacities constituting important determinants of risk for environmentally-initiated oxidatively damaged DNA and its pathological consequences.

Orientador: Fernando Braz Tangerino Hernandez / Resumo: A ocorrência de geadas nas pastagens causa perdas de alimento para os animais, reduzindo a produção de leite e carne. Existe uma diversidade de espécies forrageiras com grande potencial produtivo e ampla adaptação térmica, que podem ser cultivadas em áreas de risco desse fenômeno. No entanto é necessário caracterizar a tolerância dessas espécies a baixas temperaturas e as regiões com condições climáticas adequadas. Os métodos de avaliação de danos por baixas temperaturas são predominantemente qualitativos, baseados em critérios visuais que têm o viés da subjetividade. Esta tese teve como objetivos determinar as temperaturas mínimas de início de danos para sete espécies forrageiras e efetuar o zoneamento de risco de geadas para o estado do Paraná. Foram incluídas no estudo as forrageiras: Alfafa (Medicago sativa), Sorgo (Sorghum bicolor), Aveia Preta (Avena strigosa), Brachiaria brizantha cv. Marandu, Milheto (Pennisetum glaucum), capim Mombaça (Panicum maximum) e Tifton 85 (Cynodon spp). As plantas foram conduzidas em vasos em casa de vegetação até 60 dias e submetidas a baixas temperaturas no interior de uma câmara de crescimento com condições de luminosidade e temperatura controladas, atingindo valores mínimos de 0,2 -0,9, -1,8, -2,7, -4,1, -4,6 e -6,2oC, durante uma hora. Foram realizadas avaliações quantitativas pós testes de fluorescência da clorofila, condutividade elétrica de solução embebida com discos de folhas e atividade das enzimas ascorbato peroxidase (APX), cata... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Introdução: A fisiopatologia da doença de Parkinson está associada a lesões por estresse oxidativo/nitrosativo. O ferro encontra-se acumulado na substância negra (SN) de pacientes com DP e está relacionado com esse dano através das espécies reagentes de oxigênio (EROs) e de nitrogênio (ERNs) na reação de Fenton. EROs e ERNs são produzidas normalmente em processos celulares e inflamatórios, e controladas por sistemas antioxidantes. Objetivo: Avaliar níveis periféricos de ferro em pacientes com DP para determinar se acúmulo na SN está relacionado com níveis elevados no sangue. Determinar biomarcadores periféricos confiáveis de estresse oxidativo/nitrosativo Métodos: Selecionados 40 pacientes com DP e 46 indivíduos controles para comparar níveis séricos de ferro, ferritina e transferrina, e de biomarcadores de estresse oxidativo/nitrosativo: superóxido dismutase (SOD), catalase, óxido nítrico (NOx), substâncias reativas ao ácido tiobarbitúrico (TBARS), tióis não-proteicos, “advanced oxidation protein products” (AOPP), “ferric reducing ability of plasma” (FRAP), NTPDases, ecto-5’-nucleotidase, adenosina deaminase (ADA), mieloperoxidase, albumina modificada pela isquemia (IMA) e vitamina C. Resultados: Níveis de ferro estavam diminuídos em pacientes com DP, enquanto ferritina e transferrina não mostraram diferença. Os biomarcadores de estresse oxidativo como TBARS, AOPP, NTPDases, IMA, mieloperoxidase, FRAP, vitamina C e tiois não-proteicos encontraram-se significativamente aumentados na DP. SOD, catalase, ecto-5’-nucleotidase não foram diferentes entre os grupos e os marcadores NOx e ADA foram significativamente aumentados nos controles. Nenhuma correlação foi encontrada entre os biomarcadores e dados sociodemográficos e de características da doença. Conclusão: Níveis plasmáticos de ferro encontram-se diminuídos em pacientes com DP comparados com controles saudáveis. Os biomarcadores TBARS, AOPP, NTPDases, IMA e mieloperoxidase mostraram-se confiáveis para lesão oxidativa, enquanto tióis não-proteicos, FRAP e vitamina C demonstram diminuição da capacidade antioxidante na DP. / Background: Parkinson’s disease (PD) pathophysiology is associated with oxidative/nitrosative stress damage. Iron accumulates in the substantia nigra (SN) of PD patients and is related to this damage along with oxygen and nitrogen reactive species (ROS, RNS) through Fenton reaction. ROS and RNS are normally produced in cell and inflammatory processes, controlled by antioxidant systems. Objective: To determine peripheral levels of iron, ferritin and transferrin in PD patients to evaluate whether iron accumulation in the SN could be related to serum levels. To determine reliable peripheral biomarkers of oxidative/nitrative stress. Methods: Forty PD patients and 46 controls were selected to compared serum levels of iron, ferritin, transferrin and oxidative/nitrative stress biomarkers: superoxide dismutase (SOD), catalase, nitric oxide (NOx), thiobarbituric acid reactive substances (TBARS), non-protein thiols, advanced oxidation protein products (AOPP), ferric reducing ability of plasma (FRAP), NTPDases, ecto-5’-nucleotidase, adenosine deaminase (ADA), myeloperoxidase, ischemic-modified albumin (IMA) and vitamin C. Results: Iron levels were decreased in patients with PD, while ferritin and transferrin were not different. Oxidative stress biomarkers, TBARS, AOPP, NTPDases, IMA, myeloperoxidase, FRAP, vitamin C and non-proteic thiols were significantly higher in PD. SOD, catalase, ecto-5’-nucleotidase were not different between the groups and biomarkers NOx and ADA were significantly increased in the controls. No correlation was found between biomarkers and sociodemographic and disease data. Conclusion: Plasmatic levels of iron are decreased in patients with PD compared to healthy controls. Biomarkers TBARS, AOPP, NTPDases, IMA and myeloperoxidase presented as reliable to measure oxidative/nitrative damage, while non-proteic thiols, FRAP and vitamin C show a decrease in the antioxidant capacity in PD.

Introdução: A fisiopatologia da doença de Parkinson está associada a lesões por estresse oxidativo/nitrosativo. O ferro encontra-se acumulado na substância negra (SN) de pacientes com DP e está relacionado com esse dano através das espécies reagentes de oxigênio (EROs) e de nitrogênio (ERNs) na reação de Fenton. EROs e ERNs são produzidas normalmente em processos celulares e inflamatórios, e controladas por sistemas antioxidantes. Objetivo: Avaliar níveis periféricos de ferro em pacientes com DP para determinar se acúmulo na SN está relacionado com níveis elevados no sangue. Determinar biomarcadores periféricos confiáveis de estresse oxidativo/nitrosativo Métodos: Selecionados 40 pacientes com DP e 46 indivíduos controles para comparar níveis séricos de ferro, ferritina e transferrina, e de biomarcadores de estresse oxidativo/nitrosativo: superóxido dismutase (SOD), catalase, óxido nítrico (NOx), substâncias reativas ao ácido tiobarbitúrico (TBARS), tióis não-proteicos, “advanced oxidation protein products” (AOPP), “ferric reducing ability of plasma” (FRAP), NTPDases, ecto-5’-nucleotidase, adenosina deaminase (ADA), mieloperoxidase, albumina modificada pela isquemia (IMA) e vitamina C. Resultados: Níveis de ferro estavam diminuídos em pacientes com DP, enquanto ferritina e transferrina não mostraram diferença. Os biomarcadores de estresse oxidativo como TBARS, AOPP, NTPDases, IMA, mieloperoxidase, FRAP, vitamina C e tiois não-proteicos encontraram-se significativamente aumentados na DP. SOD, catalase, ecto-5’-nucleotidase não foram diferentes entre os grupos e os marcadores NOx e ADA foram significativamente aumentados nos controles. Nenhuma correlação foi encontrada entre os biomarcadores e dados sociodemográficos e de características da doença. Conclusão: Níveis plasmáticos de ferro encontram-se diminuídos em pacientes com DP comparados com controles saudáveis. Os biomarcadores TBARS, AOPP, NTPDases, IMA e mieloperoxidase mostraram-se confiáveis para lesão oxidativa, enquanto tióis não-proteicos, FRAP e vitamina C demonstram diminuição da capacidade antioxidante na DP. / Background: Parkinson’s disease (PD) pathophysiology is associated with oxidative/nitrosative stress damage. Iron accumulates in the substantia nigra (SN) of PD patients and is related to this damage along with oxygen and nitrogen reactive species (ROS, RNS) through Fenton reaction. ROS and RNS are normally produced in cell and inflammatory processes, controlled by antioxidant systems. Objective: To determine peripheral levels of iron, ferritin and transferrin in PD patients to evaluate whether iron accumulation in the SN could be related to serum levels. To determine reliable peripheral biomarkers of oxidative/nitrative stress. Methods: Forty PD patients and 46 controls were selected to compared serum levels of iron, ferritin, transferrin and oxidative/nitrative stress biomarkers: superoxide dismutase (SOD), catalase, nitric oxide (NOx), thiobarbituric acid reactive substances (TBARS), non-protein thiols, advanced oxidation protein products (AOPP), ferric reducing ability of plasma (FRAP), NTPDases, ecto-5’-nucleotidase, adenosine deaminase (ADA), myeloperoxidase, ischemic-modified albumin (IMA) and vitamin C. Results: Iron levels were decreased in patients with PD, while ferritin and transferrin were not different. Oxidative stress biomarkers, TBARS, AOPP, NTPDases, IMA, myeloperoxidase, FRAP, vitamin C and non-proteic thiols were significantly higher in PD. SOD, catalase, ecto-5’-nucleotidase were not different between the groups and biomarkers NOx and ADA were significantly increased in the controls. No correlation was found between biomarkers and sociodemographic and disease data. Conclusion: Plasmatic levels of iron are decreased in patients with PD compared to healthy controls. Biomarkers TBARS, AOPP, NTPDases, IMA and myeloperoxidase presented as reliable to measure oxidative/nitrative damage, while non-proteic thiols, FRAP and vitamin C show a decrease in the antioxidant capacity in PD.

Introdução: A fisiopatologia da doença de Parkinson está associada a lesões por estresse oxidativo/nitrosativo. O ferro encontra-se acumulado na substância negra (SN) de pacientes com DP e está relacionado com esse dano através das espécies reagentes de oxigênio (EROs) e de nitrogênio (ERNs) na reação de Fenton. EROs e ERNs são produzidas normalmente em processos celulares e inflamatórios, e controladas por sistemas antioxidantes. Objetivo: Avaliar níveis periféricos de ferro em pacientes com DP para determinar se acúmulo na SN está relacionado com níveis elevados no sangue. Determinar biomarcadores periféricos confiáveis de estresse oxidativo/nitrosativo Métodos: Selecionados 40 pacientes com DP e 46 indivíduos controles para comparar níveis séricos de ferro, ferritina e transferrina, e de biomarcadores de estresse oxidativo/nitrosativo: superóxido dismutase (SOD), catalase, óxido nítrico (NOx), substâncias reativas ao ácido tiobarbitúrico (TBARS), tióis não-proteicos, “advanced oxidation protein products” (AOPP), “ferric reducing ability of plasma” (FRAP), NTPDases, ecto-5’-nucleotidase, adenosina deaminase (ADA), mieloperoxidase, albumina modificada pela isquemia (IMA) e vitamina C. Resultados: Níveis de ferro estavam diminuídos em pacientes com DP, enquanto ferritina e transferrina não mostraram diferença. Os biomarcadores de estresse oxidativo como TBARS, AOPP, NTPDases, IMA, mieloperoxidase, FRAP, vitamina C e tiois não-proteicos encontraram-se significativamente aumentados na DP. SOD, catalase, ecto-5’-nucleotidase não foram diferentes entre os grupos e os marcadores NOx e ADA foram significativamente aumentados nos controles. Nenhuma correlação foi encontrada entre os biomarcadores e dados sociodemográficos e de características da doença. Conclusão: Níveis plasmáticos de ferro encontram-se diminuídos em pacientes com DP comparados com controles saudáveis. Os biomarcadores TBARS, AOPP, NTPDases, IMA e mieloperoxidase mostraram-se confiáveis para lesão oxidativa, enquanto tióis não-proteicos, FRAP e vitamina C demonstram diminuição da capacidade antioxidante na DP. / Background: Parkinson’s disease (PD) pathophysiology is associated with oxidative/nitrosative stress damage. Iron accumulates in the substantia nigra (SN) of PD patients and is related to this damage along with oxygen and nitrogen reactive species (ROS, RNS) through Fenton reaction. ROS and RNS are normally produced in cell and inflammatory processes, controlled by antioxidant systems. Objective: To determine peripheral levels of iron, ferritin and transferrin in PD patients to evaluate whether iron accumulation in the SN could be related to serum levels. To determine reliable peripheral biomarkers of oxidative/nitrative stress. Methods: Forty PD patients and 46 controls were selected to compared serum levels of iron, ferritin, transferrin and oxidative/nitrative stress biomarkers: superoxide dismutase (SOD), catalase, nitric oxide (NOx), thiobarbituric acid reactive substances (TBARS), non-protein thiols, advanced oxidation protein products (AOPP), ferric reducing ability of plasma (FRAP), NTPDases, ecto-5’-nucleotidase, adenosine deaminase (ADA), myeloperoxidase, ischemic-modified albumin (IMA) and vitamin C. Results: Iron levels were decreased in patients with PD, while ferritin and transferrin were not different. Oxidative stress biomarkers, TBARS, AOPP, NTPDases, IMA, myeloperoxidase, FRAP, vitamin C and non-proteic thiols were significantly higher in PD. SOD, catalase, ecto-5’-nucleotidase were not different between the groups and biomarkers NOx and ADA were significantly increased in the controls. No correlation was found between biomarkers and sociodemographic and disease data. Conclusion: Plasmatic levels of iron are decreased in patients with PD compared to healthy controls. Biomarkers TBARS, AOPP, NTPDases, IMA and myeloperoxidase presented as reliable to measure oxidative/nitrative damage, while non-proteic thiols, FRAP and vitamin C show a decrease in the antioxidant capacity in PD.

Le H2O2 est reconnu comme un signal dans l’activation des mécanismes de défense en réponse à divers stress, et son accumulation est donc régulée étroitement par le système antioxydant des plantes. Puisque la signalisation par le H2O2 peut être transmise par des processus thiol-dépendants, le statut du glutathion pourrait jouer un rôle important. Le rôle de ce composé en tant que molécule antioxydante est bien établi; cependant, son importance en tant que signal reste à élucider. Afin d’étudier cette question, ce travail a utilisé un mutant, cat2, ayant un défaut dans son métabolisme du H2O2 peroxysomal qui engendre, d’une manière conditionnelle, une oxydation et une accumulation du glutathion. Les modifications du glutathion dans cat2 sont accompagnées par l’activation à la fois de réponses dépendantes de l’acide salicylique (SA) ainsi que l’expression de gènes associés à l’acide jasmonique (JA). L’activation des deux voies phytohormonales par le stress oxydant intracellulaire est largement empêchée en bloquant génétiquement l’accumulation du glutathion dans un double mutant, cat2 cad2, qui porte une mutation additionnelle dans la voie de synthèse du glutathion. Les phénotypes contrastants de cat2 cad2 et cat2 gr1, dans lequel la perte de l’activité GR1 aggrave le stress oxydant, suggèrent que des processus glutathion-dépendants relient le H2O2 et l’activation des réponses de pathogenèse SA-dépendantes par un effet qui est additionnel aux fonctions antioxydantes du glutathion. Des comparaisons directes de cat2 cad2 et cat2 npr1 indiquent que les effets de bloquer l’accumulation du glutathion sur l’induction des voies SA et JA chez cat2 ne sont pas causés par une déficience dans la fonction de la NPR1. L’autophagie a été impliquée dans des processus comme la sénescence, et interagirait à la fois avec le stress oxydant et avec la signalisation par le SA. Afin d’explorer des relations entre autophagie et stress oxydant, des mutants atg ont été sélectionnés et croisés avec le cat2. Des analyses phénotypiques ont révélé que l’étendue de lésions SA-dépendantes observée chez cat2 cultivé en jours longs est similaire chez trois double mutants cat2 atg, alors que l’augmentation de la disponibilité en H2O2 peroxysomal liée à la mutation cat2 retarde la sénescence précoce observée chez les mutants atg. Dans son ensemble, le travail suggère que (1) des nouvelles fonctions glutathion-dépendantes sont importantes pour relier la disponibilité en H2O2 intracellulaire et activation des voies de signalisation SA et JA, et (2) que le H2O2 produit par la photorespiration pourrait jouer un rôle antagoniste dans les phénotypes de sénescence précoce observée chez les mutants atg. / H2O2 is a recognized signal in activation of defence mechanisms in response to various stresses, and its accumulation is thus tightly controlled by plant antioxidant systems. Because H2O2 signals may be transmitted by thiol-dependent processes, glutathione status could play an important role. While the antioxidant role of this compound is long established, the importance of glutathione in signaling remains unclear. To study this question, this work exploited a stress mimic mutant, cat2, which has a defect in metabolism of peroxisomal H2O2 that conditionally leads to oxidation and accumulation of glutathione. In cat2, changes in glutathione are accompanied by activation of both salicylic acid (SA)-dependent responses and jasmonic acid (JA)-associated genes in a time-dependent manner. This up-regulation of both phytohormone signaling pathway by intracellular oxidative stress can be largely prevented by genetically blocking glutathione accumulation in a double mutant, cat2 cad2, that additionally carries a mutation in the pathway of glutathione synthesis. Contrasting phenotypes between cat2 cad2 and cat2 gr1, in which loss of GR1 activity exacerbates oxidative stress, suggest that glutathione-dependent processes couple H2O2 to activation of SA-dependent pathogenesis responses through an effect that is additional to glutathione antioxidant functions. Direct comparison of cat2 cad2 and cat2 npr1 double mutants suggests that the effects of blocking glutathione accumulation on cat2-triggered up-regulation of both SA and JA pathways are not mediated by defective NPR1 function. Autophagy has been implicated in processes such as senescence, and may interact with oxidative stress and SA signaling. To explore relationships between autophagy and oxidative stress, selected atg mutants were crossed with cat2. Phenotypic analysis revealed that SA-dependent lesion spread observed in cat2 grown in long days is similar in three cat2 atg double mutants, whereas increased peroxisomal H2O2 availability in cat2 delays an oxidative stress related-senescence triggered by atg in short days. Overall, the work suggests that (1) novel glutathione-dependent functions are important to couple intracellular H2O2 availability to the activation of both SA and JA signaling pathways and (2) H2O2 produced through photorespiration may play an antagonistic role in the early senescence phenotype observed in atg mutants.

De nombreuses études ont bien démontré que l’activation du système rénine-angiotensine (RAS) joue un rôle important dans le développement de l’hypertension et de la néphropathie diabétique (DN). La découverte de l’enzyme de conversion de l’angiotensine-2 (ACE2) et l’identification du récepteur MAS, spécifique pour l’angiotensine 1-7 (Ang 1-7), ont permis d’identifier deux nouveaux membres du RAS. L’axe ACE2/Ang 1-7/MAS contrebalance les effets de l’axe ACE/Ang II/AT1. Plusieurs évidences impliquent la contribution du RAS intrarénal dans la DN. Des études réalisées dans notre laboratoire avec des souris transgéniques surexprimant l’angiotensinogène de rat dans les cellules de leurs tubules proximaux rénaux (RPTCs) ont permis de démontrer l’importance du RAS intrarénal dans l’induction de l’hypertension et les dommages rénaux. Nous avons également observé que l’expression rénale de l’ACE2 et les niveaux urinaires d’ANG 1-7 sont plus faibles chez les souris Akita (diabète de type 1) et qu’un traitement avec des bloqueurs du RAS permet de normaliser l’expression de l’ACE2 et de prévenir le développement de l’hypertension dans le modèle des souris Akita. Dans un milieu diabétique, à la fois la glycémie et l’angiotensine II (Ang II) peuvent induire la génération des espèces réactives de l’oxygène (ROS), contribuant ainsi aux dommages rénaux. Afin d’explorer la relation entre les ROS, ACE2 et la DN, nous avons créé des souris Akita transgéniques surexprimant la catalase (Cat) dans les RPTCs, en croisant des souris Akita diabétique de type 1 à notre modèle de souris transgéniques surexprimant la Cat de rat dans les RPTCs. Dans une seconde étude, des souris Akita ont été traitées avec l’Ang 1-7 ou une combinaison d’Ang 1-7 et de son antagoniste, A779, afin d’étudier la relation entre l’action de l’Ang 1-7, l’hypertension systolique (sHTN), le stress oxydatif, les dommages rénaux, ACE2 et l’expression du récepteur Mas. Nos résultats ont montré que la surexpression de Cat atténue le stress oxydatif rénal; prévient l’hypertension, améliore le taux de filtration glomérulaire, l’albuminurie, l’hypertrophie rénale, la fibrose tubulo-interstitielle et l’apoptose tubulaire; et supprime l’expression des gènes profibrotiques et proapoptotiques dans les RPTCs des souris Akita Cat-Tg lorsque comparées aux souris Akita. De plus, la surexpression de Cat dans les RPTC des souris Akita normalise l’expression rénale de l’ACE2 et les niveaux urinaires d’Ang 1-7. D’autre part, l’administration d’Ang 1-7 prévient l’hypertension systémique, normalise le ratio albumine/créatinine urinaire et atténue l’hyperfiltration glomérulaire des souris Akita, sans affecter la glycémie sanguine. De plus, le traitement avec l’Ang 1-7 atténue aussi le stress oxydatif et l’expression de la NADPH oxydase, Agt, ACE, TGF-β1 (transforming growth factor-β1) et collagène IV, tout en augmentant l’expression de l’ACE2 et du récepteur Mas dans les reins des souris Akita. Ces effets sont renversés par la co-admininstration d’A779. Ces résultats démontrent que la surexpression de Cat prévient l’hypertension et la progression de la néphropathie, en plus de mettre en lumière l’importance du stress oxydatif intrarénal et l’expression de l’ACE2 comme facteurs contribuant à l’hypertension et les dommages rénaux observés dans le diabète. En outre, nos données suggèrent que l’Ang 1-7 joue un rôle protecteur dans l’hypertension et les dommages aux RPTC dans le diabète, principalement en réduisant les voies de signalisations du stress oxydatif dans les reins et en normalisant l’expression de l’ACE2 et du récepteur Mas. Nos résultats indiquent aussi que l’Ang 1-7 pourrait agir comme un agent thérapeutique potentiel dans le traitement de l’hypertension systémique et les dommages rénaux observés dans le diabète. En conséquence, l’Ang 1-7 est responsable du rôle protecteur de l’ACE2 dans l’hypertension et la DN. / It is well accepted that renin-angiotensin system (RAS) activation plays an important role in the development of hypertension and diabetic nephropathy (DN). With the discovery of angiotensin-converting enzyme-2 (ACE2) and recognition of MAS as the receptor of Angiotensin 1-7 (Ang 1-7), new players in RAS, ACE2/Ang 1-7/MAS axis, have been identified to counteract the effect of ACE/Ang II/ AT1 axis. Evidence implicates the intrarenal RAS’s contribution to DN. Previous studies from our laboratory using transgenic mice overexpressing rat Angiotensinogen (Agt) in their renal proximal tubular cells (RPTCs) have demonstrated the importance of the intrarenal RAS in renal damage and the induction of hypertension. We also recently observed that renal ACE2 expression and urinary Ang 1–7 were lower in type 1 diabetic Akita mice and that treatment with RAS blockers normalized ACE2 expression and prevented hypertension development in these Akita mice. In the diabetic milieu, both glycemia and angiotensin II (Ang II) can induce reactive oxygen species (ROS) generation, which contributes to kidney injury. To explore the relationship among ROS, ACE2 and DN, we created Akita transgenic mice overexpressing catalase (Cat) in RPTCs by crossbreeding type I diabetic Akita mice with our established transgenic mice overexpressing rat Cat in RPTCs. In another study, Akita mice were treated with Ang 1-7 or combination of Ang 1-7 and its antagonist, A779, to investigate the relations between Ang 1-7 action, systolic hypertension (sHTN), oxidative stress, kidney injury, ACE2 and Mas receptor expression. Our results showed that overexpression of Cat attenuated renal oxidative stress; prevented hypertension; ameliorated glomerular filtration rate, albuminuria, kidney hypertrophy, tubulointerstitial fibrosis, and tubular apoptosis; and suppressed profibrotic and proapoptotic gene expression in RPTCs of Akita Cat-Tg mice compared with Akita mice. Furthermore, overexpression of Cat in RPTCs of Akita mice normalized renal ACE2 expression and urinary Ang 1–7 levels. On the other hand, Ang 1-7 administration prevented systemic hypertension, normalized urinary albumin/creatinine ratio and attenuated glomerular hyperfiltration without affecting blood glucose levels in Akita mice. Furthermore, Ang 1-7 treatment also attenuated oxidative stress and the expression of NADPH oxidase 4, Agt, ACE, transforming growth factor-β1 (TGF-β1) and collagen IV, and increased the expression of ACE2 and Mas receptor in Akita mouse kidneys. These effects were reversed by co-administration of A779. These data demonstrated that Cat overexpression prevents hypertension and progression of nephropathy and highlight the importance of intrarenal oxidative stress and ACE2 expression contributing to hypertension and renal injury in diabetes. Furthermore, our data suggest that Ang 1-7 plays a protective role in hypertension and RPTC injury in diabetes, predominantly through decreasing renal oxidative stress-mediated signaling and normalizing ACE2 and Mas receptor expression. Our results also indicate Ang 1-7 as a potential therapeutic agent for treatment of systemic hypertension and kidney injury in diabetes. Therefore, Ang 1-7 mediates the major protective role of ACE2 in the hypertension and DN.

No description available.

Background & Aims: Oxidative stress and apoptosis are proposed mechanisms of cellular injury in studies of xenobiotic hepatotoxicity. The aim of this work is to find early signal markers of drug-induced injury of the liver by focusing on select antioxidant/oxidant and apoptotic genes. As well, to address the relationship between conventional liver dysfunction markers and the measured mRNA and protein expressions in the D-galactosamine/lipopolysaccharide and tert-butylhydroperoxide hepatotoxicity models. Furthermore, potential hepatoprotective capabilities of antioxidant polyphenols quercetin and curcumin were evaluated in relation to its modulation of the oxidative stress and apoptotic parameters in the given xenobiotic hepatotoxicity models. Methods: Biochemical markers testing the hepatic function included aminotransferases (ALT, AST) and bilirubin. Measurements of TBARS and conjugated dienes were used to assess lipoperoxidation. Plasma levels of catalase and reduced glutathione were used as indicators of the oxidative status of the cell. Real time PCR was used to analyse the mRNA expressions of the inducible nitric oxide synthase (NOS-2), heme oxygenase-1 (HO-1), superoxide dismutase (SOD-1), glutathione peroxidase (Gpx-1), caspase 3 (Casp3), BH3 interacting domain death agonist (Bid) and Bcl-2...