Methamphetamine (MA) is a well-known, easily accessible and powerful psychostimulant, and its abuse has become a global problem. MA abuse affects millions of people worldwide and places an enormous burden on public healthcare resources. Documented consequences of MA abuse include cardiotoxic, neurotoxic and teratogenic effects, as well as long-term consequences of chronic abuse including affective disorders such as schizophrenia and major depressive disorder (MDD). MDD is a highly prevalent mood disorder in both adults and children, documented to contribute to approximately 850 000 suicides annually. This disorder is projected to become the 2nd leading disease of global burden by 2020, preceded only by ischemic heart disease. Depressive-like behaviour is documented as a symptom of chronic MA abuse and particularly during extensive MA withdrawal. Also, MA abuse during pregnancy is documented to cause neurodevelopmental changes that persist into later life. However, current understanding thereof is limited and warrants further investigation of the effects of early-life exposure to MA on outcome in adulthood, particularly in terms of mood disorders. The aim of the current study was to determine the effect of chronic exposure to MA on the depressive-like behaviour later in life in stress-sensitive (Flinders Sensitive Line) and control (Flinders Resistant Line) rats. Rats were exposed during one of the following natal day (ND) age groups: prenatal (ND-13 to ND+02), postnatal (ND+03 to ND+18), prepuberty (ND+19 to ND+34) or puberty (ND+35 to ND+50). These age groups represent different stages in neurodevelopment, as also seen in humans. For prenatal exposure, pregnant dams received 5 mg/kg daily subcutaneously (s.c.), and pups from postnatal, prepuberty and puberty age groups received an escalating dose regimen to simulate “binge-dosing” commonly seen in humans abusing MA. After MA exposure, rats were housed normally until behavioural testing on postnatal day 60 (ND+60), which included the novel object recognition test (NOR), open field test (OFT) and forced swim test (FST), measuring cognitive function, locomotor activity and depressive-like behaviour respectively. The FST data showed increased immobility behaviour of saline-treated FSL rats relative to that of FRL rats, in line with previous data validating FSL rats as a genetic rodent model of depression. Practically significant MA-induced increases in immobility behaviour were observed in all FSL and FRL treatment groups in the FST, reaching statistical significance in prenatally treated FRL rats, and in postnatally, prepuberty and puberty treated FSL rats. The data suggest that early-life MA exposure may alter neurodevelopment to predispose the rats to display depressive-like behaviour in early adulthood, and suggests that this detrimental effect of MA may be more expressed in stress-sensitive rats. Furthermore, all FSL groups plus prenatally and puberty treated FRL rats revealed practically and statistically significant decreases in swimming behaviour in the FST, whereas decreases in swimming behaviour in prepuberty treated FRL rats were practically significant but did not reach statistical significance. These data suggest that MA-induced depressive-like behaviour in FSL rats may be related to impaired serotonergic neurotransmission, and that this appears to be more robust in FSL rats. Climbing behaviour in the FST was generally not altered by early-life MA exposure, with a notable exception being a practically and statistically significant increase in puberty treated FRL rats. These data suggest that in general early-life MA exposure does not affect noradrenergic neurotransmission in early adulthood, except when normal rats were treated at puberty. The reason for the latter observation is not clear. The data from the NOR test revealed no discernible trends of MA-induced effects on memory and cognition, except for a small albeit practically significant increase in exploration time in prepuberty treated FRL rats and a practically and statistically significant decrease in exploration time in puberty-treated FRL rats. Lastly, locomotor activity in the OFT was mostly unaffected by MA treatments, except for practically significant decreases in locomotor activity in postnatally-and prepuberty-treated FRL rats and practically and statistically significant decreases in locomotor activity of prepuberty treated FSL rats. Altered locomotor activity is therefore not expected to explain any of the immobility results of the FST. In final conclusion, the study confirms that early-life MA exposure results in a depressogenic effect later in life in stress-sensitive (FSL) and control (FRL) rats, but appears to be more robust in stress-sensitive animals. Furthermore the data suggest that long-lasting MA-induced depressogenic effects may relate to impaired serotonergic neurotransmission. / MSc (Pharmacology), North-West University, Potchefstroom Campus, 2014

Humans are exposed to a variety of chemicals including environmental pollutants, cosmetics, food preservatives and drugs. Some of these substances might be harmful to the human body. Traditional toxicological and behavioural investigations performed in animal models are not suitable for the screening of a large number of compounds for potential toxic effects. There is a need for simple and robust in vitro cellular models that allow high-throughput toxicity testing of chemicals, as well as investigation of specific mechanisms of cytotoxicity. The overall aim of the thesis has been to evaluate neuronally differentiated mouse embryonal carcinoma P19 cells (P19 neurons) as a model for such testing. The model has been compared to other cellular models used for neurotoxicity assessment: retinoic acid-differentiated human neuroblastoma SH-SY5Y cells and nerve growth factor-treated rat pheochromocytoma PC12 cells. The chemicals assessed in the studies included the neurotoxicants methylmercury, okadaic acid and acrylamide, the drug of abuse MDMA (“ecstasy”) and a group of piperazine derivatives known as “party pills”. Effects of the chemicals on cell survival, neurite outgrowth and mitochondrial function have been assessed. In Paper I, we describe a fluorescence-based microplate method to detect chemical-induced effects on neurite outgrowth in P19 neurons immunostained against the neuron-specific cytoskeletal protein βIII-tubulin. In Paper II, we show that P19 neurons are more sensitive than differentiated SH-SY5Y and PC12 cells for detection of cytotoxic effects of methylmercury, okadaic acid and acrylamide. Additionally, in P19 neurons and differentiated SH-SY5Y cells, we could demonstrate that toxicity of methylmercury was attenuated by the antioxidant glutathione. In Paper III, we show a time- and temperature-dependent toxicity produced by MDMA in P19 neurons. The mechanisms of MDMA toxicity did not involve inhibition of the serotonin re-uptake transporter or monoamine oxidase, stimulation of 5-HT2A receptors, oxidative stress or loss of mitochondrial membrane potential. In Paper IV, the piperazine derivatives are evaluated for cytotoxicity in P19 neurons and differentiated SH-SY5Y cells. The most toxic compound in both cell models was TFMPP. In P19 neurons, the mechanism of action of TFMPP included loss of mitochondrial membrane potential. In conclusion, P19 neurons are a robust cellular model that may be useful in conjunction with other models for the assessment of chemical-induced neurotoxicity.

La pollution atmosphérique est omniprésente du fait de nombreuses sources émettrices de composés chimiques. Dans ce contexte, les Hydrocarbures Aromatiques Polycycliques (HAP) sont largement diffusés dans l’air et ont déjà montré des effets délétères sur la santé.Ce travail a consisté en l’évaluation de la neurotoxicité du fluorène, composé choisi comme molécule représentative de la pollution liée aux HAP, chez le rat adulte exposé par voie i.p., par voie orale ou par inhalation. Le modèle d’exposition par inhalation a ensuite été appliqué à l’étude des effets du polluant sur le développement sensori-moteur et l’activité comportementale de l’animal exposé in utero ou au cours de la lactation.Ces études ont montré que le fluorène était susceptible d’affecter le niveau d’anxiété et l’activité locomotrice du rat adulte exposé directement et indirectement au polluant, et n’avait aucun effet sur les capacités d’apprentissage. En revanche, aucune atteinte majeure de la maturation des fonctions sensori-motrices n’a été mise en évidence. L’analyse de la présence du composé et de trois de ses métabolites a par ailleurs montré que le polluant était capable de traverser la barrière hémato-encéphalique et d’être métabolisé au niveau du cerveau. Les réponses physiologiques et comportementales étant variables entre les études, les effets induits par le fluorène dépendent donc de la voie d’entrée, du niveau de contamination et du moment d’exposition.Ces résultats ont ainsi révélé chez l’animal la toxicité comportementale du fluorène à des niveaux de contamination réalistes, confirmant ainsi le risque sanitaire de l’exposition aux HAP tout au long de la vie des individus / Actually air pollution is ubiquitous due to the emission of chemical compound from many sources. In this context, Polycyclic Aromaric Hydrocarbons (PAH)related compounds are widely distributed in the air and have shown deleterious health effects.Fluorene was chosen as a representative compound of PAHs pollution. This work consisted in the evaluation of its neurotoxic effects in adult animals, exposed i.p., orally or by exposure nose-only. The inhalation model of exposure was then applied to the study of its effects on sensorimotor development and on behavioral activity of animals exposed in utero or during lactation.The results showed that fluorene is able to induce behavioral changes in adult animals exposed directly or indirectly on the level of anxiety and the locomotor activity, whereas no effect on learning and memory abilities has been observed. However, no defect on the development of motor and sensory functions was demonstrated. Furthermore, the analysis of the presence of the compound and three of its metabolites showed that the pollutant was able to cross the blood brain barrier and can be metabolized in the brain. Moreover, variations have been observed concerning behavioral and physiological responses between studies, showing that effects induced by fluorene are dependent on the way of administration, the level of contamination and the time of exposure.In conclusion, these results demonstrate in animals the behavioral toxicity of fluorene at levels of contamination corresponding to human cases of exposure, confirming so the risk of PAH exposure throughout life

O uso da cocaína na forma de crack vem crescendo nos últimos anos quando comparado às demais vias de administração. Contribuem para esse fato a obtenção quase imediata de efeitos e a maior facilidade de uso, que dispensa a necessidade de material injetável. O usuário de crack sofre os efeitos não só da cocaína, mas também de seu produto de pirólise, a metilecgonidina (AEME). Existem evidências de que a cocaína leva à neurodegeneração, entretanto a participação da AEME nesse processo ainda não foi estudada. A proposta deste estudo foi investigar a participação da AEME no processo neurodegenerativo utilizando cultura primária de hipocampo realizada a partir de fetos de ratos. Foram realizados ensaios de viabilidade celular (MTT) e da atividade da lactato desidrogenase (LDH), além da avaliação morfológica por microscopia de fluorescência. Foi estudada também a participação do dano oxidativo no processo de neurodegeneração, como a formação de aduto de DNA; atividade das enzimas antioxidantes glutationa peroxidase (GPx), glutationa redutase (GR) e glutationa S-transferase (GST); e a produção de malonaldeído (MDA), um biomarcador de peroxidação lipídica. Tanto a cocaína quanto a AEME mostraram-se neurotóxicas. A partir dos ensaios de viabilidade e da avaliação morfológica foi possível inferir que, em células hipocampais, a cocaína leva à morte celular tanto por necrose quanto por apoptose e que a provável via envolvida na neurodegeneração da AEME é a apoptose. A AEME não causou lesão direta ao DNA, uma vez que não foi observada a formação de adutos nem com a desoxiguanosina (d-G), a base nitrogenada mais reativa, nem com DNA comercial. Mais ainda, nossos resultados mostraram que a AEME e a cocaína, nas concentrações de 1 e 2 mM, respectivamente, foram equipotentes e a incubação concomitante das duas substâncias nessas concentrações apresentou efeito aditivo após 48 horas de exposição. A morte de células hopocampais evidenciada a partir de 24 horas de exposição foi precedida pela diminuição da atividade da GPx após 3 horas de incubação tanto com a AEME e a cocaína, quanto com a associação entre essas substâncias. A atividade da GST também diminuiu, no entanto, somente após 6 horas de exposição, antecedendo a morte celular. Não foi observada alteração na atividade da GR. Houve um aumento, porém, não estatisticamente significativo, de MDA após 48 horas de incubação. Nossos resultados sugerem uma maior susceptibilidade à neurodegeneração com o uso de crack do que com a cocaína isoladamente. / Smoking crack has increased in the last years when compared to the other routes of cocaine administration. Its advantage is the quicker and stronger high effects and its ease of use without the need of needles. Smoking crack involves inhaling not only cocaine, but also its pyrolysis product, methylecgonidine (AEME). There are evidences that cocaine causes neurodegeneration, but AEME involvement in this process has not been studied yet. The aim of this study was to investigate AEME participation in neurodegeneration using a primary hippocampus culture made from rat fetuses. Cellular viability assays (MTT), lactate dehydrogenase activity (LDH) and morphological evaluations with fluorescence microscopy were performed. The involvement of oxidative injury in the neurodegeneration process was also studied through DNA adduct formation; the evaluation of antioxidants enzymes activities as glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST); and the production of malonaldehyde (MDA), a lipoperoxidation biomarker. Both cocaine and AEME showed neurotoxic effects. Through viability assays and morphologic evaluations we can suggest that, in hippocampal cells, cocaine cell death mechanism involves not only necrosis, but also apoptosis and that AEME pathway involved in neurodegeneration is only apoptosis. AEME did not produce a direct DNA injury, as no DNA adduct was observed with desoxyguanosine (d-G), the most reactive nitrogenous base, nor with commercial DNA. Moreover, our results showed that 1 and 2 mM of AEME and cocaine, respectively, were equipotent and the concomitant incubation of both compounds in those concentrations showed additive effect after 48 hours of exposure. Hippocampal cell death at 24 hours was preceded by a decrease in GPx activity after 3 hours of incubation with AEME, cocaine and association between these two compounds. GST activity also decreased but only after 6 hours of exposure, also before cell death. There was no alteration in GR activity. There was an increase, although not statistically significant, in MDA after 48 hours of exposure. As smoking crack abusers are exposed to both cocaine and AEME, our results suggest a higher susceptibility to neurodegeneration in smoking crack than with cocaine alone.

O açaí é uma fruta da amazônia brasileira de consumo emergente no Brasil e outras regiões do mundo. Primordialmente consumida na sua forma de polpa e estabelecida como uma \"superfruta\" graças ao seu potencial antioxidante e anti-inflamatório. No entanto, pouco se sabe sobre sua composição em relação aos elementos químicos essenciais. No presente estudo, a análise sistemática de 12 polpas produzidas com frutos de diferentes localidades demonstrou que a polpa de açaí é naturalmente rica nos elementos Ca, Cu, Fe, Mg, Zn, com destaque para as concentrações encontradas de Mn que foram notoriamente maiores do que as comumente observadas em alimentos considerados como fonte principal deste elemento químico essencial na dieta. Cabe destacar que em valores médios, todos os elementos químicos estudados no açaí apresentaram-se em concentrações que contribuem significativamente para as suas necessidades diárias preconizadas. Entretanto, a análise do fracionamento químico da polpa de açaí demonstrou que o ferro está em uma forma potencialmente indisponível; enquanto que cerca de 40% dos elementos cálcio, magnésio, manganês e zinco estão quelados a um composto não fenólico de alta solubilidade em água e que pode aumentar suas biodisponibilidades. Diferentemente dos demais, o cobre interagiu significativamente com a fração fenólica solúvel em água do alimento. A fração fenólica eluída com metanol por SPE demonstrou ter considerável atividade antioxidante direta no ensaio de sequestro de DPPH com EC50 de 19,1 ?g/L e foi eficaz na atenuação da citotoxicidade, estresse oxidativo e alteração funcional induzidos por 500 ?M de MnCl2 em cultura primária de astrócitos, avaliados pelos ensaios de LDH e MTT, GSH/GSSG, F2-IsoPs, captação de glutamato e expressão de Nrf2. Este extrato de açaí em uma concentração ótima de 0,1 ?g/mL preveniu o estresse oxidativo induzido por Mn restaurando a razão GSH/GSSG, protegendo as membranas astrocitárias da lipoperoxidação e diminuindo a expressão de Nrf2. Uma concentração mais elevada de extrato de açaí exacerbou os efeitos do Mn nestes mesmos parâmetros, exceto na lipoperoxidação medida pela formação de F2-IsoPs. Assim, o pré-tratamento dos astrócitos com concentrações mais elevadas destas antocianinas com exposição ao Mn em sequência, pode predispor os astrócitos fazendo com que um efeito pró-oxidante prevaleça. Estes achados devem ser considerados em estudos futuros que explorem a potencialidade das antocianinas do açaí em formulações nutracêuticas para obtenção de efeitos antioxidantes e neuroprotetores ideais. Assim, conclui-se que o próprio açaí possui uma fração de compostos fenólicos capaz de atenuar os efeitos oxidativos do Mn e que portanto, pode indicar uma segurança alimentar do consumo do fruto em quantidades moderadas. / Açaí is a fruit from Brazilian Amazon with an exotic flavor possessing high antioxidant and anti-inflammatory properties. Based on these properties, the fruit is classified as one of the new \"super fruits\". However, few is known about its essential chemical elements content. In this study, the systematic analysis of 12 freeze-dried pulps processed with açaí of different locations showed that açaí pulp is naturally rich in Ca, Cu, Fe, Mg, Zn, and especially amounts of Mn, which was significantly greater than in foods considered Mn dietary source, were found. In average values, all determined essential chemical elements contribute significantly to their recommended daily intake requirements. However, fractionation analysis of açai showed that iron is potentially not bioavailable. While about 40% of calcium, magnesium, manganese, and zinc are binding to a non-phenolic compound high soluble in water, which may increase their bioavailabilities. Copper is significantly bound to water-soluble phenolic fraction in açaí. This phenolic fraction eluted with methanol by SPE provided considerable direct antioxidant activity on the DPPH scavenging assay with EC50 of 19.1 ?g/L and was effective attenuating cytotoxicity, oxidative stress, and functional alterations induced by 500 ?M of MnCl2 in primary cultured astrocytes, which was assessed by MTT and LDH assay, GSH/GSSG ratio, F2-IsoPs formation, glutamate uptake, and Nrf2 levels. This anthocyanin-rich açaí extract in an optimal concentration of 0.1 ?g/mL prevented oxidative stress induced by Mn restoring the GSH/GSSG ratio, protecting the astrocytic membrane from lipid peroxidation and decreasing levels of Nrf2. A higher concentration of the same açaí extract exacerbated the effects of Mn in these same parameters, except on lipid peroxidation assessed by F2-IsoPs. In constrast, pre-treating with high concentrations of açaí\'s anthocyanins followed by Mn exposure, pro-oxidant effects likely prevails. These findings should be considered in future studies regarding the potential of anthocyanins in açaí on nutraceutical formulations to obtain optimal antioxidant and neuroprotective effects. Thus, we conclude that the own açaí has a phenolic fraction capable of attenuates the oxidative effects of Mn. So, may indicates that moderate açaí consumption is dietary safe regarding Mn neurotoxicity.

Die Entwicklung neuer Medikamente und Therapieverfahren wie die Hochdosischemotherapie und die Möglichkeit der Stammzelletransplantation haben die Heilungschance krebskranker Kinder in den letzen Jahrzehnten enorm verbessert. Aus diesem Grund erlangt die Berücksichtigung der Spätfolgen der Therapie eine größere Bedeutung. Es wurden die Zytostatika Cyclophosphamid und Thiotepa auf ihre Neurotoxizität im infantilen Rattengehirn untersucht. Dazu wurde Ratten im Alter von 7, 14, 21 oder 28 Tagen Cyclophosphamid (200-600mg/kg) oder Thiotepa (15-45 mg/kg) intraperitoneal injiziert. Nach einer Überlebenszeit von 4-24 Stunden wurden die Tiere getötet. Die Dichte degenerierter Zellen wurde lichtmikroskopisch in den nach De Olmos gefärbten Hirnschnitten mit Hilfe des stereologischen Dissektors ermittelt. Weiterhin wurden eine TUNEL-Färbung, elektronenmikroskopische sowie eine immunhistochemische Untersuchung für Caspase 3 und den Fas Rezeptor durchgeführt. Die Unterschiede zwischen den einzelnen Versuchsgruppen wurde mit Hilfe des Student`s t-Test auf ihre Signifikanz hin überprüft. Die Untersuchungen zur Zeit und Dosisabhängigkeit wurde mit Hilfe der ermittelten Gesamtscores und der Varianzanalyse (ANOVA) überprüft. Diese Untersuchung zeigte, dass eine Exposition mit den Zytostatika Cyclophosphamid und Thiotepa altersabhängig zu ausgeprägten Zellschädigungen im Gehirn führt. Besonders ausgeprägte Zelluntergänge fanden sich im Cortex, den thalamischen Kerngebieten, und dem Hippocampus. Ultrastrukturell ließen sich bereits kurz nach der Applikation des Zytostatikums anschwellende Dendriten als Hinweis auf einen exzitotoxischen Zelltodmechanismus nachweisen. Im Gegensatz dazu zeigten sich bei Tieren mit längerer Lebensdauer nach Exposition gegenüber dem Zytostatikum typische ultrastrukturelle Veränderungen wie man sie bei apoptotischem Zelltod finden kann. Mit dieser Untersuchung konnte gezeigt werden, dass die neurotoxische Wirkung der Zytostatika Cyclophosphamid und Thiotepa eine exzitotoxische und eine apoptotische Komponente aufweist. / Survival rates for children with cancer have increased dramatically over the past few decades. The expanded use of older agents, the development of new chemotherapeutic agents, the introduction of high dose chemotherapy and stem cell transplantation regimen have had a major impact on this improvement. These positive results have also focused increased attention on post-therapeutic effects of anticancer drugs. To investigate whether common cytotoxic drugs cause neurotoxic effects in the developing rat brain the following alkylated agents were administered to 7-day-old rats: cyclophosphamide (200–600mg/kg IP) and thiotepa (15– 45mg/kg IP). The brains were analysed at 4 to 24 hours. Quantitation of brain damage was performed in De Olmos cupric silver-stained sections using the stereological dissector method. Furthermore electron microscopy on plastic sections, TUNEL staining and immunohistochemistry for activated caspase 3 and Fas receptor was performed. Statistical analysis was performed by means of Student´s t test or one-way analysis of variance with subsequent pairwise comparison (Scheffé-test). Cytotoxic drugs produced widespread lesions within cortex, thalamus, hippocampal dentate gyrus, and caudate nucleus in a dose-dependent fashion. Early histological analysis demonstrated dendritic swelling and relative preservation of axonal terminals, which are morphological features indicating excitotoxicity. After longer survival periods, degenerating neurons displayed morphological features consistent with active cell death. These results demonstrate that anticancer drugs are potent neurotoxins in vivo; they activate excitotoxic mechanisms but also trigger active neuronal death.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-12-03T16:44:19Z No. of bitstreams: 1 JEFFERSON SOARES.pdf: 2368041 bytes, checksum: d0bf3fbdf45a445f633e578aa1e4f499 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-12-03T16:45:48Z (GMT) No. of bitstreams: 1 JEFFERSON SOARES.pdf: 2368041 bytes, checksum: d0bf3fbdf45a445f633e578aa1e4f499 (MD5) / Made available in DSpace on 2018-12-03T16:45:48Z (GMT). No. of bitstreams: 1 JEFFERSON SOARES.pdf: 2368041 bytes, checksum: d0bf3fbdf45a445f633e578aa1e4f499 (MD5) Previous issue date: 2017-08-18 / O herbicida paraquat (PQ) é reconhecido como um dos principais fatores de risco para a manifestação da Doença de Parkinson (DP) por causar destruição dos neurônios dopaminérgicos, disfunção mitocondrial e estresse oxidativo. Por isso, o PQ vem sendo utilizado como neurotoxina para a indução de sintomas semelhantes à DP em diferentes modelos experimentais. A DP é caracterizada por alterações motoras oriundas principalmente da perda seletiva e progressiva dos neurônios dopaminérgicos. Como o estresse oxidativo desempenha um papel importante na patogênese da DP, o tratamento com antioxidantes sintéticos e/ou naturais pode ser útil para diminuir a ou prevenir o aparecimento de sintomas da DP. Atualmente há uma intensa busca por antioxidantes naturais oriundos principalmente de plantas medicinais que possam ser eficazes no tratamento dos sintomas da DP. Extratos preparados a partir das folhas da Bougainvillea glabra são utilizados na medicina tradicional, no entanto, suas ações no sistema nervoso ainda não foram estudadas. Assim, o objetivo deste trabalho foi avaliar o possível efeito neuroprotetor do extrato etanólico das folhas de B. glabra (EBG) sobre alterações comportamentais e bioquímicas em moscas (Drosophila melanogaster) expostas ao PQ. Um segundo objetivo desse trabalho foi desenvolver um novo método de exposição a pesticida de modo a suprir as limitações do método tradicional em meio ágar. Com relação ao primeiro objetivo, moscas do tipo selvagem (macho, 14 dias de idade) foram concomitantemente expostas a uma dieta contendo PQ (3,5 mM) e EBG (120 μg/mL) por 4 dias. Após o tratamento foram realizadas as análises comportamentais e bioquímicas. As moscas expostas ao PQ tiveram uma diminuição da capacidade locomotora e apresentaram uma maior mortalidade que o grupo controle. A neurotoxicidade do PQ também foi associada a uma diminuição dos níveis de dopamina, aumento da atividade da enzima acetilcolinesterase (AChE) além de um aumento da produção de espécies reativas e peroxidação lipídica. A coexposição com EBG protegeu contra a mortalidade, melhorou o desempenho locomotor, impediu a redução dos níveis de dopamina e diminuiu a atividade da AChE, produção de espécies reativas e peroxidação lipídica. As análises fitoquímicas do EBG revelaram a presença de importantes compostos antioxidantes como fitol, esqualeno, α,γtocoferol, stigmasterol, geranilgeraniol, quercetina e ácidos cafeico, vanílico, cumárico e ferúlico. Nossos resultados mostram que as folhas de B. glabra podem ser consideradas um agente eficaz na prevenção de distúrbios neurológicos, onde a depleção de dopamina e/ou o estresse oxidativo estão envolvidos com a DP. Com relação ao segundo objetivo, nós desenvolvemos um novo método de exposição da Drosophila melanogaster a pesticidas denominado “alimentação líquida contínua” (ALC) visando suprir as limitações existentes no método tradicional de exposição em meio ágar como, incerteza quanto à biodisponibilidade e quantidade de pesticidas ingeridos além da elevada manipulação das moscas em tratamento. Nesse método, a alimentação (junto com o pesticida) é disponível na forma líquida para as moscas através de capilares suspensos no frasco de tratamento. O método ALC apresentou várias vantagens em relação ao método com ágar, tais como: melhor biodisponibilidade da alimentação, menor consumo de reagentes, e pouca manipulação das moscas em tratamento. Nós esperamos que o método ALC possa ser útil em futuras investigações sobre a toxicidade de pesticidas e que também possa ser utilizado em outras áreas que utilizam a D. melanogaster como modelo experimental. / The paraquat herbicide (PQ) is recognized as one of the main risk factors for the manifestation of Parkinson's disease (PD). PQ has already been shown to cause destruction of dopaminergic neurons, mitochondrial dysfunction and oxidative stress, being used as neurotoxin for the induction of PDlike symptoms in different experimental models. PD is characterized by motor alterations originating mainly from the selective and progressive loss of dopaminergic neurons. As oxidative stress plays an important role in the pathogenesis of PD, treatment with synthetic and / or natural antioxidants may be useful in slowing or preventing the onset of PD symptoms. Therefore, there is currently an intense search for natural antioxidants derived mainly from medicinal plants that may be effective in the treatment of PD symptoms. Extracts prepared from the leaves of Bougainvillea glabra are used in traditional medicine, however, their actions in the nervous system have not yet been studied. Thus, the objective of this work was to evaluate the possible neuroprotective effect of ethanol extract from B. glabra leaves (BG extract) on behavioral and biochemical changes in flies (Drosophila melanogaster) exposed to PQ. Wild type flies (male, 14 days old) were concomitantly exposed to a diet containing PQ (3.5 mM) and BG extract (120 μg /mL) for 4 days. After the treatment, behavioral and biochemical analyzes were performed. Flies exposed to PQ had a decrease in locomotor capacity and had a higher mortality than the control group. The neurotoxicity of PQ was also associated with a decrease in dopamine levels, increased activity of the acetylcholinesterase enzyme (AChE), and increased production of reactive species and lipid peroxidation. Coexposure with BG extract prevented mortality, improved locomotor performance, prevented depletion of dopamine levels, and decreased AChE activity, reactive species production and lipid peroxidation. The phytochemical analyzes of BG extract revealed the presence of important antioxidant compounds such as phytol, squalene, α, γtocopherol, stigmasterol, geranilgeraniol, quercetin and caffeic, vanillic, coumaric and ferulic acids. Our results show that B. glabra leaves can be considered an effective agent in the prevention of neurological disorders, where dopamine depletion and / or oxidative stress are involved in PD. Generally in pesticide toxicity studies with D. melanogaster, exposure to pesticides occurs by introducing them into the feed medium in agar medium. Unfortunately, this type of exposure has several limitations such as: uncertainty about the bioavailability and amount of pesticides ingested besides the high manipulation of the flies being treated due to the need of medium exchange. Thus, this work also aimed to develop a new method of exposure to pesticides in order to overcome the limitations of the method using the agar medium. We developed the method called "Continuous Liquid Feeding" (CLF). In this method, feed (along with the pesticide) is available in liquid form to the flies through suspended capillaries in the treatment vial. The CLF method presented several advantages over the agar method, such as: better feed bioavailability, lower reagent consumption, and little handling of the flies under treatment. We hope that the CLF method may be useful in future investigations on the toxicity of pesticides and that it can also be used in other areas that use D. melanogaster as an experimental model.

A Doença de Alzheimer (DA) é o tipo mais comum de demência em idosos, a etiologia é multifatorial e a fisiopatologia da doença é complexa, com um novo caso acontecendo a cada sete segundos; globalmente, a doença está se tornando em uma lenta pandemia. Bioquimicamente, a DA é caracterizada pela presença das placas neuríticas (PNs) e os novelos neurofibrilares (NNFs). O peptídeo beta peptide1-42 (A?(1-42)) é o principal componente das placas neuríticas e tem sido fortemente associado ao estresse oxidativo, desregulação colinérgica e morte celular. Os múltiplos mecanismos envolvidos na patogênese criam consideráveis dificuldades para identificar alvos terapêuticos apropriados. As abordagens terapêuticas atuais melhoram temporariamente os sintomas da DA; no entanto, apesar de esforços intensivos, nenhum dos tratamentos disponíveis hoje conseguiu alterar o curso da doença. Porém, algumas das terapias mais relevantes para o tratamento da doença estão baseadas na atividade inibidora da acetilcolinesterase (AChE). Nos últimos anos, os alcaloides pertencentes à família Amaryllidaceae têm recebido muita atenção devido à atividade anticolinérgica e antioxidante. A galantamina foi o primeiro alcaloide isolado a partir de diferentes espécies de Amaryllidaceas e é o mais recente inibidor da AChE aprovado para o tratamento sintomático da DA. Este fato tem motivado a pesquisa de outros alcaloides como possíveis moduladores da doença em adição à atividade inibitória da AChE. Diante disso, o objetivo deste estudo foi investigar se o extrato de Caliphruria subedentata e a galantamina modulam a neurotoxicidade induzida pelo A?(1-42) na linhagem celular SH-SY5Y indiferenciada. Para compreender os mecanismos de neuroproteção, um conjunto de ensaios foi realizado tais como atividade inibitória da AChE, ensaios clonogênico, micronúcleos com bloqueio na citocinese celular (CBMNcyt), cometa; análises por microscopia eletrônica de transmissão (MET) e de metilação. Os resultados mostraram que tanto o extrato quanto a galantamina diminuíram significativamente a citotoxicidade e genotoxicidade induzida pelo A?(1-42). Além disso, ambos os tratamentos modularam alterações morfológicas mitocondriais induzidas pelo peptídeo. Em conclusão, os resultados deste estudo demonstraram que, em adição à atividade inibitória da AChE, tanto o extrato de C. subedentata quanto a galantamina exercem propriedades antigenotóxicas. Essas propriedades relevantes da Amaryllidaceaes e o fármaco tornam-se um potencial valioso para continuar sendo explorado. / Alzheimer´s disease (AD) is the most common type of dementia in elderly population, the etiology is multifactorial and the pathophysiology of the disease is complex, with a new case occurring every seven seconds; globally, the disease itself is becoming a slowly pandemic. Biochemically, the AD is characterized by presence of the neuritic plaques and neurofibrillary tangles. Amyloid beta peptide1-42 (A?(1-42)) is the principal component of neuritic plaques and it has been strongly associated with oxidative stress, cholinergic deregulation and cell death. The multiple mechanisms involved in the pathogenesis create considerable difficulty to identify appropriate targets. The current therapeutics approaches for AD improve temporally the symptoms; and despite intensive efforts, none of the treatments available today alter the course of disease. Nervertheless, some of the most relevant therapies for the treatment of disease are based on acetylcholinesterase (AChE) inhibitor activity. In recent years, alkaloids belonging Amaryllidaceae family have received great attention due to the well-known anticholinergic and antioxidant activity and the galanthamine was the first alkaloid isolated from different species of Amaryllidacea and it is the most recently AChE inhibitor approved for the symptomatic treatment of AD. This fact has motivated the screening of other alkaloids as possible modulators of disease in addition acetylcholinesterase activity. Purpose this study was to investigate whether C. subedentata extract and galanthamine modulate A?(1-42)- induced neurotoxicity in the undifferentiated SH-SY5Y cell line. To understand the mechanisms of the neuroprotection, a set of biomarkers such as AChE activity, clonogenic, cytokinesis block micronucleus cytome (CBMNcyt) and comet assays; beside transmission electron microscope (TEM) and methylation analyses were realized. The results showed that C. subedentata extract and galanthamine were capable to significantly reduce the A?(1-42)- induced cytotoxicity and genotoxicity. Furthermore both treatments modulated A?(1-42)- induced mitochondrial morphological alterations. In conclusion, this study demonstrated that in addition to inhibition of acetylcholinesterase (AChE), the extract of C. subedentata and galanthamine exert antigenotoxic properties. This relevant property of Amaryllidaceaes and galanthamine are worthwhile exploring further which may improve the development of new diseases-modifying agents.

Astrócitos são células gliais com fundamental importância no sistema nervoso central (SNC), tanto em condições fisiológicas quanto patológicas. Estas células são essenciais na plasticidade neural, no metabolismo de neurotransmissores, na defesa antioxidante, na regulação do metabolismo energético, na homeostase iônica, na resposta inflamatória, na manutenção da barreira sangue-cérebro, na migração neuronal e na estabilização da comunicação entre as células. Assim, alterações em funções astrocitárias (como as que ocorrem no envelhecimento) estão relacionadas a importantes alterações na funcionalidade cerebral. Desta forma, esta tese teve por objetivo demonstrar que a cultura de astrócitos derivada de ratos Wistar adultos, desenvolvida e caracterizada pelo nosso grupo de pesquisa, pode ser um modelo de estudo fidedigno e versátil das propriedades celulares astrocitárias. Nossos resultados apontam que esta metodologia pode ser utilizada para elucidar o perfil de aminoácidos e gliotransmissores assim como da atividade enzimática glial e gerenciamento de neurotransmissores. Também demonstramos que a cultura adulta não é derivada de progenitores neurais e que parâmetros mitocondriais observados no cérebro adulto foram reproduzidos in vitro. A análise de respostas a estímulos demonstrou ser variável, dependendo da idade dos animais. Da mesma forma, o uso de culturas de diferentes idades revelou o efeito antienvelhecimento da guanosina, sugerindo sua atividade glioprotetora. Finalmente, demonstramos que culturas preparadas a partir de animais neonatos submetidas a um modelo de senescência in vitro apresentam respostas diferentes das apresentadas por culturas preparadas a partir de animais adultos e/ou envelhecidos, demonstrando que o modelo mais adequado para elucidar propriedades astrocitárias do cérebro maduro é o derivado de animais adultos. Portanto, demonstramos com este estudo a importância da disponibilidade de uma ferramenta como a cultura de astrócitos adultos e elucidamos características bioquímicas, celulares e moleculares desta ferramenta, evidenciando algumas de suas diferenças em comparação à cultura preparada a partir de ratos neonatos. Assim, ampliamos a compreensão das propriedades e funções celulares desta ferramenta, fornecendo respostas mais aproximadas às respostas fornecidas por astrócitos do cérebro maduro in vivo, especialmente no estudo do envelhecimento e das doenças neurodegenerativas. / Astrocytes are glial cells of pivotal importance in the central nervous system (CNS), both in physiological and pathological conditions. Some of their roles include neural plasticity, neurotransmitter metabolism, antioxidant defenses, control of energy metabolism, ionic homeostasis, inflammatory response, formation and maintenance of blood-brain barrier, neuronal migration and cellular communication. Thus, changes in astrocytic function (such as occurs in aging) are related to changes in brain function. The aim of this thesis was to demonstrate that astrocyte cultures from adult Wistar rats (developed and characterized by our research group) might be a reliable and versatile tool for studying astrocytic cellular properties. Our results suggest that this culture model is suitable to study the amino acids content and gliotransmitters, as well as glial enzymatic activity and neurotransmitter management. Next, we showed that the astrocyte cultures are not derived from neural progenitors and tissue mitochondrial parameters were reproduced in in vitro cultures. Responses to stimulus were variable, depending on the animals’ age. Accordingly, guanosine presented an anti-aging effect, indicating its glioprotective activity. Finally, we showed that cultures prepared from newborn rats submitted to an in vitro senescence model presented different responses when compared with mature animals, indicating that our culture model is the most suitable model to represent astrocytic properties in the mature brain. Therefore, this study demonstrates the relevance of this tool to understanding the biochemical, cellular and molecular properties of adult astrocytes, showing some differences related to the culture prepared from newborn animals. Thus, we amplify the comprehension about cellular functions of this tool, providing closer responses related to mature brain in vivo, especially regarding studies about aging and neurodegenerative diseases.

O Bisfenol A (BPA) é usado na fabricação de plásticos de policarbonato e resinas epóxi. A exposição pré-natal a esse agente pode causar diversos efeitos, tais como: antecipação da puberdade, hiperplasia de próstata, diminuição do número de espermatozoides, diminuição dos níveis de testosterona, alteração do desenvolvimento e organização tecidual da glândula mamária, diminuição da resposta celular induzida por hormônios, câncer de mama, diabetes, doenças cardiovasculares, alterações das funções de enzimas hepáticas, além de efeitos sobre o desenvolvimento cognitivo. Poucos estudos avaliam os efeitos do BPA sobre as células neuronais, porém existem evidências de que este agente induza a apoptose. O presente trabalho tem como objetivo estudar a neurotoxicidade do BPA, avaliando vias de sinalização que levam a indução da apoptose em cultura primária de hipocampo. As células foram expostas ao BPA nas concentrações de 50, 100, 150, 200, e 250 µM (0,1% DMSO v/v) pelos períodos de 6, 12, 24, e 48 horas para a realização dos ensaios da atividade mitocondrial (MTT) e citotoxicidade pela liberação da enzima Lactato Desidrogenase (LDH). A partir dos resultados de MTT e LDH, foram adotados novos horários de exposição (3, 6 e 9 horas) utilizando somente as concentrações de 200 e 250 µM. Neste novo desenho experimental, foi realizada a quantificação da concentração de BPA na cultura primária por HPLC-PDA, determinação da concentração de Ca2+ intracelular pela quantificação da fluorescência do Fluo-4 AM, caracterização dos mecanismos envolvidos na morte celular por citometria de fluxo e Western Blotting, e avaliação dos receptores de estrógeno ER-α e ER-β por Western Blotting. Nossos resultados apontam que aproximadamente 20% de BPA na concentração de 250 µM após 6 horas de exposição e 18% para a concentração de 200 µM com 9 horas de exposição foram absorvidos pela cultura celular. O ensaio do MTT mostrou que as células expostas a 200 e 250 µM de BPA, por 12, 24 e 48 horas, apresentaram diminuição significativa da função mitocondrial em relação ao controle. Porém, não houve liberação de LDH para o meio de cultura para nenhuma das concentrações de BPA em nenhum dos períodos de incubação, o que sugere que não houve rompimento da membrana plasmática. Foi observada atividade apoptótica somente com a concentração de 250 µM no período de exposição de 6 horas por citometria de fluxo. Não foram encontradas células em necrose, nem alteração na concentração de cálcio intracelular em nenhuma das condições estudadas. Na avaliação dos marcadores de morte celular, observamos aumento da razão de Bax/Bcl-2 para a concentração de 250 µM em todos os períodos de exposição e aumento das caspases 8, 9 e 3 para a concentração de 250 µM no período de exposição de 6 horas, indicando que o BPA deve ativar tanto a via intrínseca como a extrínseca no processo de apoptose. Verificamos ainda, por Western Blotting, que a cultura primária de hipocampo apresenta os receptores de estrógeno ER-α e ER-β. A exposição ao BPA aumentou os ER-α e ER-β avaliados por Western Blotting para as duas concentrações estudadas no período de 6 horas de exposição e, para o período de exposição de 9 horas, houve um aumento do ER-α para a concentração de 250 µM e do ER-β para a concentração de 200 µM. É possível concluir que o BPA pode levar a morte das células neuronais hipocampais por apoptose por ambas as vias intrínseca e extrínseca, sendo o processo de morte celular mais evidente para a concentração de 250 µM no período de 6 horas de exposição. Sugerimos ainda que o aumento observado em ambos os receptores de estrógeno possa representar uma tentativa de interrupção ou reversão do processo de morte celular. / Bisphenol A (BPA) is used in the manufacture of polycarbonate plastics and epoxy resins. The prenatal exposure to this agent may cause several effects, such as anticipation of puberty, prostate hyperplasia, reduced number of sperm, reduced testosterone levels, alteration in the development and tissue organization of the mammary gland, decreased cellular response induced by hormones, breast cancer, diabetes, cardiovascular disease, changes in the functions of liver enzymes, and effects on cognitive development. Few studies have evaluated the effects of BPA in neuronal cells, however there are evidences that this agent may induce apoptosis. This work aims to study the neurotoxicity of BPA, by analyzing the signaling pathways of apoptosis in hippocampus primary culture. Cells were exposed to BPA at 50, 100, 150, 200, and 250 µM (0.1% DMSO v/v) for 6, 12, 24, and 48 hours for the assay of mitochondrial activity (MTT) and the release of the enzyme lactate dehydrogenase (LDH). From the results of MTT and LDH, new exposure times (3, 6 and 9 hours) and only 200 and 250 µM were used. In this new experimental design we performed the quantification of the BPA concentration in the primary culture by HPLC-PDA, intracellular Ca2+ quantification by Fluo-4 AM assay and the characterization of the mechanisms involved in cell death by flow cytometry and Western Blotting assays. Furthermore, evaluation of the estrogen receptor ER-α and ER-β was done by Western Blotting. Our results demonstrate that about 20% of the BPA concentration of 250 µM after 6 hours of exposure and 18% for the concentration of 200 µM with 9 hours of exposure were absorbed by the cell culture. Cells exposed to 200 and 250 µM of BPA for 12, 24 and 48 hours, showed a significant decrease in mitochondrial function, by the MTT assay, compared to control. However, there was no release of LDH into the culture medium for any of the BPA concentrations in any of incubation times studied, which suggests no rupture of the plasma membrane by BPA. Apoptotic activity was observed after 6 hours of exposure to 250µM BPA by flow cytometry. It was not observed cell necrosis and changes in intracellular calcium concentration in any of the studied conditions. Regarding the cell death markers, exposure to 250 µM BPA in all periods of exposure resulted in an increased Bax/Bcl-2 ratio; moreover, an increase in caspase 8, 9 and 3 was detected after exposure to 250 µM BPA for 6 hours. Taken together, these findings indicate that BPA activates both the intrinsic and the extrinsic pathway during the apoptotic process. We also verified by Western Blotting the presence of the estrogen receptors ER-α and ER-β at the primary culture of hippocampus, and that they can be modulated by BPA. The exposure to 200 and 250 µM BPA for 6 hours caused an increase of ER-α and ER-β, however, 9 hours of exposure to 200 µM and 250 µM BPA increased the expression of ER-α and ER-β, respectively. In conclusion, BPA can lead hippocampal neuronal cells to death by both, intrinsic and extrinsic, apoptotic pathways and this process is more evident at 250 µM BPA after 6 hours of exposure. Furthermore, we suggest that the increase of both estrogen receptors might represent an attempt to interrupt or reverse the cell death process.