O sistema antioxidante da glautationa como alvo molecular na neurotoxicidade induzida por metilmercúrio: efeitos protetores de compostos naturais

Tese (doutorado) - Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Programa de Pós-Graduação em NeurociênciasO mercurio, em suas formas organicas e inorganicas, e um toxicante ambiental capaz de provocar deficits neurologicos severos em animais e humanos. O sistema nervoso em desenvolvimento apresenta uma maior suscetibilidade aos efeitos deleterios induzidos por mercuriais. Neste contexto, exposicoes durante o periodo pre#]natal e lactacional representam importantes questoes do ponto de vista toxicologico. De fato, pouco se conhece a respeito da contribuicao lactacional para o desenvolvimento de neurotoxicidade frente a intoxicacoes com mercuriais. Alem disso, nao existem tratamentos efetivos capazes de reverter completamente os efeitos toxicos do mercurio. No presente estudo, foi demonstrado que a exposicao exclusivamente lactacional de camundongos jovens ao metilmercurio, uma forma organica de mercurio, causou deficits motores que foram correlacionados com um desequilibrio no estado tiolico cerebelar. Observamos tambem, que o mercurio inorganico causou neurotoxicidade em camundongos expostos a este metal atraves do leite materno. O cerebelo de animais jovens expostos ao mercurio inorganico apresentou uma atividade glutationa redutase aumentada, um efeito que pode estar relacionado a uma resposta de defesa celular frente a acao pro#]oxidativa do mercurio sobre tiois endogenos. Em contrapartida, os camundongos jovens expostos ao metilmercurio nao demonstraram esta resposta, o que aponta para mecanismos moleculares de resposta distintos para as diferentes formas de mercurio, sendo este fenomeno importante do ponto de vista toxicologico. O tratamento de animais adultos com metilmercurio por via oral causou uma diminuicao significativa na atividade glutationa peroxidase cerebral, uma enzima primaria no processo de detoxificacao de peroxidos produzidos endogenamente. Este efeito ocorreu em paralelo a um aumento na proteolise da enzima poli(ADP ribose) polimerase, um indice de apoptose. Em estudos in vitro, nossos dados demonstraram uma participacao importante do peroxido de hidrogenio como mediador da neurotoxicidade induzida por mercuriais e este fato foi acompanhado por uma marcante inibicao da enzima antioxidante glutationa peroxidase mitocondrial. O bloqueio da atividade desta enzima com acido mercaptosuccinico aumentou o estresse oxidativo e a disfuncao mitocondrial induzida pelos mercuriais in vitro. A incubacao de celulas de neuroblastoma humano SH#]SY5Y com metilmercurio causou uma diminuicao significativa na atividade glutationa peroxidase destas celulas. Este efeito tornou as celulas menos eficientes em degradar peroxido de hidrogenio e ocasionou um aumento na expressao de marcadores de dano oxidativo. Alem disso, a inibicao da lutationa peroxidase com acido mercaptosuccinico potencializou a perda de viabilidade celular e aumentou significativamente a expressao de marcadores de apoptose. O tratamento de animais adultos com extrato hidroalcoolico da planta Polygala paniculata preveniu os efeitos comportamentais e relacionados ao estresse oxidativo induzidos pelo tratamento com metilmercurio administrado por via oral. Este efeito se deu provavelmente devido a acao antioxidante do extrato. Em estudos in vitro, demonstramos a participacao do flavonoide quercetina como um importante mediador da neuroprotecao induzida pelo extrato de Polygala frente aos efeitos toxicos do metilmercurio. Alem disso, o flavonoide miricetina tambem apresentou efeito benefico contra a disfuncao mitocondrial e estresse oxidativo induzidos por metilmercurio in vitro. Em conclusao, nossos dados apontam para o sistema antioxidante da glutationa como um importante alvo molecular na neurotoxicidade induzida por mercuriais, onde disturbios no sistema antioxidante, causados por estes agentes, levam a um quadro de estresse oxidativo associado a disfuncao mitocondrial e aumentam a expressao de marcadores de morte celular. O extrato hidroalcoolico da planta Polygala paniculata, bem como os flavonoides quercetina e miricetina, representam potenciais agentes neuroprotetores contra a toxicidade induzida por mercuriais Mercury, in its organic and inorganic forms, is a potent environmental toxicant able to induce neurological deficits in both animals and humans. The developing central nervous system is particularly susceptible to the deleterious effects of mercury compounds. In this regard, mercury exposures during the pre#]natal and lactational periods are main factors from a toxicological point of view. In fact, little is known about the exclusive contribution of the lactational exposure to mercury and its relationship with neurotoxicity. In addition, there are no available treatments able to totally block mercurials#]induced toxicity. In the present study, we demonstrate that the exposure of young mice to methylmercury, an organic form of mercury, exclusively via maternal milk induces motor deficits associated with disturbs in the cerebellar thiol homeostasis. We also show that inorganic mercury caused neurotoxicity by exposure of young mice exclusively through the lactational route. The pups exposed to inorganic mercury presented an increased activity of the antioxidant enzyme glutathione reductase in the cerebellum. This effect appears to be related to an antioxidant response against the well known pro#]oxidative actions of mercury towards endogenous thiols. On the other hand, pups exposed to methylmercury through maternal milk did not show such adaptative response, pointing to different molecular mechanisms of action of the distinct forms of mercury. From a toxicological point of view, this is an important finding. Treatment of adult mice with methylmercury via oral route caused a significant decrease on the glutathione peroxidase activity in the brain. This enzyme is a main antioxidant defence against the deleterious effects of indogenous peroxides. This phenomenon was concomitant with an increase in poly(ADP ribose) polymerase proteolysis, an index of apoptosis. During in vitro studies, our data shows the central role of hydrogen peroxide as an important contributor to the neurotoxicity induced by mercuric compounds. We also show the inhibitory effect of methylmercury on mitochondrial glutathione peroxidase. The blockade of this enzyme with mercaptosuccinic acid caused an increase of oxidative stress markers and mitochondrial dysfunction induced by mercurials in vitro. The incubation of human neuroblastoma cells SH#]SY5Y with methylmercury caused a significant decrease on glutathione peroxidase activity. This effect resulted in a decreased ability of cells to remove hydrogen peroxide from the culture medium, causing an increase in the endogenous formation of peroxides. In addition, the inhibition of glutathione peroxidase improved the methylmercury#]induced anti#]viability effect and potentiated apoptosis. Treatment of adult animals with Polygala paniculata extract prevented the effects of methylmercury in inducing motor deficits and oxidative stress. This effect is probably related to the antioxidant actions of the extract. During in vitro studies, we demonstrated the participation of the flavonoid quercetin as an important mediator on the neuroprotection induced by Polygala extract. In addition, the flavonoid myricetin also displayed beneficial effects against methylmercury induced mitochondrial dysfunction and oxidative stress in vitro. In summary, our data point to the glutathione antioxidant system as an important molecular target for mercury#]induced neurotoxicity. Disturbances in this cellular defence system can induce oxidative stress associated with mitochondrial dysfunction and apoptosis. The Polygala paniculata extract, as well as the flavonoids quercetin and myricetin, may be potential therapeutic agents against mercury induced neurotoxicity

Determinação dos efeitos de dois suplementos alimentares em Drosophila melanogaster

Pleurotus spp. are considered extremely rich mushroom species from the nutritional pointof view, providing immune-enhancement effects when consumed. However, few mushroomshave been tested for their phenotypic and genotypic responses in animal modelsto ensure the proper dosage for their use. This study aimed to evaluate the effects of twomushroom species on the reproductive capacity of Drosophila melanogaster. Pleurotuscitrinopileatus Singer and Lentinus sajor-caju (Fr.) Fr. were provided separately, in specificconcentrations, as feed supplements to the fruit flies. The total numbers of developedlarvae, pupae, and adults were then measured for each treatment. Inter simple sequencerepeats marker analysis was carried out to infer genotypic changes in the mushroom-fedflies. Our results suggest that Pleurotus spp. can cause positive changes to the flies’ reproductivecapacity, since Pleurotus citrinopileatus in particular accelerated the life cycle andrevealed a higher genetic dissimilarity of the diet-supplemented flies.Keywords: fruitflies, reproductive fitness, edible mushrooms.Pleurotus spp. são consideradas espécies de cogumelo extremamente ricas do ponto de vista nutricional, proporcionando reforço imunológico quando consumidas. No entanto, poucos cogumelos foram testados quanto à sua interferência fenotípica e genotípica em modelos animais para garantir a dosagem adequada para seu uso. Este estudo teve como objetivo avaliar os efeitos de duas espécies de cogumelos sobre a capacidade reprodutiva de Drosophila melanogaster. Pleurotus citrinopileatus Singer e Lentinus sajor-caju (Fr.) Fr. foram fornecidos separadamente, em concentrações específicas, como suplementos alimentares às moscas da fruta. O número total de larvas, pupas e adultos desenvolvidos foram então avaliados para cada tratamento. Análises de marcadores moleculares do tipo Inter simple sequence repeats foram realizadas para inferir mudanças genotípicas nas moscas alimentadas com os cogumelos. Nossos resultados sugerem que Pleurotus spp. podem causar mudanças positivas na capacidade reprodutiva das moscas, uma vez que Pleurotus citrinopileatus, em particular, acelerou o ciclo de vida e revelou uma maior dissimilaridade genética das moscas suplementadas com esse fungo em sua dieta.Palavras-chave: moscas das frutas, estímulo reprodutivo, cogumelos comestíveis

Fumigant Activity of the Psidium guajava

The guava fruit, Psidium guajava var. pomifera (Myrtaceae family), is a native plant from South America. Its leaves and fruits are widely used in popular medicine in tropical and subtropical countries. Drosophila melanogaster has been used as one of the main model organisms in genetic studies since the 1900s. The extensive knowledge about this species makes it one of the most suitable organisms to study many aspects of toxic compound effects. Due to the lack of studies on the effects of the bioactive compounds present in the P. guajava var. pomifera essential oil, we performed a phytochemical characterization by CG-MS and evaluated the toxicity induced by the essential oil in the D. melanogaster insect model. In order to understand the biochemical mechanisms of toxicity, changes on the Nrf2 signaling as well as hallmarks of oxidative stress response were followed in the exposed flies. Our results showed that exposure of insects to the P. guajava oil increased mortality and locomotor deficits in parallel with an oxidative stress response signaling. Therefore, it suggested a bioinsecticidal activity for P. guajava volatile compounds by means of oxidative stress. Further studies are ongoing to identify which oil compounds are responsible for such effect

Phytochemical Composition, Antifungal and Antioxidant Activity of Duguetia furfuracea

Background. Duguetia furfuracea is popular plant used in popular medicine. Hypothesis/Purpose. This claim evaluated the phytochemical composition of the hydroethanolic extract (HEDF), fractions of Duguetia furfuracea, and antioxidant and antifungal activity. Methods. The chemical profile was carried out by HPLC-DAD. The total phenolic contents and flavonoid components were determined by Folin-Ciocalteu and aluminium chloride reaction. The antioxidant activity was measured by scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical and ferric reducing ability of plasma (FRAP) methods. The antifungal activity was determined by microdilution assay. Results. HPLC analysis revealed caffeic acid and rutin as major compounds (HEDF), caffeic acid and quercitrin (Mt-OH fraction), and quercitrin and isoquercitrin (Ac-OEt fraction). The highest levels of phenols and total flavonoids were found for Ac-OEt fraction, and the crude extract showed higher in vitro antioxidant potential. The antifungal activity showed synergic effect with fluconazole and EHDF against C. krusei, fluconazole and Mt-OH against C. krusei and C. tropicalis, and Ac-OE and fluconazole against C. albicans. Conclusion. The highest levels of phenols and total flavonoids were marked with antioxidant effect. This is the first report of bioactivity of the synergic effect of HEDF and fractions. More studies would be required to better clarify its mechanism of synergic action

Phytochemical Constituents and Toxicity of Duguetia furfuracea

Duguetia furfuracea is frequently used as a medicinal plant in Brazil. However, studies have evidenced its cytotoxic, bactericide, and antitumor activities. In the present study we aimed to evaluate the potential toxicity of hydroalcoholic leaves extracts of D. furfuracea (HEDF) in a Drosophila melanogaster model. Toxicity was assessed as changes in locomotor performance, mitochondrial activity, oxidative stress, MAPKs phosphorylation, and apoptosis induction after exposure to HEDF concentrations (1–50 mg/mL) for 7 days. The phytoconstituents of the plant were screened for the presence of alkaloids, tannins, xanthones, chalcones, flavonoids, aurones, and phenolic acids. Exposure of adult flies to HEDF caused mitochondrial dysfunction, overproduction of ROS, and alterations in the activity of detoxifying enzymes GST, SOD and CAT. Induction of ERK phosphorylation and PARP cleavage was also observed, indicating occurrence of HEDF-induced cell stress and apoptotic cell death. In parallel, alterations in cholinesterase activity and impairments in negative geotaxis behavior were observed. Our study draws attention to the indiscriminate use of this plant by population and suggests oxidative stress as a major mechanism underlying its toxicity

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Human neuroblastoma cells transfected with tyrosine hydroxylase gain increased resistance to methylmercury-induced cell death

In a previous study we demonstrated that human neuroblastoma SH-SY5Y cells transfected with human tyrosine hydroxylase isoform 1 (SH + TH cells) were substantially more resistant to cell death induced by pro-oxidants than wild type SH-SY5Y cells (SH cells). In the present communication we used methylmercury as a model of cell stress in order to test whether SH + TH cells would behave in a similar manner in response to this stressor. Incubation with methylmercury (0.1–3 μM) for 24 h caused a significant reduction in cell viability and increased apoptotic markers in both cell types. However, the effects were significantly reduced in the SH + TH cells when compared to the SH cells. Activation of p38ᴹᴬᴾᴷ was also reduced in the SH + TH compared to the SH cells after methylmercury exposure. Since p38ᴹᴬᴾᴷ is known to participate in signal transduction pathways during cell stress, our data suggest that SH + TH cells develop an increased resistance to environmental stress caused by neurotoxins such as methylmercury. In conclusion our results show that insertion of the human TH gene in cells that originally do not express this protein leads to alterations in cell homeostasis and triggers defense mechanisms against pro-oxidative insults

Ethnobotany and antioxidant evaluation of commercialized medicinal plants from the Brazilian Pampa

Despite the importance of medicinal plants for the healthcare of local people, the knowledge about medicinal species used in the Pampa biome has been neglected over the years. In this study, an ethnobotanical survey was employed aiming to characterize the species richness and diversity of commercialized medicinal plant species in five cities within the Brazilian Pampa. Additionally, among the listed plants, ten species were selected for in vitro testing of their potential antioxidant activity. A total of 56 plant species belonging to 33 botanical families were listed by the 115 interviewees. No significant difference in commercialized medicinal plant species, and very similar species richness was observed among the cities, indicating that the local knowledge is consistently preserved across the studied cities. According to the biochemical analysis, Sphagneticola trilobata, Malva parviflora and Struthanthus flexicaulis emerged as very promising species for antioxidant activity. Further studies are recommended to advance our knowledge about the richness of medicinal plant species in the Brazilian Pampa, and to assess their therapeutic potential