Made available in DSpace on 2014-07-29T16:11:52Z (GMT). No. of bitstreams: 1 dissertacao guilherme.pdf: 2090110 bytes, checksum: a1c991816c551ebd8ad069c1df1f9f72 (MD5) Previous issue date: 2008-09-25 / The araticum (Annona coriacea Mart.) is a typical fruit of the brazilian cerrado used popularly to overcome inflammatory processes. The family Annonaceae presents as the main constituents acetogenins, a class of substances with great cytotoxic and genotoxic potential, and cited as responsible for a disease similar to parkinson in a Caribbean population that used the fruit as much as food and for medicinal purposes. The aim of this study was to evaluate the putative activity of crude ethanolic extract of seeds of A. coriacea on the cerebral cortex of mice exposed at doses of 12.5, 25, 50 and 100 mg / kg, and evaluate its activity on different areas of the brain, the liver and kidneys. We used 30 adult male Swiss mice divided into groups control, solvent and treated (12.5, 25, 50 and 100 mg / kg). The extract was administered orally for four days. The target organs were extirpated, fixed in 70% ethanol (v / v) and processed for histological method - hematoxylin and eosin. The analysis of the slides was performed by image processing system for counting cells and other morphometric analysis. The morphological studies showed no significant changes to the brain in different areas, just as no changes were detected in the kidneys. On the other hand, was found a reduction on the frequency of cells per area of the liver, like as an reduction on the consumption of food, water and production of excreta. We conclude with this work a possible hepatotoxic activity induced by exposure to crude ethanol extract of seeds of A. Coriacea Mart., observed by the decrease in frequency of cells per area of the liver, correlated with reductions in consumption of food / water and production of excreta by animals. / O Araticum (Annona coriacea Mart.) é um fruto típico do cerrado brasileiro utilizado popularmente para remediar processos inflamatórios. A família Annonaceae apresenta como principais constituintes as acetogeninas, uma classe de substâncias com grande potencial citotóxico, genotóxico e ainda citado como o responsável por uma doença similar ao parkinsonismo em uma população caribenha que utilizava o fruto tanto como alimento quanto para fins medicinais. O objetivo deste estudo foi avaliar a putativa atividade do extrato bruto etanólico das sementes da A. coriacea sobre o córtex cerebral de camundongos expostos nas doses de 12,5; 25; 50 e 100 mg/kg, além de avaliar sua atividade sobre diferentes áreas do encéfalo, sobre o fígado e rins. Foram utilizados 30 camundongos Swiss machos adultos divididos em grupos controle, solvente e tratados (12,5; 25; 50 e 100 mg/kg). O extrato foi administrado por via oral durante quatro dias. Os órgãos-alvo foram extirpados, fixados em etanol 70% (v/v) e processados para método histológico - hematoxilina e eosina. A análise das lâminas foi executada via sistema de processamento de imagens para a contagem das células e demais análises morfométricas. Os estudos morfológicos não demonstraram alterações significativas para o cérebro em suas diferentes áreas, da mesma forma que não foram detectadas alterações nos rins. Por outro lado, foi detectado uma redução da freqüência de células por área do fígado, assim como verificamos uma diminiução no consumo de ração, água e produção de excreta. Concluimos com este trabalho uma possível atividade hepatotóxica induzida pela exposição ao extrato bruto etanólico das sementes da A. Coriacea Mart., observada pela diminuição da freqüência de células por área do fígado, correlacionado com reduções de consumo de ração/água e produção de excretas pelos animais.

A cocaína é considerada a principal droga de abuso utilizada na América do Sul, sendo que o crack é a via de administração que mais cresceu nos últimos anos. Cabe salientar que o usuário do crack sofre ação tanto da cocaína quanto das substâncias advindas da sua pirólise, dentre elas a metilecgonidina (AEME). Trabalho publicado pelo nosso grupo demonstrou que a AEME é mais neurotóxica que a cocaína em cultura primária de hipocampo. Além disso, dados da literatura têm mostrado uma possível ação da AEME em receptores colinérgicos muscarínicos no sistema nervoso periférico. Na tentativa de elucidar se essa ação ocorre no sistema nervoso central, a AEME foi incubada na presença e na ausência de atropina, um antagonista de receptores colinérgicos muscarínicos. Nossos resultados em cultura primária de hipocampo mostraram que a atropina foi capaz de prevenir os efeitos neurotóxicos causados pela AEME, sugerindo uma afinidade aos receptores colinérgicos muscarínicos. Contudo, o mesmo efeito não foi observado após a incubação com a cocaína e a associação (AEME 1 mM /cocaína 2 mM). Pode-se pressupor que a AEME age preferencialmente em receptores colinérgicos muscarínicos subtipos M1, M3 e M5, uma vez que houve a formação de IP3 e aumento de cálcio intracelular, sendo esse último observado também nos grupos incubados com cocaína e associação (AEME 1 mM /cocaína 2 mM). Com a finalidade de verificar se a apoptose era uma das vias de morte neuronal, foi avaliada a expressão das proteínas mitoncondriais (Bax e Bcl-2), a atividade da caspase-3 e a análise da fragmentação do DNA, bem como a integridade da membrana celular. Foi observado que a AEME aumentou a razão das proteínas mitocondriais Bax/Bcl-2, a atividade da caspase-3 e o DNA fragmentado, bem como a perda da integridade da membrana. A cocaína aumentou a atividade da caspase 3, a fragmentação do DNA e a perda da integridade da membrana celular, mas não alterou a razão da expressão das proteínas mitocondriais Bax/Bcl-2. Apesar de apresentar uma diminuição da atividade da caspase-3, a associação (AEME 1 mM /cocaína 2 mM) apresentou um aumento do DNA fragmentado e do rompimento da membrana, bem como um aumento da razão Bax/Bcl-2. Estes dados sugerem que estas substâncias estimulam vias de morte neuronal tanto de apoptose quanto de necrose. Mais ainda, nas vias estudas neste trabalho, parece que a associação (AEME 1 mM /cocaína 2 mM) desencadeia os efeitos neurotóxicos mais rápido, estimulando, possivelmente, vias diferentes das encontradas com as substâncias isoladamente. / Cocaine is the main illicit drug used in South America, and the crack cocaine is the administration route that grown more than any other route in the last years. The user of crack cocaine suffers the action of both cocaine and its pyrolysis products, which methylecgonidine (AEME) is the main compound. Published work by our group demonstrated that AEME is more neurotoxic than cocaine in rat primary hippocampal cell culture. Moreover, published data have shown a possible muscarinic cholinergic action of AEME in the peripheral nervous system. To verify if this action occurs in the central nervous system, AEME was incubated in the presence and absence of atropine, a muscarinic cholinergic receptor antagonist. Our results in rat primary hippocampal cell culture showed that atropine was able to prevent AEME-induced neurotoxic effects, suggesting its affinity for muscarinic cholinergic receptors. However, this effect was not observed after incubation with cocaine and association (AEME 1 mM /cocaine 2 mM). It is suggestive that AEME acts, with preference, on subtypes M1, M3 and M5 muscarinic cholinergic receptors, once there was the formation of IP3 and the increase of intracellular calcium. It is important to mention that the intracellular calcium was also increased in both cocaine and association (AEME 1 mM /cocaine 2 mM) groups. In order to know whether apoptosis was a neuronal death pathway, it was evaluated the expression of mitochondrial proteins (Bax and Bcl-2), the capase-3 activity and the DNA fragmentation, as well as the loss of membrane integrity. It was observed that AEME increased the ratio of mitochondrial proteins Bax/Bcl-2, the activity of caspase-3, the fragmentation of DNA and the loss of membrane integrity. Cocaine increased the activity of caspase-3, the DNA fragmentation and the loss of cell membrane integrity, but did not affect the ratio expression of mitochondrial proteins Bax/Bcl-2. Although it was observed a decrease in caspase-3 activity, the association (AEME 1 mM / cocaine 2 mM) showed an increase in the DNA fragmentation and the cell membrane disruption, as well as an increase in Bax/Bcl-2 ratio. These data suggest that these substances stimulate neuronal death pathways of both apoptosis and necrosis. Moreover, in the pathways studied in this work, it seems that the association (AEME 1 mM /cocaine 2 mM) has the fastest neurotoxic effects, stimulating, possibly, different neuronal death pathways when compared to substances isolated.

No description available.

Over the past 60 years, a large number of selective neurotoxins were discovered and developed, making it possible to animal-model a broad range of human neuropsychiatric and neurodevelopmental disorders. In this paper, we highlight those neurotoxins that are most commonly used as neuroteratologic agents, to either produce lifelong destruction of neurons of a particular phenotype, or a group of neurons linked by a specific class of transporter proteins (i.e., dopamine transporter) or body of receptors for a specific neurotransmitter (i.e., NMDA class of glutamate receptors). Actions of a range of neurotoxins are described: 6-hydroxydopamine (6-OHDA), 6-hydroxydopa, DSP-4, MPTP, methamphetamine, IgG-saporin, domoate, NMDA receptor antagonists, and valproate. Their neuroteratologic features are outlined, as well as those of nerve growth factor, epidermal growth factor, and that of stress. The value of each of these neurotoxins in animal modeling of human neurologic, neurodegenerative, and neuropsychiatric disorders is discussed in terms of the respective value as well as limitations of the derived animal model. Neuroteratologic agents have proven to be of immense importance for understanding how associated neural systems in human neural disorders may be better targeted by new therapeutic agents.

Indiana University-Purdue University Indianapolis (IUPUI). / Calcium/calmodulin-dependent protein kinase II (CaMKII) is a critical modulator of synaptic function, plasticity, and learning and memory. In neurons and astrocytes, CaMKII regulates cellular excitability, cytoskeletal structure, and cell metabolism. A rapid increase in CaMKII activity is observed within the first few minutes of ischemic stroke in vivo; this calcium-dependent process is also observed following glutamate stimulation in vitro. Activation of CaMKII during pathological conditions is immediately followed by inactivation and aggregation of the kinase. The extent of CaMKII inactivation is directly correlated with the extent of neuronal damage. The studies presented here show that these fluctuations in CaMKII activity are not correlated with neuronal death; rather, they play a causal role in neuronal death. Pharmacological inhibition of CaMKII in the time immediately surrounding glutamate insult protects cultured cortical neurons from excitotoxicity. Interestingly, pharmacological inhibition of CaMKII during excitotoxic insult also prevents the aggregation and prolonged inactivation of the kinase, suggesting that CaMKII activity during excitotoxic glutamate signaling is detrimental to neuronal viability because it leads to a prolonged loss of CaMKII activity, culminating in neuronal death. In support of this, CaMKII inhibition in the absence of excitotoxic insult induces cortical neuron apoptosis by dysregulating intracellular calcium homeostasis and increasing excitatory glutamate signaling. Blockade of the NMDA-receptors and enzymatic degradation of the extracellular glutamate signal affords neuroprotection from CaMKII inhibition-induced toxicity. Co-cultures of neurons and glutamate-buffering astrocytes also exhibit this slow-induced excitotoxicity, as CaMKII inhibitors reduce glutamate uptake within the astrocytes. CaMKII inhibition also dysregulates calcium homeostasis in astrocytes and leads to increased ATP release, which was neurotoxic when applied to naïve cortical neurons. Together, these findings indicate that during aberrant calcium signaling, the activation of CaMKII is toxic because it supports aggregation and prolonged inactivation of the kinase. Without CaMKII activity, neurons and astrocytes release stores of transmitters that further exacerbate neuronal toxicity.

The risks of damage to the developing nervous system of many chemicals are not known because these studies often require costly and time-consuming multi-generational animal experiments. Pluripotent stem cell-based systems can facilitate developmental neurotoxicity studies because disturbances in nervous system development can be modeled in vitro. In this study, neurons derived from embryonal carcinoma (EC) and induced pluripotent stem (iPS) cells, were first characterized to establish their suitability for developmental neurotoxicity studies. The EC stem cell line, TERA2.cl.SP-12, was differentiated into neurons that expressed voltage-gated sodium and potassium channels as well as ionotropic GABA and glutamate receptors. These cells could also fire action potentials when stimulated electronically. However spontaneous action potentials were not observed. In contrast, pre-differentiated neurons derived from iPS cells fired evoked and spontaneous action potentials. Furthermore, iPS cell-derived neurons also expressed a wide array of functional voltage- and ligand-gated ion channels. Antiepileptic drugs (AEDs) are associated with developmental neurotoxicity. These agents can cause congenital malformations, cognitive deficits and behavioral impairment in children as a result of in utero exposure. The impact of four major AEDs, namely phenobarbital, valproic acid, carbamazepine and lamotrigine, on cell viability, cell cycle and differentiation of TERA2.cl.SP-12 into neurons was studied. All AEDs tested reduced differentiating stem cell viability. Valproic acid and carbamazepine increased apoptosis and reduced cell proliferation. A brief exposure to phenobarbital, valproic acid and lamotrigine at the start of differentiation impaired the subsequent generation of neurons. Additionally, the effect of transient exposure to phenobarbital and carbamazepine on neuronal maturation of iPS-derived neurons was investigated. Exposure to both AEDs resulted in diminished membrane potentials and reduced the proportion of cells that were able to fire action potentials spontaneously in culture. The data from these studies suggest that impairments in proliferation, differentiation and maturation of neurons derived from human stem cells may be sensitive indicators of neurodevelopmental disruption by these drugs that can result from in utero exposure. Furthermore, these findings suggest that the use of human pluripotent stem cells and neurons derived from them can reduce the time, cost and the number of animals used in toxicological research.

科学研究費補助金 研究種目:基盤研究(C)(2) 課題番号:10670348 研究代表者:柴田 英治 研究期間:1998-1999年度