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No. of bitstreams: 2 Tese - Fernanda Craveiro Franco - 2017.pdf: 4803005 bytes, checksum: 35b5c5c99f5a0689a4c30ebe8ea513cd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-09-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The interaction of some compounds and DNA molecule can cause genotoxic changes. The tests used to evaluate genotoxicity have evolved over the years, and they have great efficiency. Currently, several traditional methodologies are used, such as the comet assay, and some more modern, based on molecular biology. Considering the importance of detecting the effects caused by exposure to potentially genotoxic agents, this thesis presents three chapters with different approaches to this topic. The first chapter aimed at evaluating published studies on the mutagenic, genotoxic and oxidative effects caused by occupational exposure to pesticides. For this, a systematic review of articles published between the years of 2011 and 2015 was carried out, focusing on these exposure conditions. The results showed that 44% of the studies addressed only men and 44% approached men and women, and 78% used rural workers as an exposed population. The most evaluated class of pesticide was organophosphorus. The studies were separated by methodologies of analysis used and all showed some type of alteration in the populations occupationally exposed to pesticides. The second chapter, published in the journal Environmental Science and Pollution Research, considered the dengue scene in the state of Goias and techniques of mosquitoes control, and sought to understand the genotoxic effects caused by exposure of endemic agents to pesticides used against Aedes aegypti, being the first study published with this group of workers. The main pesticides used were insecticides and larvicides and an increase in DNA damage was observed in the exposed population, without influence of the lifestyle factors. In the whole transcriptome assay the differential expression of genes related to various chronic diseases was observed. Finally, the third chapter was carried out in collaboration with the Institute of Experimental Medicine of the Czech Academy of Sciences, aiming the determination of cytotoxic and genotoxic effects caused by in vitro exposure to different types of metallic nanomaterials. In nanomaterials that showed cell viability reduction (cytotoxic), an increase in genotoxicity was also identified. In addition, three of the four non-cytotoxic nanomaterials evaluated showed genotoxic effects. Studies aimed at detecting the compounds genotoxic potential are extremely important for the regulatory definition and identification of risks caused by exposure. In this study, the toxicological and genotoxic effects of different compounds and mixtures were evaluated using traditional and modern techniques, in vitro or by human biomonitoring, seeking to understand and determine the risks involved in exposure to these substances. / Compostos que interagem com a molécula de DNA podem provocar alterações genotóxicas. Os testes utilizados para avaliação de genotoxicidade evoluíram ao longo dos anos, apresentando grande eficiência. Atualmente, utilizam-se várias metodologias tradicionais, como o ensaio cometa, e também algumas mais modernas, baseadas na biologia molecular. Considerando a importância de detectar os efeitos causados pela exposição a agentes potencialmente genotóxicos, essa tese apresenta três capítulos com formas de abordagem distintas, referente a essa temática. O primeiro capítulo visou a avaliação de estudos publicados acerca dos efeitos mutagênicos, genotóxicos e oxidativos causados pela exposição ocupacional a pesticidas pela realização uma revisão sistemática de artigos publicados entre os anos de 2011 a 2015. Observou-se que 44% dos estudos abordaram exclusivamente indivíduos do sexo masculino e 44% abordaram ambos os sexos, e 78% utilizaram trabalhadores rurais como população exposta. A classe de pesticida mais avaliada foi a de organofosforado. Os estudos foram separados por metodologias de análise utilizada e todos apresentaram algum tipo de alteração nas populações expostas a pesticidas no ambiente de trabalho. O segundo capítulo, publicado na revista Environmental Science and Pollution Research, considerou o cenário da dengue no estado de Goiás e as formas de combate ao mosquito, e buscou compreender os efeitos genotóxicos causados pela exposição de Agentes de combate a endemias aos pesticidas utilizados contra o mosquito, sendo o primeiro estudo publicado com esse grupo de trabalhadores na região centro-oeste. Os principais pesticidas utilizados foram inseticidas e larvicidas e foi observado um aumento no dano ao DNA na população exposta, sem influência dos fatores relacionados ao estilo de vida. No ensaio de transcritoma foi observada a expressão diferencial de genes relacionados a diversas doenças crônicas. Finalmente, o terceiro capítulo foi realizado em colaboração com o Instituto de Medicina Experimental, da Academia Tcheca de Ciências, visando a determinação de efeitos citotóxicos e genotóxicos, causados pela exposição in vitro a diferentes tipos de nanomateriais metálicos. Nos nanomateriais que apresentaram redução na viabilidade celular (citotóxicos) também foi identificado aumento na genotoxicidade. Além disso, três dos quatro nanomateriais não citotóxicos avaliados apresentaram efeitos genotóxicos. Estudos que visam detectar o potencial gentoxicológico de compostos são de extrema importância para definição de normas e regulamentações de aplicação e identificação dos riscos causados pela exposição. Nesse estudo, foram avaliados os efeitos toxicológicos e genotóxicos de diferentes compostos e misturas, com a utilização de técnicas tradicionais e modernas, in vitro ou por biomonitoramento humano, buscando compreender e determinar os riscos envolvidos na exposição a essas substâncias.

Philosophiae Doctor - PhD / Cancer is one of the leading causes of death worldwide. According to the WHO, cancer accounted for 7.4 million deaths world wide in 2004. The metallo-compound cisplatin has been used for years as an effective antitumor agent for treating solid tumours such as breast, bladder, lung, oesophageal, and head and neck carcinomas. However, the use of cisplatin as an antitumor agent has been limited because of its association with problems such as lack of selectivity for cancer cells over normal cells, development of resistance to cisplatin treatment, and side effects such as nephrotoxicity. Recent studies on anticancer drugs have focussed on alternative anticancer agents such as gold compounds in both Au(I) and (III) oxidation states, which have shown to be potential anticancer drug agents because of their ability to induce apoptosis in several human cancer cells. Some gold complexes have shown to be able to selectively kill cancer cells over normal cells.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Acetaminophen (APAP) is the analgesic most used in world, in therapeutic doses does not show toxicity, but in elevated doses can cause hepatic damage due the formation of his metabolic toxic N-acetyl-p-benzoquinonimine (NAPQI). The brain damage can occurs because hepatic damage a condition called hepatic encephalopathy, since the liver already show his function altered, like transform ammonia in urea, causing accumulation of ammonia in the brain, which is toxic for this organ. Furthermore, the NAPQI can cause oxidative damage and mitochondrial dysfunction on brain tissue. The diphenyl diselenide [(PhSe)2] is an organoselenium compound that exhibit antioxidant activity and potential pharmacological. The aim of this study is investigated the ability of (PhSe)2 in reversing the oxidative brain damage and mitochondrial dysfunction induced by a toxic dose of APAP. Mice received a APAP (600mg/kg), followed by a dose of (PhSe)2 (15,6 mg/kg) 1 hour latter. Four hours after APAP administration, plasma was withdrawn from the mice and used for biochemical assays of aspastate aminotransferase (AST) and alanine aminotransferase (ALT) confirming the hepatic damage. The APAP administration increase lipid peroxidation, production of reactive oxygen species and decrease in activity of Na+, K+ - ATPase enzyme. Similary, APAP caused alteration on parameters of mitochondrial function. The treatment with (PhSe)2 revert the cerebral damage induced by a single dose of APAP. / O paracetamol é o analgésico mais usado no mundo, em doses terapêuticas não apresenta nenhuma toxicidade, porém em doses elevadas pode causar dano hepático pela formação do seu metabólito tóxico N-acetil-p-benzoquinoneimina (NAPQI). O dano cerebral pode ocorrer em decorrência ao dano no fígado, uma condição chamada de encefalopatia hepática, já que o fígado pode apresentar um comprometimento das suas funções, como transformar amônia em uréia, causando um acúmulo de amônia no organismo, sendo esta tóxica para o cérebro. Além disso, o NAPQI pode causar estresse oxidativo e disfunção mitocondrial no cérebro. O disseleneto de difenila [(PhSe)2] é um composto orgânico de selênio que apresenta atividade antioxidante e potencial farmacológico. O objetivo desse estudo é verificar a capacidade do (PhSe)2 em reverter o dano cerebral e disfunção mitocondrial causada por uma dose tóxica de paracetamol. Os camundongos receberam paracetamol (600 mg/kg) e 1h após, disseleneto de difenila (15,6 mg/kg). Após 4h da administração do paracetamol, coletou-se o soro para as análises bioquímicas de aspartato aminotransferase (AST) e alanina aminotransferase (ALT) que confirmaram o dano hepático. A administração de APAP aumentou a peroxidação lipídica, a produção de espécies reativas de oxigênio e diminuiu a atividade da enzima Na+, K+ - ATPase. Da mesma forma, o APAP alterou os parâmetros de funcionalidade mitocondrial. O tratamento com (PhSe)2 reverteu o dano cerebral induzido por uma dose única de APAP.

Secondary metabolites are compounds that are not necessary for the survival of the organism, but which are to be related to the organism's interaction with its environment. This work studied compounds of secondary metabolism through the study of chemical and biological extracts and fractions Simaba Maiana Casar. and the involvement of candidate genes (TPS10, TPS14, and CYP71B31 CYP76C3) in the metabolism of monoterpenes in Arabidopsis thaliana. The extracts from roots and stems of Simaba Maiana were tested in antioxidant activity, molluscicide, inhibition of lymphocyte proliferation, inhibition of NO production, anti-Leishmania amazonensis and anti-Trypanosoma cruzi. The four candidate genes (CYP76C3, CYP71B31, TPS10 and TPS14) were selected with the CYPedia, which calculates the coexpression between genes based on the Arabidopsis Affymetrix ATH1 microarray. The extracts and fractions Simaba Maiana showed a lower antioxidant activity by DPPH method, low concentrations of total phenols measured by Folin-Ciocalteu, however, a good antioxidant activity by TBARS method, using three agents of oxidative damage (AAPH, FeSO4 and H2O2). The extract showed cytotoxic activity and molluscicidal concentration of 100 mg/mL. The crude extract of the stem was not active for leishmanicidal and trypanocidal. This extract did not inhibit NO production, but showed a high percentage of inhibition of lymphoproliferation. The skimmianine furoquinoline alkaloid and pellopterin furanocoumarin were isolated from chloroform fraction. The candidate genes showed a similar pattern of expression of the stamen, more specifically the top of the filaments. Heterologous expression in transiently expressed in leaves of Nicotiana benthamiana, in the volatiles of TPS10 TPS14 (alone) were found enantiomers R-(-)-linalool and S-(+)-linalool. In the extraction buffer leaf discs were found that CYP76C3 converts linalool to E-8-hydroxy-linalool and E-8-oxo-linalool, and CYP71B31 in 1,2-epoxy-linalool. The analysis of the methanol extract of the discs incubated in S-(+)-linalool showed the use of this substrate by P450s converting to lilac alcohol for both P450s. Analysis of flowers of Arabidopsis mutants showed minor differences, analyzes with extracts of fresh flowers in UPLC-MS/MS MRM mode, has been found a compound having the same signature as linalool, however with different retention time and may be is an indication of linalool bound. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os metabólitos secundários são compostos que não são necessários para a sobrevivência do organismo, mas que apresentam-se relacionados com a interação do organismo com o seu ambiente. O presente trabalho estudou compostos do metabolismo secundário através do estudo químico e biológico dos extratos e frações Simaba maiana Casar. e a envolvimento dos genes candidatos (TPS10, TPS14, CYP76C3 e CYP71B31) no metabolismo de monoterpenóides em Arabidopsis thaliana. Os extratos das raízes e caule de Simaba maiana foram testadas em ensaios de atividade antioxidante, moluscicida, inibição da linfoproliferação, inibição da produção de NO, anti-Leishmania amazonensis e anti-Trypanosoma cruzi. Os quatro genes candidatos (CYP76C3, CYP71B31, TPS10 e TPS14) foram selecionados com o CYPedia, que calcula a co-expressão entre os genes de Arabidopsis com base no Affymetrix ATH1 microarray. Os extratos e frações de Simaba maiana mostraram uma baixa atividade antioxidante pelo método do DPPH, baixas concentrações de fenóis totais avaliados pelo método de Folin-Ciocalteu, entretanto, uma boa atividade antioxidante pelo método de TBARS, usando três agentes de danos oxidativos (AAPH, FeSO4 e H2O2). Os extratos mostraram atividade moluscicida e citotóxica na concentração de 100 mg/mL. O extrato bruto do caule não foi ativo para as atividades anti-Leishmania e anti-Trypanosoma. Este extrato não inibiu a produção de NO, mas apresentou uma alta porcentagem da inibição da linfoproliferação. O alcalóide furoquinolínico esquiamina e a furanocumarina felopterina foram isolados da fração clorofórmica. Os genes candidatos mostraram um similar padrão de expressão nos estames, mais especificamente na parte superior dos filamentos. Na expressão heteróloga transitoriamente expressa em folhas de Nicotiana benthamiana, nos voláteis de TPS10 e TPS14 (sozinho) foram encontrados os enantiômeros R-(-)-linalol e S-(+)-linalol. No tampão de extração de discos de folhas, verificou-se que CYP76C3 converte linalol em E-8-hidroxi-linalol e E-8-oxo-linalol, e CYP71B31 em 1,2-epoxilinalol. A análise do extrato metanólico dos discos foliares incubados em S-(+)-linalol mostrou a utilização deste substrato por P450s convertendo para lilac álcool para ambos os P450s. Análises de flores em plantas mutantes de Arabidopsis thaliana mostraram pequenas diferenças, em que análises com extratos de flores frescas em UPLC-MS/MS no modo MRM, foi encontrado um composto com a mesma assinatura que linalol, no entanto, com tempo de retenção diferente e pode ser uma indicação de forma ligada do linalol.

The rate of cancer patients is increasing as the development of science and technology. Twenty million cancer survivors are estimated living in the United States by 2025. However, many cancer survivors show cognitive dysfunction, negatively affecting the quality of life. These cognitive impairments are recognized as chemotherapy-induced cognitive impairment (CICI), also called "chemo brain" by cancer survivors, including the diminished ability of memory and learning, hard to concentrate and focus, as well as diminution of executive function and processing speed. The etiologies and pathologies of CICI are complicated, especially in most cases the anti-cancer drug cannot cross the blood-brain barrier (BBB). One of the significant candidate mechanisms underlying CICI is chemotherapy-induced, oxidative damage-mediated tumor necrosis factor-alpha (TNF-a) elevation. One of the prototypes of reactive oxygen species (ROS)-generating chemotherapeutic agents is Doxorubicin, normally used as part of multi-drug chemotherapeutic regimens to treat solid tumors and lymphomas. In this dissertation, TNF-a null (TNFKO) mice were used to investigate the role of TNF-a in Dox-induced, oxidative damage-mediated alterations in brain. Dox-induced oxidative damage in brain is ameliorated and brain mitochondrial function is preserved in brains of TNFKO mice. Both Dox-decreased levels of hippocampal choline-containing compounds and activities of brain phospholipases are partially protected in the TNFKO group. It is shown in this dissertation that Dox-targeted mitochondrial damage and levels of brain choline-containing metabolites, as well as changes in the activity of phospholipases, including both phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD), are decreased in the CNS and associated with oxidative damage mediated by TNF-a. The results are discussed with respect to identifying a potential therapeutic target to protect against cognitive problems after chemotherapy and thereby improve the quality of life of cancer survivors. We also tested the effect of a chemotherapy drug adjuvant, 2-mercaptoethane sulfonate sodium (MESNA), on CICI in this dissertation research. MESNA ameliorated Dox-induced oxidative protein damage in plasma and led to decreased oxidative damage in brain. MESNA was demonstrated to rescue the memory deficits caused by Dox in the novel object recognition test. The activity of PC-PLC was preserved when MESNA was co-administered with Dox. This study is the first evidence for demonstrating the protective effects of MESNA on Dox-related protein oxidation, cognitive decline, phosphocholine levels, and PC-PLC activity in brain and suggests novel potential therapeutic targets and strategies to mitigate CICI. Parkinson Disease (PD) is considered as the second most neurodegenerative disease, associated with aging and gender. Although the detailed mechanisms remain unknown, inflammation and oxidative damage are two main etiological factors of PD. Certain genetic factors have been discovered related to this disease. Thus, using rodent models with relative gene mutations are the main strategies to investigate PD. However, few rodent models showed same clinical and biochemical features of PD. PTEN-induced putative kinase -1 (PINK1) knockout (KO) rat is the rodent model used in this dissertation research. The oxidative damage in the brain of PINK1 KO rats, the ventricle sizes, and neurochemical metabolite profiles in these rats as a function of age and gender were measured. Distinct gender- and age-related alterations were found, many consistent with those in PD. The proteome of brain of PINK1 KO rat as a function of age and gender also was studied. Based on the collected data, the suitability of this unique rat as a faithful model of known characteristics of PD with our results is discussed.

N5-deoxyribosyl derivatives of 2,5-diaminoimidazolone formed by oxidative damage to the guanine bases in 2-deoxyguanosine and highly polymerized DNA readily undergo nucleophilic substitution at C5 in reaction with primary amines in neutral aqueous solutions at 37–70 °C, as it was found in a kinetic study using reverse-phase HPLC. The reaction of 2-amino-5-[(2′-deoxy-β-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz) with excess of ethanolamine, alanine and γ-aminobutyric acid (0.2–1 M) is a pseudo-first-order process that proceeds with 45–80 % yields depending on the nature of the amine, its concentration, and the reaction temperature. In the case of ethanolamine, the corresponding bimolecular rate constant has a pre-exponential factor and activation energy of 1.1 × 105 s−1 and 47 kJ mol−1, respectively. The reaction is highly competitive with the previously described hydrolysis of dIz into 2,2-diamino-4-[(2-deoxy-β-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone under biologically relevant conditions. A similar reaction with the same lesion in polymeric DNA results in the release of a low-molecular-weight analog of dIz, presumably producing an abasic site as the second reaction product. Kinetic characteristics of this process make it a potentially important source of abasic sites in oxidatively damaged DNA, formed through the reaction of 2,5-diaminoimidazolone lesions with naturally abundant DNA-affinic amines and proteins. The release of low-molecular-weight analogs of dIz can potentially be employed for quantification of imidazolone lesions in oxidized DNA. The half-life of imidazolone lesions in double-stranded DNA evaluated using this approach was found to be 154 min at 37 °C.

The first cases of patients with pneumonia which grew into an acute respiratory distress syndrome and caused breathing problems began to appear in December 2019. Coronavirus disease 2019 (COVID-19) is the cause of a global pandemic and it is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A complex interplay of factors is responsible for the progression of the disease. Some studies suggest that it promotes oxidative stress and thus may lead to oxidative damage to cells and DNA. The purpose of this study was to observe the relationship between oxidative DNA damage and a critical condition caused by COVID-19 using a comet assay technique. The basic principle of the used method consists in fixation of lymphocytes in an agarose gel, removal of the membrane and cytoplasm of cells, incubation with specific enzymes and electrophoresis. In the process of electrophoresis, negatively charged DNA fragments migrates towards the anode and the cell thus acquires the typical shape of a comet. Comets are visualized using the DNA intercalation dye ethidium bromide. We quantified single - strand breaks and oxidized pyrimidines and purines by using specific enzymes (modification of the method for detecting specific lesions). Results are reported as % tail DNA, thus the percentage of DNA in the...

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Oxidative stress is caused by overproduction and overaccumulation of ROS (reactive oxygen species). This state is responsible for cellular damage during unfavorable environmental conditions such as drought, low temperatures, salinity. In order to directly study oxidative stress at tobacco plants (Nicotiana tabacum cv. Xanthi) I used specific herbicides, MV (methyl viologen) and 3-AT (3- aminotriazole). There were several markers used for monitoring oxidative damage to cellular components: DNA damage detected by a comet assay, lipid peroxidation, carbonylated proteins and modification of activities of antioxidant enzymes CAT (catalase) and APX (ascorbate peroxidase). Fluorescent microscopy documented changes in a redox state of tobacco cells and a specific signal for peroxisomes was observed after treatment with higher concentrations of MV and 3-AT. Application of both herbicides caused significant DNA damage, while they worked in a different concentrations, MV in µM and 3-AT in mM. Another convincing oxidative stress marker for MV was protein carbonylation. The inhibition of antioxidant enzymes CAT and APX was less significant when compared to the effects of 3-AT. Decreasing membrane stability proved to be an universal oxidative stress marker for both herbicides. On the other hand, lipid...