A cidade de Ribeirão Preto é abastecida com água proveniente do Aquífero Guarani, sendo apenas fluoretada e clorada antes da distribuição. O objetivo desse trabalho foi avaliar a qualidade da água que a população está consumindo com relação às concentrações de fluoreto, chumbo e ferro. Além disso, foi investigada se a concentração de ferro poderia ser utilizada como marcadora da contaminação por chumbo na água potável. Foram investigadas 145 residências de 4 bairros antigos de Ribeirão Preto (casas com mais de 40 anos), havendo pelo menos uma criança moradora por residência. Um bairro novo (menos de 10 anos) foi escolhido para controle. Amostras de água foram coletadas na torneira da cozinha e do quintal, sendo a 1ª alíquota coletada após pelo menos 6 h de estagnação e a 2ª alíquota após 5 minutos de escoamento. A concentração dos íons fluoreto foi determinada por eletrodo combinado íon-seletivo e cromatografia iônica; chumbo por ET AAS e ICP-MS; e ferro por espectrofotometria. As concentrações de fluoreto nas amostras (n = 281) variaram de 0,052 a 1,01 mg L-1. Considerando apenas 1 algarismo significativo e as amostras provenientes da cozinha, observou-se que 30% das casas estiveram fora do padrão de potabilidade estabelecido pela Resolução SS-65 (2005), isto é, de 0,6 a 0,8 mg L-1. Considerado 2 algarismos, essa percentagem passou para 45%. Um baixo consumo de fluoreto pelas crianças pode não prevenir eficientemente a formação de cáries, enquanto que concentrações elevadas podem levar à fluorose dentária. As concentrações de chumbo variaram de 0,025 a 58,4 µg L-1 (n = 562), sendo que a concentração máxima permitida pela Portaria 2.914 (Ministério da Saúde) é de 10,0 µg L-1. Para todas as casas avaliadas, as concentrações de chumbo na água da torneira da cozinha não ultrapassaram 5,00 µg L-1, caracterizando um baixo risco de contaminação por chumbo por essa via. Para 2 alíquotas de água coletadas na torneira do quintal, a concentração de chumbo ultrapassou o limite tolerável para chumbo, porém, o uso dessa água para ingestão é pouco usual. Possivelmente a contaminação observada seja oriunda da própria torneira e outras partes metálicas, pois após escoamento da água por 5 minutos, a concentração de chumbo caiu dentro dos valores permitidos. As concentrações de ferro variaram de 13,40 a 4.119 µg L-1 (n = 189; 1ª alíquota), sendo a concentração máxima permitida de 300 µg L-1 (Portaria 2.914). Em 4 casas a água do quintal apresentou concentração superior a permitida, mas na água da cozinha a concentração foi de 8 a 66 vezes menor. Isso indica que a torneira do quintal pode ser uma grande fonte de contaminação de ferro. Apesar do coeficiente de correlação linear entre a concentração de ferro e chumbo na água ser estatisticamente significativo (r = 0,410; n = 184), essa correlação foi relativamente baixa para sugerir o uso do ferro como marcador da contaminação por chumbo. / The city of Ribeirão Preto is supplied with water from the Guarani Aquifer, that is only fluoridated and chlorinated before distribution. The objective of this study was to evaluate the quality of drinking water that the population is consuming with respect to the concentrations of fluoride, lead and iron. Furthermore, it was investigated if the concentration of iron could be used as a marker to infer lead contamination in drinking water. We investigated 145 residences with more than 40 years of age in 4 districts of Ribeirão Preto, with at least one resident child. One new district (less than 10 years) was selected as a control group. Water samples were collected from the kitchen and the yard taps. The 1st aliquot was collected after at least 6 h of stagnation and the 2nd after leaving the water running for 5 minutes. The concentration of fluoride ions was determined by combined ion-selective electrode and ion chromatography; lead by ET AAS and ICP-MS; and iron by spectrophotometry. The fluoride concentrations in the samples varied from 0.052 to 1.01 mg L-1 (n = 281). Considering only 1 significant digit and the kitchen samples, it was observed that 30% of the homes did not attend the legislation for fluoride in drinking water (Resolution SS-65, 2005), that is, from 0.6 to 0.8 mg L-1. Considering 2 significant digits, this percentage increased to 45%. A low intake of fluoride by children cannot effectively prevent the formation of dental caries, while high concentrations can cause dental fluorosis. Lead concentrations ranged from 0.025 to 58.4 µg L-1 (n = 562), and the maximum permited by the law 2,914 (Ministry of Health) is 10.0 µg L-1. For all homes investigated, lead concentrations from the kitchens tap water did not exceed 5.00 µg L-1, showing there is a low risk of lead contamination through this pathway. For 2 aliquots of water collected from the yards tap, the concentration of lead exceeded the tolerable limit, however, the use of this water for ingestion is unusual. Possibly this contamination might come from the tap itself and from others metal parts, because after running the water for 5 minutes the lead concentration fell below the permitted value. Iron concentrations varied from 13.40 to 4,119 µg L-1 (n = 189, 1st aliquot), and the tolerable limit is 300 µg L-1 (law 2.914). In 4 houses the water from the year tap had concentrations above the limit, while in the kitchen water iron concentrations were from 8 to 66 times lower. This indicates that the tap material from the yard can be a large source of iron contamination. Although the linear correlation coefficient between iron and lead concentrations in the drinking water was statistically significant (r = 0.410; n = 184), this correlation was relatively low to suggest the use of iron as a marker of contamination by lead.

O gênero Crotalus pertence a família Viperidae e é popularmente conhecido como cascavél. Seu veneno é reconhecido como fonte biológica de moléculas capaz de produzir danos irreversíveis na saúde dos seres humanos. O presente trabalho foi desenvolvido com a finalidade de estudar os mitos e as crenças, o perfil epidemiológico e toxicológico do veneno das serpentes do gênero Crotalus, para o estado do Tocantins. Para isso, foi realizado levantamento bibliográfico para avaliar os mitos e as crenças que cercam esses animais. Os agravos por serpentes do gênero Crotalus durissus foram obtidos da plataforma TabWin- SINAN, dos registros disponibilizados para os últimos cinco anos – 2012 a 2016, com informações sobre sexo, idade, soroterapia, evolução do caso e município de ocorrência. Também foi analisada a precipitação pluviométrica e temperatura, para avaliar variáveis facilitadoras do encontro com esses animais. O veneno obtido de cascavéis Crotalus durissus collilineatus foi obtido de espécimes coletadas no Tocantins, para análise do seu perfil tóxico. Foram realizados ensaios proteicos, determinada atividade fosfolipásica, dose mínima coagulante, verificada a toxicidade (DL50) e o potencial anti-ofídico do extrato da planta Chiococca alba. Todos os ensaios foram conduzidos em paralelo com o veneno referência, cedido pelo Instituto Butantan – SP, da mesma espécie de serpente em estudo. Diante dos resultados foi possível avaliar que as crenças e os mitos sobre serpentes precisam ser desmistificados, para que, o medo possa ser minimizado e substituído pelo respeito a estes animais. Quanto ao aumento dos acidentes crotálicos no estado, deve-se considerar o alto grau de letalidade para a região norte do país, sendo a sazonalidade um importante fator que influencia nesses índices. Para o veneno de Crotalus durissus collilineatus os experimentos demonstraram ausência de crotamina para nos animais coletados no Tocantins, e a indicação de atividade antiofídica para o estrato da planta Chiococca alba. / The genus Crotalus belongs to the family Viperidae and is popularly known as rattlesnake. Its venom is recognized as a biological source of molecules capable of producing irreversible damage to human health. The present work was developed with the purpose of studying the myths and beliefs, the epidemiological and toxicological profile of venom of snakes of the genus Crotalus, to the state of Tocantins. For this, a bibliographic survey was conducted to evaluate the myths and beliefs surrounding these animals. Crotalus durissus snake complaints were obtained from the TabWin-SINAN platform, from the records available for the last five years - 2012 to 2016, with information on sex, age, serum therapy, case evolution and municipality of occurrence. Rainfall and temperature were also analyzed to evaluate variables that facilitated the encounter with these animals. The venom obtained from rattlesnakes Crotalus durissus collilineatus was obtained from specimens collected in Tocantins, to analyze its toxic profile. Protein assays, phospholipase activity, minimal coagulant dose, toxicity (LD50) and anti-odor potential of the extract of the Chiococca alba plant were performed. All the tests were conducted in parallel with the reference venom, provided by the Butantan Institute - SP, of the same snake species under study. In view of the results it was possible to evaluate that the beliefs and myths about snakes need to be demystified, so that fear can be minimized and replaced by respect for these animals. As for the increase in crotalic accidents in the state, the high degree of lethality for the northern region of the country should be considered, with seasonality being an important factor that influences these indices, the highest incidence occurring on colder days and with higher rainfall. For the venom of Crotalus durissus collilineatus, the experiments demonstrated the absence of crotamine in the animals collected in Tocantins and the indication of antiofidic activity for the stratum of the Chiococca alba plant.

Os parabenos utilizados como conservantes nas indústrias de cosméticos, alimentos e fármacos não são removidos por completo nas estações de tratamento de água e esgoto, além disso, podem causar danos a biota aquática. O presente estudo teve como finalidade aplicar um método analítico novo para quantificar o metil (MP), etil (EP), propil (PP), butil (BP), benzilparabeno (BzP) e a mistura (metil e propilparabeno) em amostras de água dos aquários com tilápias do Nilo (Oreochromis niloticus). A técnica analítica usada foi a cromatografia líquida com detector de arranjo de diodo (HPLC-DAD). Avaliou-se a toxicidade dos parabenos em tilápias e os efeitos nos biomarcadores bioquímicos dos animais após 6 e 12 dias dos testes de exposição e por administração via injeção intraperitoneal. A concentração dos parabenos utilizada em todos os testes foi de 4,0 mg L-1 (de cada parabeno individualmente) e de 6,0 mg L-1 do metil e de 1,7 mg L-1 do propilparabeno para a mistura. Foram feitas análises nos biomarcadores superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx), glutationa redutase (GR), glutationa reduzida(GSH-t) e peroxidação lipídica (MDA). O limite de detecção dos parabenos foi de 0,03 mg L-1 (MP e EP), 0,05 mg L-1 (PP) e 0,10 mg L-1 (BP e BzP), e o limite de quantificação foi de 0,13 mg L-1 (MP, EP, PP), 0,18 mg L-1 (BP) e 0,25 mg L-1 (BzP). Foi possível quantificar somente o PP e a mistura (MP + PP) nas amostras de água dos aquários que continham peixe, no máximo 30 h após a exposição. Nas amostras de água sem a presença dos peixes, foi possível quantificar o BP e a mistura, metil e propilparabeno, durante os 12 dias de exposição. Os testes de toxicidade mostraram que a concentração letal para 50% dos indivíduos após 48 h de exposição foi de 67,11 mg L-1 do MP, 24,08 mg L-1 do EP, 17,34 mg L-1 do PP, 7,98 mg L-1 do BzP e 7,80 mg L-1 do BP, sendo que estes dois últimos compostos podem ser considerados os mais tóxicos da classe. Outro modo de ação tóxica também observada dos parabenos foi a narcose, ou seja, a perda temporária da consciência e da mobilidade. À medida que aumenta o comprimento da cadeia, aumenta a lipofilicidade destas substâncias, que está relacionada com o coeficiente de partição octanol/água (Kow) das mesmas e consequentemente aumenta a toxicidade. Estes dados indicaram que quanto mais lipofílico mais tóxico é o composto. Relacionando as atividades enzimáticas testadas com os níveis de peroxidação lipídica, o metilparabeno foi o único composto capaz de provocar danos aos tecidos testados por meio das espécies reativas de oxigênio. Isso foi comprovado através da inibição da atividade das enzimas analisadas com o aumento nos níveis de MDA. Por outro lado, mesmo com as enzimas antioxidantes apresentando atividades elevadas isso não foi suficiente para impedir a redução nos níveis de GSH-t. Tais resultados indicam que os parabenos podem agir negativamente nas tilápias. / Parabens, used as preservatives in cosmetics, foodstuffs and pharmaceuticals are not completely removed from water in sewage treatments, which may cause damage to aquatic biota. The present study addresses a new methodology to measure the quantity of methyl (MP), ethyl (EP), propyl (PP), butyl (BP), benzyl (BzP) parabens and a mixture of methyl and propylparaben in water. Aquarium water of experiments with tilapia samples was analyzed for 12 days by liquid chromatography with a doide array detector (HPLC-DAD). The toxicity of parabens in Nile tilapia (Oreochromis niloticus) and its effects on biochemical biomarkers were also evaluated after 6 and 12 days of exposure and intraperitoneal injection. The concentrations of parabens used in all tests were 4.0 mg L-1 (alone) and to mixture was 6.0 mg L-1 of methyl and 1.7 mg L-1 of propylparaben. Biomarkers, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione reduced (GSH-t) and lipid peroxidation (MDA) were analyzed. The results show the detection limits for the analysis of parabens were 0.03 mg L-1 (MP and EP), 0.05 mg L-1 (PP) and 0.10 mg L-1 (BP and BzP), and the quantification limits were 0.13 mg L-1 (MP, EP, PP), 0.18 mg L-1 (BP) and 0.25 mg L-1 (BzP). In the water sample with fish, the compounds PP and the mixture (MP + PP) could be quantified up to 30h after exposure. In the water sample without fish, the compounds BP and the mixture were quantified for 12 days of exposure. Toxicity test revealed the lethal concentrations for 50% of individuals after 48 h of exposure were 67.11 mg L-1 for MP, 24.08 mg L-1 for EP, 17.34 mg L-1 for PP, 7.98 mg L-1 for BzP and 7.80 mg L-1 for BP. Therefore, BzP and BP can be considered the most toxic of the class. As the chain length grows, the lipophilicity of the substances increases. Such an increase is related to their octanol/water (Kow) partition coefficient and, consequently, increases toxicity. Another toxic action observed for the parabens was the temporary loss of consciousness and mobility of the organisms. According to the enzymatic activity tested and the lipid peroxidation levels, the methylparaben was the only compound that caused damage by reative oxidative species, supported by the inhibition of the activities of the enzymes and the increase in the MDA levels. However, the high activity of the antioxidant enzymes to exposure and intraperitoneal injections could not prevent the reduction in the levels of GSH-t. Such results indicate parabens can cause negative effects on tilapia.

by Josephine Wing Sze Lau. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 84-112). / Abstracts in English and Chinese. / Acknowledgment --- p.iii / Abstract --- p.iv / List of Abbreviations --- p.viii / Chapter CHAPTER ONE: --- INTRODUCTION / Chapter 1.1 --- Methamphetamine (METH) --- p.1 / Chapter 1.1.1 --- Historical Background and Epidemiology --- p.1 / Chapter 1.1.2 --- Physical Effects of METH --- p.4 / Chapter 1.1.3 --- Neurochemical Alternation of METH --- p.6 / Chapter 1.2 --- Mechanisms of METH Toxicity / Chapter 1.2.1 --- Oxidative Stress --- p.8 / Chapter 1.2.1.1 --- Superoxide (O2-) and Superoxide Dismutase (SOD) --- p.10 / Chapter 1.2.1.2 --- "Hydrogen Peroxide (H202), Catalase and Glutathione (GSH)" --- p.11 / Chapter 1.2.1.3 --- Hydroxyl Radical (OH.) --- p.12 / Chapter 1.2.1.4 --- Nitric Oxide (NO) --- p.13 / Chapter 1.2.2 --- Apoptosis --- p.16 / Chapter 1.2.3 --- Excitotoxicity --- p.17 / Chapter 1.2.4 --- Mitochondrial Dysfunction --- p.18 / Chapter 1.2.5 --- Hyperthermia --- p.21 / Chapter 1.2.5.1 --- Cyclooxygenase-2 (COX-2) --- p.23 / Chapter 1.2.5.2 --- Heme-oxygenase-1 (HO-1) --- p.25 / Chapter 1.2.5.3 --- The Effects of Nitric Oxide (NO) on COX-2 and HO-1 Expressions --- p.27 / Chapter 1.3 --- Astrocytes / Chapter 1.3.1 --- Characteristics of Astrocytes --- p.29 / Chapter 1.3.2 --- Astrocyte Functions --- p.30 / Chapter 1.3.3 --- The Role of Astrocytes in METH-induced Neurotoxicity --- p.34 / Chapter 1.4 --- Aim of Project --- p.37 / Chapter CHAPTER TWO: --- MATERIALS AND METHODS / Chapter 2.1 --- Cell Cultures / Chapter 2.1.1 --- Astrocyte Cultures --- p.42 / Chapter 2.1.2 --- CATH.a Cell line and Astrocytes Co-cultures --- p.43 / Chapter 2.2 --- Treatment / Chapter 2.2.1 --- METH Treatment --- p.44 / Chapter 2.2.2 --- Inhibition of Cyclooxygenase-2 (COX-2) and Inducible Nitric Oxide Synthase (iNOS) --- p.44 / Chapter 2.3 --- Lactate Dehydrogenase (LDH) Assay --- p.45 / Chapter 2.4 --- Assay for Reactive Oxygen Species (ROS) Formation --- p.47 / Chapter 2.5 --- Assay for Adenosine Triphosphate (ATP) Content --- p.48 / Chapter 2.6 --- Determination of Mitochondrial Membrane Potential (Δ Ψm) --- p.50 / Chapter 2.7 --- Determination of Nitrite Levels in Cultured Astrocytes --- p.51 / Chapter 2.8 --- Western Blot Analysis --- p.52 / Chapter 2.8.1 --- COX-2 --- p.53 / Chapter 2.8.2 --- HO-1 --- p.53 / Chapter 2.9 --- Viability Assay of CATH.a-Astrocyte Cocultures --- p.54 / Chapter 2.10 --- Statistics --- p.55 / Chapter CHAPTER THREE: --- RESULTS / Chapter 3.1 --- The Effects of METH Treatment on Cultured Astrocytes / Chapter 3.1.1 --- Lactate Dehydrogenase (LDH) Activities --- p.56 / Chapter 3.1.2 --- Morphological Changes --- p.56 / Chapter 3.1.3 --- The Production of Reactive Oxygen Species / Chapter 3.1.3.1 --- Rate of change (0-120 min) --- p.57 / Chapter 3.1.3.2 --- Time course (0 - 48 h) --- p.57 / Chapter 3.1.4 --- Change in ATP Content --- p.58 / Chapter 3.1.5 --- Change in Mitochondrial Membrane Potential (Δ Ψm) --- p.59 / Chapter 3.1.6 --- Nitrite levels after METH treatment / Chapter a) --- Striatal astrocytes --- p.59 / Chapter b) --- Mesencephalic astrocytes --- p.60 / Chapter c) --- Cortical astrocytes --- p.60 / Chapter 3.1.7 --- The Effects of Aminoguanidine (AG) on Nitrite Levels / Chapter a) --- Striatal astrocytes --- p.61 / Chapter b) --- Mesencephalic astrocytes --- p.62 / Chapter c) --- Cortical astrocytes --- p.62 / Chapter 3.1.8 --- The Effects of Indomethacin (INDO) on Nitrite Levels / Chapter a) --- Striatal astrocytes --- p.63 / Chapter b) --- Mesencephalic astrocytes --- p.64 / Chapter c) --- Cortical astrocytes --- p.64 / Chapter 3.1.9 --- Change in Cyclooxygenase-2 (COX-2) Protein Levels / Chapter a) --- Striatal astrocytes --- p.65 / Chapter b) --- Mesencephalic astrocytes --- p.65 / Chapter c) --- Cortical astrocytes --- p.66 / Chapter 3.1.10 --- Change in Heme-oxygenase-1 (HO-1) Protein Levels / Chapter a) --- Striatal astrocytes --- p.66 / Chapter b) --- Mesencephalic astrocytes --- p.66 / Chapter c) --- Cortical astrocytes --- p.67 / Chapter 3.2 --- Cell Viability on CATH.a-Astrocyte Cocultures After METH Treatment --- p.67 / Chapter CHAPTER FOUR: --- DISCUSSION AND CONCLUSION --- p.69 / REFERENCES --- p.84

by Chan Hoi Tak. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 129-137). / Abstracts in English and Chinese. / Acknowledgment --- p.i / Abstract --- p.ii / Abstract (Chinese Version) --- p.iv / Abbreviations --- p.vi / Contents --- p.viii / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Aflatoxins --- p.1 / Chapter 1.1.1 --- AFB1 Metabolism --- p.5 / Chapter 1.1.2 --- Bioactivation --- p.5 / Chapter 1.1.3 --- Detoxification --- p.8 / Chapter 1.1.4 --- Toxicity of AFB1 --- p.9 / Chapter 1.2 --- Licorice Plants --- p.11 / Chapter 1.2.1 --- Glycyrrhizic Acid (GA) --- p.13 / Chapter 1.2.2 --- Metabolism of GA --- p.16 / Chapter 1.2.3 --- Adverse Effects of GA --- p.19 / Chapter 1.3 --- Aim of Research --- p.19 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.21 / Chapter 2.1 --- Cell cultures --- p.21 / Chapter 2.1.1 --- Isolation of splenocytes --- p.21 / Chapter 2.1.2 --- Culture of cell lines --- p.22 / Chapter 2.1.3 --- Trypsinziation of cells --- p.22 / Chapter 2.2 --- Preparation of drugs --- p.23 / Chapter 2.2.1 --- Preparation of commercially available chemicals for cell culture --- p.23 / Chapter 2.2.2 --- Preparation of Licorice extracts for cell culture --- p.23 / Chapter 2.2.3 --- Preparation of chemicals for enzymatic reaction --- p.24 / Chapter 2.3 --- Cytotoxicity assay Methods --- p.24 / Chapter 2.3.1 --- MTT assay --- p.25 / Chapter 2.3.2 --- Neutral red assay --- p.25 / Chapter 2.4 --- Cytotoxicity Assay --- p.32 / Chapter 2.5 --- Chemoprotection assay --- p.32 / Chapter 2.5.1 --- Direct addition of drugs --- p.33 / Chapter 2.5.2 --- Pretreatment of cells --- p.33 / Chapter 2.5.3 --- Culture of cells with drugs --- p.34 / Chapter 2.6 --- Preparation of enzymes --- p.36 / Chapter 2.6.1 --- Preparation of cells --- p.36 / Chapter 2.6.2 --- Pretreatment of rat microsomes --- p.36 / Chapter 2.7 --- Bradford Assay --- p.37 / Chapter 2.8 --- Alkoxyresorufin O-Dealkylase assay --- p.39 / Chapter 2.8.1 --- Effects of AFB1 on P450 --- p.44 / Chapter 2.8.2 --- Effects of drugs on P450 --- p.44 / Chapter 2.9 --- Glutathione-S-transferase assay --- p.44 / Chapter 2.9.1 --- GST assay in rat microsomal fractions --- p.48 / Chapter 2.9.1.1 --- Effects of AFB1 on GST --- p.48 / Chapter 2.9.1.2 --- Effects of drugs on GST --- p.48 / Chapter 2.9.2 --- GST assay in crude fractions from cells --- p.48 / Chapter 2.9.2.1 --- Direct addition effects of drugs on GST --- p.48 / Chapter 2.9.2.2 --- Effects of drug-containing medium on GST --- p.49 / RESULTS / Chapter CHAPTER THREE --- CELL CULTURES --- p.50 / Chapter 3.1 --- Cytotoxicity assay --- p.51 / Chapter 3.1.1 --- Cytotoxicity of AFB1 --- p.51 / Chapter 3.1.1.1 --- Cytotoxicity of AFB1 to splenocytes --- p.51 / Chapter 3.1.1.2 --- Cytotoxicity of AFB1 to cell lines --- p.57 / Chapter 3.1.2 --- Cytotoxicity of drugs to cell lines --- p.62 / Chapter 3.2 --- Chemoprotection assay --- p.73 / Chapter 3.2.1 --- Direct addition of drugs --- p.73 / Chapter 3.2.2 --- Pretreatment of cells with drugs --- p.78 / Chapter 3.2.3 --- Culture of cells with drugs --- p.83 / Chapter CHAPTER FOUR --- ENZYMATIC ASSAYS --- p.87 / Chapter 4.1 --- Alkoxyresorufin O-dealkylase assay --- p.88 / Chapter 4.1.1 --- Effects of AFB1 on P450s --- p.88 / Chapter 4.1.2 --- Effects of GA on P450s --- p.92 / Chapter 4.1.3 --- Effects of EX on P450s --- p.95 / Chapter 4.2 --- Glutathione -S- transferase assay --- p.98 / Chapter 4.2.1 --- Effects of drugs on GST in cells --- p.100 / Chapter 4.2.2 --- Effects of AFB1 and drugs on GST from rats --- p.108 / Chapter 4.2.2.1 --- Effects of AFB1 on GST from rats --- p.108 / Chapter 4.2.2.2 --- Effects of drugs on GST from rats --- p.112 / Chapter CHAPTER FIVE --- DISCUSSION --- p.115 / Chapter 5.1 --- Cytotoxicity of AFB1 --- p.118 / Chapter 5.2 --- "Cytotoxicity of EX, GA and AA" --- p.119 / Chapter 5.3 --- Chemoprotection of GA and EX on AFB1 cytotoxicity --- p.121 / Chapter 5.4 --- Effects of GA and EX on enzymes involved in AFB1 metabolism --- p.123 / REFERENCES --- p.129

Embryonic stem cells are derived from the inner cell mass of a blastocyst-stage embryo and are characterized by the ability to self-renew and differentiate into all cell types of an adult organism, as demonstrated by their transplantation into embryos in the mouse. Isolation of cells with similar properties from human embryos has permitted the study of human cell differentiation in vitro as might occur during development. As such, human ES cells may be useful to assess and predict the developmental toxicity of environmental compounds capable of epigenetic alterations of the genome and its expression. The first objective of my research was to validate the functional significance to maintenance of an undifferentiated human ES cell state of expressed genes whose epigenetic modification is conserved across diverse lines and/or likely to be deterministic of an embryo stem cell associated epigenetic state. The second goal was to determine the sensitivity and relationship of the expression of these genes to environmental factors known to perturb the epigenome, specifically subcytotoxic exposure to diverse organic and metallic compounds and the availability of atmospheric oxygen. siRNA-mediated knockdown of genes previously identified on the basis of the conserved methylation status of gene associated Cytosine-Guanine islands (i.e. GLIS2, HMGA1, PFDN5, TET1 and JMJD2C) and two related family members (TET2 & 3) resulted in induction of cell differentiation in two independent human ES cell lines (RH1 and H9). Differentiation was reflected by morphological changes, reduction or loss of pluripotency associated markers, qualitative and quantitative reduction in genomic 5-hmC and upregulation of diverse germinal lineage markers. Subcytotoxic exposure of the same human ES cell lines to diverse compounds known to alter the epigenome (i.e. 5-azacytidine, sodium arsenite, cadmium chloride and valproic acid) generally induced downregulation of the aforementioned genes, loss of genomic hydroxymethylation and differentiation when applied under normoxia (20% O2), the exception being valproic acid. The same treatment applied under hypoxia (0.5% O2), did not induce differentiation, with the exception of cadmium chloride. Hypoxia is a general feature of developing embryos prior to the establishment of a maternal/fetal placental interface and fetal cardiovasculature. The protective effect of hypoxia was associated with elevation of ROS, expression of the dioxygenases TET1 and JMJD2C, and genomic hydroxymethylation. This research has demonstrated that genes identified on the basis of a conserved pattern of epigenetic modification function in the maintenance of an undifferentiated human ES cell phenotype. Furthermore, a human ES cell-based toxicology test system has been developed with which one can assess the subcytotoxic effects of compounds known to disrupt the epigenome and affect development by assessing their impact on maintenance of an undifferentiated human ES cell state. This is reflected by alterations in pluripotency markers, epigenetically-defined biomarkers and changes in global 5-hmC levels and the expression of genes responsible for this epigenetic modification (TET1-3). The epigenetically-defined biomarkers of pluripotent human ES cell identity (GLIS2, HMGA1, PFDN5, JMJD2C and TET1) could serve as biomarkers for screenings of compounds at an epigenetic level as their expression has been shown to be altered upon compound exposure along with monitoring the expression of 5-hmC.

O manganês é um elemento traço essencial, porém existe uma preocupação com seus potenciais efeitos neurotóxicos associados à exposição a níveis excessivos, podendo provocar uma síndrome conhecida como manganismo, cujos sintomas são semelhantes aos da doença de Parkinson. A maioria dos trabalhos envolvendo manganês usa espécies de Mn(II), mas sabe-se que Mn(III) é acumulado em maior quantidade no cérebro. Nesse sentido, foi feito um estudo dos danos oxidativos e de toxicidade provocados por três complexos de Mn(III): citrato, pirofosfato e salicilenodiamina (respectivamente MnCit, MnPPi e EUK8). Para tanto, as três espécies foram sintetizadas e caracterizadas por métodos espectroscópicos. Em seguida foram determinadas suas capacidades pró-oxidantes sobre os seguintes marcadores: dihidrorodamina (DHR), tirosina (Tyr), albumina (BSA) e dopamina (DA). Finalmente, seu efeito sobre células cerebelares e da cepa HeLa estressada por meio de irradiação UV também foi avaliado, e foi usado ascorbato na tentativa de tratar o dano sobre células HeLa. O teste com a DHR também foi feito em presença de H2O2 e ascorbato. A capacidade pró-oxidante testada por fluorescência da DHR sugere que o ascorbato atua como anti-oxidante. Além disso, MnCit e MnPPi (mas não EUK8), quando na presença de H2O2, são menos oxidantes. O mesmo comportamento foi percebido nas medidas de fluorescência de Tyr. A carbonilação da BSA, verificada pela absorbância do seu marcador (DNPH), seria indício de capacidade oxidante dos complexos, mas não percebeu-se variação significativa de grupos C=O na proteína após tratamento com espécies de Mn(III), mesmo em amostras com H2O2, embora notem-se as mesmas tendências apresentadas pelos complexos com DHR e Tyr. Estudos de oxidação de DA por luminescência tiveram resultados inconclusivos, mas dados mais concretos em testes com medidas de absorbância de soluções de DA e fluorescência de misturas de DA com DHR indicaram que DA é preferencialmente oxidada por todos os compostos. A viabilidade celular de culturas de células neuronais granulares (CGC) mostrou pouca diferença entre as toxicidades dos compostos, mas verifica-se uma relação inversamente proporcional entre as toxicidades e lipofilicidades dos complexos. O mesmo não ocorre nos experimentos com HeLa, cuja viabilidade foi avaliada por contagem de colônias após fixação e coloração das células, pois nesse caso o EUK8 se mostrou o mais tóxico dos três. Além disso, ao contrário do observado com a DHR, o ascorbato teve ação pró-oxidante, e, aparentemente, houve um efeito sinérgico negativo entre os complexos e a radiação UV. Tratamento com o quelante p-aminossalicilato só foi eficaz na recuperação das culturas para amostras não irradiadas. / Manganese is an essential trace element, however there is considerable concern regarding its neurological effects when in excess, giving rise to a condition termed manganism which is characterized by Parkinson disease-like symptoms. Most evaluations of manganese toxicity use poorly defined Mn(II) species, although Mn(III) is known to accumulate preferentially in the brain. Therefore, in this work we proposed a study of oxidative damage and citotoxicity of Mn(III) derivatives of citrate, pyrophosphate and salycilenediamine (respectively, MnCit, MnPPi and EUK8). The species were synthesized and characterized by spectroscopic methods. Their pro-oxidant abilities were assessed over markers of oxidant activity dihydrorhodamine (DHR), tyrosine (Tyr), albumin (BSA) and dopamine (DA). In addition, their effect over granular cerebral cells (CGC) and HeLa cells stressed by ultraviolet irradiation was studied, and treated with ascorbate. Tests with DHR were repeated treating the samples with H2O2 and ascorbate. Pro-oxidant ability tested by both DHR and Tyr fluorescence suggest that ascorbate is antioxidant towards Mn(III)-induced oxidative damage. MnCit and MnPPi (but not EUK8), when in presence of peroxide, are less oxidants. An analogous trend was observed for BSA, although without statistical significance. Evaluation of DA formation by luminescence was inconclusive, but competition studies of DA+DHR mixtures indicated that DA is preferentially oxidized by all the complexes. To CGC, little difference was observed for the toxicities of the complexes. An inverse relationship of toxicity and lipophilicity has been observed. However this was not observed for HeLa cells, to which EUK8 was more toxic. In addition, and in opposition to the DHR solution study, ascorbate was found to be pro-oxidant. A negative synergic effect was observed between complex doses and irradiation. Treatment of the cells with paraaminosalicylate was beneficial only for non-irradiated cells.

Ruan, Jianqing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 162-175). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Les écosystèmes et les personnes sont exposés chaque jour à de multiples facteurs de stress chimiques par l'intermédiaire de multiples voies et voies en raison de la croissance économique et démographique. Considérant que l'utilisation de pesticides est particulièrement susceptible de nuire à la santé sur le lieu de travail, nous avons décidé de concevoir une méthode dédiée. Ce projet de thèse vise à développer une méthode pour examiner les effets comparatifs de différents systèmes de production agricole sur la santé des opérateurs à cause des pesticides. L'objectif est de permettre l'évaluation d'un ensemble complet d'informations à des fins d'aide à la décision, en tenant compte des bonnes et mauvaises pratiques de travail réelles. En outre, un outil d'aide à la décision pour les différentes parties prenantes (les gestionnaires de plantations dans un premier temps) pour faire la différence entre plusieurs systèmes de production concernant leur impact potentiel sur la santé des travailleurs agricoles, est en cours de développement. Cette méthode doit être basée sur des données facilement disponibles dans un délai raisonnable, et correspondant à des données significatives pour les gestionnaires, afin de leur fournir des occasions d'action dans l'innovation des cultures. Nous avons suivi quatre étapes : demander l'aide d’experts ; construire des arbres de connaissances ; développer des arbres de décision ; calculer l'impact potentiel sur la santé des travailleurs agricoles dû aux pesticides pour les opérateurs à différentes échelles, et concernant un système agricole donné dans un test de faisabilité.Les premières phases ont été mises en œuvre grâce à la Delphi experts consensus method qui a permis d'obtenir des connaissances d'agronomes, d'économistes et de spécialistes de l'évaluation des expositions afin de cartographier les différents flux de production et les origines des bonnes et mauvaises pratiques. Après avoir obtenu et interprété les résultats du calcul de l'indicateur dans le test de faisabilité, nous devrions proposer des améliorations de la méthode.Nous avons décidé de réaliser un test de faisabilité à travers des entretiens semi-structurés. Nous flanquons ces entretiens avec l'observation directe des opérateurs et des travailleurs accomplissant les différentes étapes de la production. Le but de cette comparaison est de vérifier à la fois ce que les experts ont référé dans leur narration (informations présentes dans les arbres) et ce que les personnes interrogées ont déclaré. Les producteurs conventionnels (non biologiques) ont été sélectionnés pour des entrevues. Ce travail de recherche contribue à l'étude de la manière dont le capital humain (considéré comme une entrée dans l'approche MCM) pourrait être consommée pour atteindre des résultats déterminés en termes de marché d'exportation. Le présent travail vise à encourager le travail décent en identifiant des choix possibles moins nocifs pour la santé et en garantissant un environnement et une sécurité sains. Le résultat de l'ensemble de ce travail de thèse est formalisé dans une voie méso-échelle, appelée « Wesseling pathway ». / Ecosystems and people are exposed every day to multiple chemical stressors via multiple pathways and routes due to economic and population growth. Considering that the use of pesticides is especially prone to potentially damage health in the workplace, we decided to design a dedicated method.This thesis project aims to develop a method to examine the comparative effects of different agricultural production systems on operators’ health because of pesticide. The objective is to enable the assessment of a comprehensive set of information for decision support purposes, considering the actual (good and bad) work practices. Furthermore, a decision support tool for different stakeholders (plantation managers at first) to discriminate between several production systems regarding their potential impact on farmworkers health, is under development. This method has to be based on readily available data in a reasonable time, and corresponding to meaningful data for managers, to provide them opportunities of action in crop innovation. We followed four steps: seeking help of experts; constructing knowledge trees; developing decision trees; calculating the potential impact on farmworkers health due to pesticides for operators at different scales, and regarding one given farming system in a feasibility test.The first phases were implemented through a Delphi expert consensus method eliciting knowledge from agronomists, economists and exposure assessment specialists, in order to map the different “banana workflows” and the origins of good and bad practices.After getting and interpreting results of the indicator calculation in the feasibility test, we ought to propose improvements of the method.We decided to carry out a feasibility test through semi-structured interviews. We flank these interviews with direct observation of operators and workers accomplishing the different steps of the production. The aim of this comparison is to cross- check both what experts referred in their narration (information present in trees), and what the interviewed persons declared. Conventional (non-organic) producers were selected for interviews.This research work contributes to the study of the way in which human capital (considered like an input in the MCM approach) could be consummate to reach determined results in terms of export market.The present work aims encouraging decent work through identification of possible choices less harmful to health and ensuring healthy environment and safety. The result of this entire thesis work is formalized in a meso-scale pathway, named “Wesseling pathway”.

O tecido ósseo é o maior sítio de acúmulo de flúor (F) do nosso organismo. Parte do F ingerido pode transitar através de um mecanismo de estado estacionário entre cristais da superfície óssea e plasma, ou quando da remodelação óssea, mantendo constantes os níveis plasmáticos algum tempo após a diminuição da ingestão de F. O objetivo deste estudo foi avaliar o período de tempo para que o F acumulado no osso de ratos, após uma exposição aguda, seja removido. Três grupos experimentais com 10 ratos Wistar (70 dias de idade) receberam, por via gástrica, dose única de 50 mg F/Kg peso corporal (como NaF) e 3 grupos controle, também com 10 animais cada, receberam água deionizada. A eutanásia dos animais ocorreu após 30, 90 ou 180 dias da administração de F. Os ratos foram anestesiados, sangue e fêmures removidos e analisados quanto à concentração de F. Para o fêmur, foi analisado o F solúvel em ácido removido por biópsia da superfície óssea, além do F presente no osso total, após calcinação. As cinzas e o plasma foram analisados para o F com o eletrodo, após difusão facilitada por HMDS. Os dados foram analisados pelo teste de Kruskal-Wallis, seguido pelo teste de Dunn (p<0,05). Os níveis plasmáticos de F do grupo controle foram constantes ao longo do tempo, e similares aos do grupo experimental eutanasiado aos 30 dias. Para os grupos experimentais eutanasiados após 90 e 180 dias, houve uma redução significativa nos níveis plasmáticos de F em relação ao grupo controle. A concentração de F na superfície do fêmur para o grupo experimental foi significativamente maior em relação ao grupo controle no tempo de eutanásia de 30 dias apenas, sendo que para os demais períodos experimentais, apesar de a concentração de F ser maior no grupo experimental quando comparado ao controle, esta diferença não foi estatisticamente significativa. Para ambos os grupos pôde ser observado um aumento nos níveis de F na superfície óssea longo do tempo. Para o osso total, o grupo experimental apresentou concentrações similares de F nos diferentes tempos de morte, e estes níveis foram similares aos encontrados para o grupo controle, no tempo de eutanásia de 180 dias. O grupo controle, nos tempos de morte 30 e 90 dias, apresentou valores significativamente menores quando comparado às demais situações. Os dados sugerem que o F incorporado à superfície óssea de ratos a partir de uma exposição aguda não é irreversivelmente ligado, mas vai sendo perdido ao longo do tempo, parecendo ser completamente perdido entre 90 e 180 dias após a exposição. Já os níveis de F incorporados no osso total 30 dias após uma ingestão aguda subletal não parecem ser perdidos ao longo do tempo. A não ocorrência de aumento ao longo do tempo nos níveis de F no osso total do grupo experimental sugere que existe um limite para a incorporação de F no osso total, provavelmente relacionado ao número de sítios possíveis para ligação irreversível do F. / Bone is the major site for fluoride (F) accumulation in the body. Part of the ingested F can transit between bone surface crystals and plasma, through a steady-state mechanism, which can also occur during bone remodeling. This could maintain constant plasma F levels for some time after the F intake is reduced. The aim of this study was to evaluate the lag time required to remove F accumulated in bone of rats after acute exposure. Three experimental groups, containing ten 70-day-old Wistar rats each, received, by gastrogavage, a single dose of 50 mgF/Kg body weight (as NaF), while 3 control groups received deionized water. The animals were euthanized 30, 90 or 180 days after F administration. The animals were anesthetized, blood and femurs were collected and analyzed for F. F on the femur surface was removed through an acid biopsy and F in whole bone was analyzed after ashing. Ash and plasma F were analyzed with the electrode following HMDS-facilitated diffusion. Data were analyzed by Kruskal-Wallis and Dunn\'s tests (p<0.05). Plasma F levels in control group were constant along time and similar to the levels found for the experimental group euthanized after 30 days. For the experimental groups euthanized after 90 and 180 days, a significant reduction in plasma F levels in respect to control was found. F in bone surface for the experimental group was significantly higher than for the control group only 30 days after F administration. For the other experimental times, despite F concentration was higher in the experimental group when compared to control, this difference was not significant. For both groups an increase in bone surface F levels along time was seen. For whole bone, the experimental group had similar F concentrations for all times of euthanasia and these levels were similar to those found for control group 30 days after F administration. Control group, 30 and 90 days after F administration had values significantly lower when compared to the other situations. Data suggest that F incorporated into bone surface of rats after acute exposure does not seem to be irreversibly bound and is lost along time. It seems to be completely lost between 90 and 180 days after F administration. On the other hand, F levels incorporated in whole bone 30 days after acute exposure to F do not seem to be lost along time. The lack of increase in whole bone F levels in the experimental group along time suggests that there is a limit for F uptake in whole bone, probably related to the number sites available to bind F irreversibly.