Wong Chun-kwan. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 127-137). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgments --- p.viii / Tables of Contents --- p.ix / List of Figures --- p.xv / List of Tables --- p.xxvi / Chapter Chapter 1: --- INTRODUCTION --- p.1 / Chapter Chapter 2: --- LITERATURE REVIEW --- p.8 / Chapter 2.1 --- Toxicology --- p.8 / Chapter 2.1.1 --- Acute toxicity test --- p.8 / Chapter 2.1.2 --- Biochemical Analysis --- p.9 / Chapter 2.1.3 --- Organ weights --- p.10 / Chapter 2.2 --- Histology --- p.11 / Chapter 2.2.1 --- Light Microscope --- p.11 / Chapter 2.2.2 --- Electron Microscopy --- p.11 / Chapter 2.3 --- Tissue injury --- p.12 / Chapter 2.3.1 --- Free-radical mechanisms --- p.12 / Chapter 2.3.2 --- Lipid peroxidation --- p.13 / Chapter 2.4 --- Carbon tetrachloride (CC14) --- p.14 / Chapter 2.4.1 --- Mechanisms of carbon tetrachloride toxicity --- p.15 / Chapter 2.5 --- Trichloroethylene (TCE) --- p.18 / Chapter 2.5.1 --- Mechanisms of trichloroethylene toxicity --- p.21 / Chapter 2.6 --- Dimethyl sulfoxide (DMSO) --- p.25 / Chapter 2.7 --- N-acetylcysteine (NAC) --- p.27 / Chapter Chapter 3: --- MATERIALS AND METHODS --- p.28 / Chapter 3.1 --- Materials --- p.28 / Chapter 3.2 --- Methods --- p.31 / Chapter 3.2.1 --- Acute hepatotoxicity test on aqueous seaweed extracts --- p.31 / Chapter 3.2.1.1 --- Preparation of aqueous extracts of seaweed --- p.31 / Chapter 3.2.1.2 --- Experimental protocol --- p.31 / Chapter 3.2.1.3 --- Biochemical assays --- p.32 / Chapter 3.2.1.4 --- Organ weights --- p.36 / Chapter 3.2.1.5 --- Histopathological examination --- p.36 / Chapter 3.2.1.6 --- Statistical analysis --- p.36 / Chapter 3.2.2 --- Curative and preventive tests of seaweed aqueous extracts against the CCl4-induced hepatotoxicity --- p.37 / Chapter 3.2.2.1 --- Preparation of aqueous extracts of seaweed --- p.37 / Chapter 3.2.2.2 --- Experimental protocol --- p.37 / Chapter 3.2.2.3 --- Biochemical assays --- p.39 / Chapter 3.2.2.4 --- Organ weights --- p.39 / Chapter 3.2.2.5 --- Histopathological examination --- p.40 / Chapter 3.2.2.6 --- Statistical analysis --- p.41 / Chapter 3.2.3 --- Acute hepatotoxicity test of TCE in rats by oral and intraperitoneal routes --- p.42 / Chapter 3.2.3.1 --- Experimental protocol --- p.42 / Chapter 3.2.3.2 --- Biochemical assays --- p.43 / Chapter 3.2.3.3 --- Organ weights --- p.43 / Chapter 3.2.3.4 --- Histopathological examination --- p.44 / Chapter 3.2.3.5 --- Statistical analysis --- p.44 / Chapter 3.2.4 --- Curative and preventive tests of seaweed aqueous extracts against the TCE effective dose-induced toxicity --- p.44 / Chapter 3.2.4.1 --- Preparation of aqueous extracts of seaweed --- p.44 / Chapter 3.2.4.2 --- Experimental protocol --- p.45 / Chapter 3.2.4.3 --- Biochemical assays --- p.46 / Chapter 3.2.4.4 --- Organ weights --- p.46 / Chapter 3.2.4.5 --- Histopathological examination --- p.46 / Chapter 3.2.5 --- Antidotal effects of dimethyl sulfoxide (DMSO) and N-acetylcysteine (NAC) against CC14- and TCE- induced poisoning in rats --- p.47 / Chapter 3.2.5.1 --- Experimental protocol --- p.47 / Chapter 3.2.5.2 --- Biochemical assays --- p.48 / Chapter 3.2.5.3 --- Organ weights --- p.48 / Chapter 3.2.5.4 --- Histopathological examination --- p.49 / Chapter 3.2.6 --- Hepatoprotective effect of seaweeds' methanol extract against CC14- and TCE-induced poisoning in rats --- p.49 / Chapter 3.2.6.1 --- Preparation of methanol extracts of seaweed --- p.49 / Chapter 3.2.6.2 --- Experimental protocol --- p.50 / Chapter 3.2.6.3 --- Biochemical assays --- p.52 / Chapter 3.2.6.4 --- Organ weights --- p.52 / Chapter 3.2.6.5 --- Histopathological examination --- p.53 / Chapter Chapter 4 --- RESULTS --- p.54 / Chapter 4.1 --- Acute hepatotoxicity test on aqueous seaweed extracts --- p.54 / Chapter 4.1.1 --- The biochemical assays of the serum transaminase activity --- p.54 / Chapter 4.1.2 --- The organ weight (Aqueous seaweed crude extracts) --- p.56 / Chapter 4.2 --- Curative and preventive tests of seaweed aqueous extracts against the CCl4-induced hepatotoxicity --- p.58 / Chapter 4.2.1 --- The biochemical assays of the serum transaminase activity (Curative) --- p.58 / Chapter 4.2.2 --- The organ weight (Curative) --- p.60 / Chapter 4.2.3 --- The biochemical assays of the serum transaminase activity (Preventive) --- p.62 / Chapter 4.2.4 --- The organ weight (Preventive) --- p.64 / Chapter 4.3 --- Acute hepatotoxicity test of TCE in rats by oral and intraperitoneal routes --- p.66 / Chapter 4.3.1 --- Oral route --- p.66 / Chapter 4.3.1.1 --- One-time oral route --- p.66 / Chapter 4.3.1.2 --- Two-time oral route --- p.66 / Chapter 4.3.2 --- Intraperitoneal route --- p.66 / Chapter 4.3.3 --- Time course of the effective dose of 20% TCE in i.p. route --- p.67 / Chapter 4.4 --- Curative and preventive tests of seaweed aqueous extracts against the TCE effective dose-induced toxicity --- p.12 / Chapter 4.4.1 --- The biochemical assays of the serum transaminase activity (Curative) --- p.72 / Chapter 4.4.2 --- The organ weight (Curative) --- p.74 / Chapter 4.4.3 --- The biochemical assays of the serum transaminase activity (Preventive) --- p.76 / Chapter 4.4.4 --- The organ weight (Preventive) --- p.78 / Chapter 4.5 --- Antidotal effects of dimethyl sulfoxide (DMSO) and N-acetylcysteine (NAC) against CC14- and TCE-induced poisoning in rats --- p.80 / Chapter 4.5.1 --- The biochemical assays of the serum transaminase activity (Curative) --- p.80 / Chapter 4.5.2 --- The organ weight (Curative) --- p.82 / Chapter 4.5.3 --- The biochemical assays of the serum transaminase activity (Preventive) --- p.84 / Chapter 4.5.4 --- The organ weight (Preventive) --- p.86 / Chapter 4.6 --- Hepatoprotective effect of methanol extract of seaweed against CC14- and TCE-induced poisoning in rats --- p.88 / Chapter 4.6.1 --- The biochemical assays of the serum transaminase activity (Curative) --- p.88 / Chapter 4.6.2 --- The organ weight (Curative) --- p.89 / Chapter 4.7 --- Histopathological examinations --- p.90 / Chapter 4.7.1 --- Acute hepatotoxicity test on aqueous seaweed extracts --- p.91 / Chapter 4.7.2 --- Curative and preventive tests of seaweed aqueous extracts against the CC14-induced hepatotoxicity --- p.92 / Chapter 4.7.3 --- Acute hepatotoxicity test of TCE in rats by oral and intraperitoneal routes --- p.99 / Chapter 4.7.4 --- Curative and preventive tests of seaweed aqueous extracts against the TCE effective dose-induced toxicity --- p.100 / Chapter 4.7.5 --- Antidotal effects of dimethyl sulfoxide (DMSO) and N-acetylcysteine (NAC) against CC14- and TCE-induced poisoning in rats --- p.100 / Chapter 4.7.6 --- Hepatoprotective effect of methanol extract of seaweed against CC14- and TCE-induced poisoning in rats --- p.102 / Chapter Chapter 5 --- DISCUSSION --- p.106 / Chapter Chapter 6 --- CONCLUSION --- p.124 / REFERENCES --- p.127 / APPENDIX --- p.138

The primary goal of spectroscopy is to obtain molecularly detailed information about the system under study. Sum frequency generation (SFG) vibrational spectroscopy is a nonlinear optical technique that is highly interface specific, and is therefore a powerful tool for understanding interfacial structure and dynamics. SFG is a second order, electronically nonresonant, polarization experiment and is consequently dipole forbidden in isotropic media such as a bulk liquid. Interfaces, however, serve to break the symmetry and produce a signal. Theoretical approximations to vibrational spectra of O-H stretching at aqueous interfaces are constructed using time correlation function (TCF) and instantaneous normal mode (INM) methods. Detailed comparisons of theoretical models and spectra are made with those obtained experimentally in an effort to establish that our molecular dynamics (MD) methods can reliably depict the system of interest. The computational results presented demonstrate the potential of these methods to accurately describe fundamentally important systems on a molecular level.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-08-29T20:29:44Z No. of bitstreams: 1 Sheilla Andrade deOliveira. Estudo do potencial terapêutico de células mononucleares de medula óssea em lesões hepáticas crônicas em camundongos - UFBA-FIOCRUZ-CPqGM - Tese Doutorado.pdf: 16762445 bytes, checksum: f804f2624774de877295c8841f4ff9c1 (MD5) / Made available in DSpace on 2012-08-29T20:29:44Z (GMT). No. of bitstreams: 1 Sheilla Andrade deOliveira. Estudo do potencial terapêutico de células mononucleares de medula óssea em lesões hepáticas crônicas em camundongos - UFBA-FIOCRUZ-CPqGM - Tese Doutorado.pdf: 16762445 bytes, checksum: f804f2624774de877295c8841f4ff9c1 (MD5) Previous issue date: 2007 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / O potencial terapêutico das células-tronco em doenças hepáticas vem sendo investigado. Estudos em modelos animais têm servido de base para a realização de ensaios clínicos utilizando células de medula óssea. Embora os resultados iniciais venham sendo bastante promissores, os mecanismos envolvidos na melhora hepática ainda não estão claros e novas avaliações em modelos experimentais se fazem necessárias. O objetivo da presente investigação foi avaliar a eficácia terapêutica de células mononucleares de medula óssea em doenças hepáticas crônicas. Inicialmente estabeleceu-se cirrose hepática no camundongo C57Bl/6, pela administração de uma solução de 20% de tetracloreto de carbono (CCI4) diluído em azeite de oliva combinado com 5% de etanol (EtOR) na água. Neste estudo observamos que, após seis meses de administração dos agentes hepatotóxicos, ocorre o desenvolvimento de lesões crônicas características de cirrose hepática. O segundo modelo utilizado foi o da infecção crônica pelo Schistosoma mansoni. Uma vez estabelecidas as lesões hepáticas crônicas nos animais, os estímulos "lesivos foram retirados e células mononucleares de medula óssea (3xl07) obtidas de tíbia e fêmures de camundongos transgênicos para proteína fluorescente verde (GFP) foram infundidas. A avaliação da capacidade migratória das células infundidas, bem como das alterações ocorridas após a terapia celular foi avaliada por meio de métodos histológicos, de imunofluorescência, morfológicos e imunológicos no tecido hepático. Após a terapia celular, observamos, nos dois modelos de fibrose hepática analisados, que as células GFP+ infundidas foram capazes de migrar e permanecer nas áreas de lesão do fígado. Inicialmente estas células apresentam formas arredondadas sem adentrar o tecido fibroso. Posteriormente, estas células tendem a invadir o tecido fibroso e assumem formas fusiformes. Uma redução do tecido fibroso foi verificada no 2° mês pós-terapia, em todos os grupos que receberam a terapia celular tanto nas áreas de fibrose septal e portal, quanto nas áreas dos granulomas. Os níveis de TGF-B no tecido hepático estavam diminuídos, após a terapia celular. O número de células ovais, conhecidas como células-tronco do fígado, estava aumentado no parênquima hepático após terapia celular. Os animais esquistossomóticos tratados com células de medula óssea recuperaram parcialmente a produção de albumina, quando comparado aos animais infectados. As observações sobre a terapia celular nos dois modelos de lesão crônica dos fígados avaliados nos permitem concluir que o transplante de células mononucleares de medula óssea diminui as alterações teciduais decorrentes de agressões crônicas ao fígado e reforçam a utilização deste tratamento para pacientes portadores de lesões hepáticas crônicas. / The therapeutic potential of stem cells on chronic liver diseases has been widely investigated. Based on results obtained mainly in studies using experimental models of acute liver injury, phase I clinical trials have already been carried out in patients with chronic liver diseases using therapy with bone marrow cells. Although the initial results were promising, the mechanisms involved on improvement of hepatic function are not clear and new evaluations with experimental models akin to human chronic liver diseases are needed. The object of the present investigation was the evaluation of the therapeutic efficacy of bone marrow mononuclear cells in chronic hepatic disease models. InitialIy, a mouse model of hepatic cirrhosis was induced in C57BI/6 mice by administration of 20% carbon tetrachloride solution (CCI4) diluted in olive oil combined with 5% ethanol (EtOH) in water was established. In this study we observed the development of chronic lesions characteristic of cirrhosis afier at least 6 months of administration of the hepatotoxic agents. The second model used was hepatic fibrosis caused by chronic infection by Schistosoma mansoni. Once the chronic lesions were established, the lesion stimulus was suspended and bone marrow mononuclear cells (3x I 07) obtained from tibiae and femurs of green fluorescence protein (GFP) transgenic mice were administered. The migratory capacity of the administered celIs, as well as the alterations occurring afier the celI therapy, was evaluated by histological, immunofluorescence, morphological and immunological methods in the liver tissue. After cell therapy in both hepatic fibrosis models it was observed that administered GFP+ cells were able to migrate and remain on hepatic lesion areas. Initially those celIs presented an oval shape outside thc fibrous tissue. At latter timepoints, those cells invadcd the fibrous tissue and acquired a fusifonll shape. A reduction of fibrosis was verified after the second month oftherapy in alI groups that received the celI therapy in the granuloma areas, as welI as in areas of septal and frontal fibrosis. The levels of TGF-~ were lower after the cell therapy, with statistical significance only in schistossomotic animaIs. The number of oval cells, known as liver stem cells, was increased in hepatic parenchyma afier cell therapy. The schistossomotic animaIs treated with bone marrow celIs recovered partialIy the production of albumin when compared to infected controls. Based on our observations about cell therapy in both models of chronic liver diseases evaluated we conc1ude that bone marrow mononuclear cell transplantation decreases tissue alterations caused by chronic aggressions to the liver and reinforce the use of this therapy in patients with chronic liver lesions.

Ácidos graxos conjugados (AGCs) é o termo geral para descrever os isômeros posicionais e geométricos dos ácidos graxos poliinsaturados com duplas ligações conjugadas. Inúmeros efeitos benéficos para a saúde como: anti-cancerígeno, anti-aterogênicos, anti-adipogênicos e anti-inflamatórios, tem sido atribuídos ao consumo dos AGCs. Entretanto, estudos sobre os efeitos dos AGCs no organismo ainda são inconclusivos e por isso o interesse em pesquisas visando a sua participação em processos fisiológicos. O objetivo deste estudo foi avaliar em ratos o efeito dos isômeros conjugados do ácido linoleico (9cis, 11trans e 10trans, 12cis) sobre o perfil lipídico tecidual e sua influência sobre parâmetros bioquímicos em processos oxidativos. Não foi possível detectar a atividade antioxidante in vitro dos CLAs. Os resultados obtidos demonstraram que o FFA-CLA foi capaz de reagir apenas com o radical DPPH. No experimento in vivo uma mistura comercial dos isômeros (9cis, 11trans e 10trans, 12 cis) foi utilizada como fonte de CLAs, e a influência da suplementação desses isômeros, foi avaliada e comparada com um grupo controle suplementado com água e com grupos experimentais suplementados com óleo de soja. O primeiro experimento foi realizado com animais Wistar saudáveis e teve duração de 40 dias, as amostras de ácidos graxos livres de CLAs, (FFACLAs) e óleo de soja foram fornecidas aos animais por meio de entubação orogástrica nas concentrações de 1%, 2% e 4% em relação ao consumo diário de dieta, o grupo controle recebeu 1% de água. A suplementação da dieta dos animais com os CLAs leva ao aumento nos níveis de triglicérides, mas não interfere nos parâmetros de estresse oxidativo, embora haja incorporação dose-dependente nos tecidos hepático, muscular e adiposo, não foram observadas alterações no volume das células adiposas, e na área e diâmetro do tecido muscular. No segundo experimento, com duração de 21 dias, ao avaliar o efeito da suplementação na dose de 2% de CLAs em ratos Wistar induzidos a peroxidação, pelo tratamento com tetracloreto de carbono. Pôde-se observar efeitos hepatoprotetor, dos CLAs atribuídos a sua prevenção na peroxidação lipídica e ao aumento da atividade das enzimas catalase (CAT), glutatina redutase (GR) e glutationa reduzida (GSH). / Conjugated fatty acids (CFAs) is the general term to describe positional and geometric isomers of polyunsaturated fatty acids with conjugated double bonds. Many beneficial health effects such as anti-cancer, anti-atherogenic, anti-adipogenic and anti-inflammatory, has been attributed to the consumption of the CFAs. However, studies on the effects of the CFAs in the body are still inconclusive and therefore the interest in research aimed at their participation in processes physiological. The objective of this study was to evaluate in rats the effect of conjugated linoleic acid isomers (9cis, 11trans and 10trans, 12cis) on lipid profile tissue and its effect on biochemical parameters in oxidative processes. It was not possible to detect the in vitro antioxidant activity of CLAs. The results showed that the free fatty acids of CLAs (FFA-CLAs) was able to react only with the DPPH radical. In in vivo experiment a commercial mixture of isomers (9cis, 11trans and 10trans, 12cis) was used as a source of CLAs and the influence of supplementation of these isomers was evaluated and compared with a control group supplemented with water and experimental groups supplemented with soybean oil. The first experiment was conducted with healthy Wistar and lasted 40 days, samples of FFA-CLA and soybean oil were delivered to animals via orogastric intubation at concentrations of 1%, 2 % and 4% in relation to daily diet, the control group received 1% water. Supplementing the diet with CLAs leads to increased levels of triglycerides, but does not interfere with oxidative stress parameters, although there is a dose-dependent incorporation into liver tissue, muscle and fat, no changes were observed in the volume of fat cells, and in the area and diameter of muscle tissue. In the second experiment, lasting 21 days, to evaluate the effect of supplementation at a dose of 2% of CLAs in Wistar, tha peroxidation induced by treatment with carbon tetrachloride. Could be observed hepatoprotective effects, attributed to the CLAs prevention lipid peroxidation and increased activity of the enzymes catalase (CAT), glutathione reductase (GR) and glutathione reduzida (GSH).

Submitted by Adriana Martinez (amartinez@unoeste.br) on 2018-05-10T22:56:59Z No. of bitstreams: 1 Cristiane.pdf: 2390262 bytes, checksum: 25e1f95e171ca5f09f8ad8d90cc4b3d9 (MD5) / Made available in DSpace on 2018-05-10T22:56:59Z (GMT). No. of bitstreams: 1 Cristiane.pdf: 2390262 bytes, checksum: 25e1f95e171ca5f09f8ad8d90cc4b3d9 (MD5) Previous issue date: 2018-03-05 / Carbon tetrachloride (CCl4) is a xenobiotic that can induce injury by the formation of free radicals. When administered it is distributed and deposited in organs such as liver, brain, kidney, lung and heart. Since ancient times, medicinal plants have been of great importance in human health. According to the World Health Organization, about 80% of the world's population uses medicinal herbs as the first health care. Bidens pilosa (BP), popularly known as “Picão”, is a plant native to South America. BP has been used over the years to treat many diseases, such as inflammation, immune disorders, digestive diseases, infectious diseases, cancers, metabolic syndrome, wounds and liver diseases. BP has constituents with antioxidant activity, such as flavonoids. Objective: The aim of this study was to evaluate if the topical and oral treatment with BP have a protective effect against the carbon tetrachloride (CCl4)-induced toxicity. Material and methods: We used 56 male Wistar rats divided into 6 groups: A (n = 10): CCl4 only; B (n = 10): CCl4 + oral aqueous extract of BP; C (n = 10): CCl4 and topical BP (daily shower/1minute); D (n = 10): CCl4 and oral and topical aqueous extract (daily bath/1minute) BP; E (n = 8): oral aqueous extract of BP; F (n = 8): olive oil. The dose of CCl4 and olive oil was 1 mL solution/kg intraperitoneally 2 weight times/week and oral BP dose was 0.5mL/100g by gavage daily for 10 weeks. Blood samples were collected for hepatic and renal function tests and fragments of liver, spleen, pancreas, kidney and small and large intestine for histopathological analysis. Results: Statistical differences between group A and the other groups were observed in the following parameters: GGT (gamma glutamyl transpeptidase) (p = 0.017), albumin (p = 0.002), urea (p = 0.009) and creatinine 0.009), hepatic inflammation (p = 0.0001), renal tubular lesion (p <0.05) and inflammation of the small intestinal mucosa (p = 0.0001) between the BP groups and the untreated group. Conclusion: BP was effective in protecting the hepatic, renal and intestinal toxic effects of carbon tetrachloride, and combined (oral and topical) use showed the best protective effect. / O tetracloreto de carbono (CCl4) é um xenobiótico que pode induzir lesão pela formação de radicais livres. Quando administrado é distribuído e depositado em órgãos como: fígado, cérebro, rim, pulmão e coração. Desde a antiguidade, as plantas medicinais têm apresentado grande importância na saúde humana. Segundo a Organização Mundial de Saúde, cerca de 80% da população mundial utilizam ervas medicinais como primeiros cuidados de saúde. A Bidens pilosa (BP), popularmente denominada de “Picão”, é uma planta originária da América do Sul. A BP, ao longo dos anos, tem sido empregada no tratamento de muitas doenças, tais como: inflamação, distúrbios imunológicos, doenças digestivas, doenças infecciosas, cânceres, síndrome metabólica, feridas e hepatopatias. A BP possui constituintes com atividade antioxidante, como os flavonoides. Objetivo: O objetivo deste estudo foi avaliar se o tratamento tópico e por via oral com BP tem efeito protetor contra a intoxicação induzida pelo CCl4. Material e métodos: Utilizaram-se 56 ratos Wistar, machos, distribuídos em 6 grupos: A (n=10): CCl4; B (n=10): CCl4 + extrato aquoso oral de BP; C (n=10): CCl4 e BP tópica (banho diário/1minuto); D (n=10): CCl4 e extrato aquoso oral e tópico (banho diário/1minuto) de BP; E (n=8): extrato aquoso oral de BP; F (n=8): óleo de oliva. A dose utilizada de CCl4 e de óleo de oliva foi de 1mL de solução/Kg de peso intraperitoneal 2 vezes/semana e a de BP oral foi 0,5mL/100g de peso por gavagem diariamente por 10 semanas. Foi coletado sangue para realização de provas de função hepática e renal e fragmentos de: fígado, baço, pâncreas, rim e intestino delgado e grosso para análise histopatológica. Resultados: Observou-se diferença estatística entre o grupo A e os demais grupos estudados nos seguintes parâmetros: GGT (gama glutamil transpeptidase) (p= 0,017), albumina (p=0,002), uréia (p= 0,009) e creatinina (p= 0,009), inflamação hepática (p=0,0001), lesão tubular renal (p<0,05) e inflamação da mucosa do intestino delgado (p=0,0001) entre os grupos tratados com BP e o não tratado. Conclusão: A BP se mostrou efetiva para proteção aos efeitos tóxicos hepáticos, renais e intestinais do tetracloreto de carbono, e o uso combinado (oral e tópico), mostrou o melhor efeito protetor.

<p>Lipid peroxidation is thought to be an important factor in the pathophysiology of a number of diseases and in the process of ageing, but its measurement <i>in vivo</i> has been difficult. The aim of this thesis was to evaluate methods for measurement of lipid peroxidation <i>in vivo</i> that are suitable for clinical investigations, and to apply these methods in animal and human studies investigating basal conditions and situations associated with increased lipid peroxidation.</p><p>The ferrous oxidation in xylenol orange assay for quantification of total plasma lipid hydroperoxides was re-evaluated regarding sample handling and storage. It was shown to be a useful tool for analyses of fresh but not stored plasma samples.</p><p>A methodology for measurement of the total amount (sum of free and esterified) of an F₂-isoprostane, 8-iso-prostaglandin F_2α, in tissues using alkaline hydrolysis in combination with an existing radioimmunoassay was developed. High levels of 8-iso-prostaglandin F_2α in rat liver tissue were quantified by this technique both at basal conditions and in an experimental model of increased lipid peroxidation induced by carbon tetrachloride.</p><p>Supplementation with vitamin E to rats decreased both non-enzymatic and enzymatic lipid peroxidation as measured by 8-iso-prostaglandin F_2α and a major prostaglandin F_2α metabolite. This was verified both in the urine at basal conditions, and in the urine and liver tissue after carbon tetrachloride induced lipid peroxidation.</p><p>In a randomised cross-over study in humans, a rapeseed oil-based diet with an increased proportion of easily oxidised polyunsaturated fatty acids was compared to a control diet rich in saturated fats. The rapeseed oil-based diet did not seem to increase the degree of lipid peroxidation in plasma and urine as measured by 8-iso-prostaglandin F_2α, hydroperoxides and malondialdehyde, presumably due to a sufficient content of antioxidants in the rapeseed oil diet.</p><p>In conclusion, the simultaneous measurement of several biomarkers of lipid peroxidation is a promising approach for future studies investigating the possible role of lipid peroxidation <i>in vivo</i> under basal conditions and in the pathology of diseases.</p>

Lipid peroxidation is thought to be an important factor in the pathophysiology of a number of diseases and in the process of ageing, but its measurement in vivo has been difficult. The aim of this thesis was to evaluate methods for measurement of lipid peroxidation in vivo that are suitable for clinical investigations, and to apply these methods in animal and human studies investigating basal conditions and situations associated with increased lipid peroxidation. The ferrous oxidation in xylenol orange assay for quantification of total plasma lipid hydroperoxides was re-evaluated regarding sample handling and storage. It was shown to be a useful tool for analyses of fresh but not stored plasma samples. A methodology for measurement of the total amount (sum of free and esterified) of an F2-isoprostane, 8-iso-prostaglandin F2α, in tissues using alkaline hydrolysis in combination with an existing radioimmunoassay was developed. High levels of 8-iso-prostaglandin F2α in rat liver tissue were quantified by this technique both at basal conditions and in an experimental model of increased lipid peroxidation induced by carbon tetrachloride. Supplementation with vitamin E to rats decreased both non-enzymatic and enzymatic lipid peroxidation as measured by 8-iso-prostaglandin F2α and a major prostaglandin F2α metabolite. This was verified both in the urine at basal conditions, and in the urine and liver tissue after carbon tetrachloride induced lipid peroxidation. In a randomised cross-over study in humans, a rapeseed oil-based diet with an increased proportion of easily oxidised polyunsaturated fatty acids was compared to a control diet rich in saturated fats. The rapeseed oil-based diet did not seem to increase the degree of lipid peroxidation in plasma and urine as measured by 8-iso-prostaglandin F2α, hydroperoxides and malondialdehyde, presumably due to a sufficient content of antioxidants in the rapeseed oil diet. In conclusion, the simultaneous measurement of several biomarkers of lipid peroxidation is a promising approach for future studies investigating the possible role of lipid peroxidation in vivo under basal conditions and in the pathology of diseases.

The liver presented exceptional characteristics, like controlling energy production, immunological defenses, and blood reserve. In the environment like in the work place, the human is exposed to a different kind of hepatotoxic compounds, for example, on inks and derivatives (2-nitropropane), chemical reagents (carbon tetrachloride) and in tobacco smoke (2-nitropropane and cadmium). In fact, is interesting studies of therapies which protect or ameliorated the damage induced by these compounds. Considering the growing interesting around organochalcogens, in special interest, diphenyl diselenide (PhSe)2, which posses important pharmacological properties, such as: anti-ulcer, antiinflammatory, antinociceptive, anti-hyperglycemic, protected against orofacial diskinesia induced by reserpine and halopheridol and may act on memory facilitation in mice, the hepatoprotective properties of this compound induced by different models of liver damage (2-nitropropane, cadmium and carbon tetrachloride) were examined. The results demonstrated that (PhSe)2 (100 µmol/kg) significantly reduced hepatic markers levels when compared to 2-nitropropane (2-NP) group. Treatment with diphenyl diselenide, at all doses, effectively protects against the increase of lipid peroxidation when compared to 2-NP group. In addition, histological examination revealed that 2-NP treatment causes a moderate swelling and degenerative alterations on hepatocytes and (PhSe)2 protects against these alterations. This study evidences the protective effect of diphenyl diselenide by 2-NP-induced acute hepatic damage. In addition the effect of post-treatment with (PhSe)2 on liver damage induced by 2-NP was also examined. (PhSe)2 effectively restored the increase of aminotransferase activities and urea level when compared to the 2-NP group. At the highest dose (100 mol/kg), (PhSe)2 decreased -glutamyl transferase activity (GGT) and ameliorated the increase of hepatic and renal lipid peroxidation when compared to 2-NP group. 2-NP reduced catalase activity (CAT) and did not alter superoxide dismutase activity (SOD) nor ascorbic acid level. This study points out the involvement of CAT activity in 2-NP-induced acute liver damage and suggests that the post-treatment with diphenyl diselenide was effective in restoring the hepatic damage induced by 2-NP. Similar results were obtained with cadmium (Cd), an environmental toxic metal implicated in human diseases. Cadmium content determined in the tissue of rats exposed to cadmium chloride (CdCl2) provides evidence that the liver is the major cadmium target. The concentration of cadmium in liver was about three fold higher than that in kidney, and (PhSe)2 reduced about six fold the levels of this metal in liver of rats exposed. Rats exposed to CdCl2 showed histological alterations abolished by (PhSe)2 administration. In addition, (PhSe)2 administration ameliorated plasma malondialdehyde (MDA) levels, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and GGT activities increased by CdCl2 exposure. In conclusion, this study demonstrated that co-treatment with (PhSe)2 ameliorated hepatotoxicity and cellular damage in rat liver after sub-chronic exposure with CdCl2. The proposed mechanisms by which (PhSe)2 acts in this experimental protocol are its antioxidant properties and its capacity to form a complex with Cd. On the contrary, the administration of (PhSe)2 potentiated acute hepatic damage induced by carbon tetrachloride (CCl4), as manifested by an increase in biochemical parameters (AST, ALT, ALP, GGT and BT) and severe alteration in histopathology. This study also demonstrated a potentiation of lipid peroxidation levels and a consequent depletion of important antioxidant defenses including catalase and ascorbic acid, suggesting that the oxidative damage is related to the potentiation effect induced by (PhSe)2. Considering the results obtained, could be suggested that (PhSe)2 present a hepatoprotective effect depending of experimental protocol. / O fígado apresenta extraordinária pluralidade funcional, destacando-se no controle de produção de energia, defesa imunológica e reserva volêmica. No meio ambiente e ocupacionalmente, o ser humano está exposto a uma variedade de compostos hepatotóxicos, como por exemplo, no uso de tintas e seus derivados (2-nitropropano), reagentes químicos (tetracloreto de carbono) e na exposição ao cigarro (cádmio e 2-nitropropano). Portanto, é interessante o estudo de terapias que previnam ou até mesmo revertam à intoxicação causada por estes compostos. Considerando o crescente interesse por compostos orgânicos de selênio, em especial o disseleneto de difenila ((PhSe)2) que possui propriedades farmacológicas mais amplas como: efeitos anti-úlcera, antiinflamatório e antinociceptivo, anti- hiperglicemiante, protege contra a discinesia orofacial induzida por reserpina e haloperidol e pode atuar na facilitação da formação de memória em camundongos. Deste modo, os efeitos hepatoprotetores deste composto frente a diferentes modelos de dano hepático (2-nitropropano, cádmio e tetracloreto de carbono) foram examinados. Os resultados obtidos neste estudo demonstraram que a administração de (PhSe)2 (100 µmol/kg) reduziu os níveis de marcadores hepáticos e os níveis de peroxidação lipídica quando comparado ao grupo tratado com 2-nitropropano (2-NP). Além disso, os exames histológicos revelaram que o tratamento com 2-NP causou alterações degenerativas nos hepatócitos e que o (PhSe)2 foi capaz de proteger, evidenciando o efeito hepatoprotetor desse composto sobre o dano hepático induzido por 2-NP. O efeito do pós-tratamento com (PhSe)2 sobre o dano hepático induzido com 2-NP também foi investigado. Este composto restaurou a atividade plasmática das enzimas aminotransferases e os níveis de uréia quando comparado ao grupo tratado com 2-NP. Na maior dose (100 mol/kg), o (PhSe)2 causou uma diminuição na atividade da enzima -glutamil transferase (GGT) e restituiu o aumento nos níveis de peroxidação lipídica hepáticos e renais quando comparado ao grupo tratado com 2-NP. O tratamento com 2-NP reduziu a atividade hepática da catalase, entretanto não alterou a atividade da superóxido dismutase (SOD) e os níveis de ácido ascórbico, sugerindo que a inibição da CAT pode estar relacionada com o aumento nos níveis de peroxidação lipídica hepática nos ratos tratados com 2-NP. Resultados similares foram encontrados quando o dano hepático foi induzido por cádmio (Cd), um contaminante ambiental implicado em várias doenças. O conteúdo de Cd determinado nos ratos expostos ao cloreto de cádmio (CdCl2) provêm evidências de que o fígado é o maior alvo da toxicidade deste metal. A concentração de cádmio no fígado foi em torno de 3 vezes maiores que os níveis encontrados no rim. O (PhSe)2 reduziu em torno de 6 vezes os níveis deste metal no fígado dos ratos expostos ao CdCl2. Além disso, a administração de (PhSe)2 causou uma redução nos níveis de malondialdeído plasmáticos (MDA), na atividade das aminotransferases, na fosfatase alcalina (ALP), lactato desidrogenase (LDH) e GGT quando comparado ao grupo tratado com cádmio. Em conclusão, esse estudo demonstrou que o tratamento concomitante com (PhSe)2 reduziu a hepatotoxicidade e o dano celular em fígado de ratos expostos ao cádmio. O mecanismo proposto para ação do (PhSe)2 pode ser devido as suas propriedades antioxidantes ou pela sua capacidade de formar um complexo com Cd. Em contraste, a administração de (PhSe)2 potencializou o dano induzido por tetracloreto de carbono (CCl4), o que foi demonstrado pelo aumento dos níveis de marcadores bioquímicos (AST, ALT, ALP, GGT and BT) e pela severa alteração na histologia. Esses estudos também demonstraram que a administração de (PhSe)2 potencializou os níveis de peroxidação lipídica com consequente depleção das defesas antioxidantes, como a catalase e o ácido ascórbico, sugerindo que o dano oxidativo está relacionado com este efeito. Considerando os resultados obtidos, podemos sugerir que o disseleneto de difenila apresenta um efeito hepatoprotetor dependendo do modelo experimental.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The liver presents extraordinary functional diversity, particularly in the control of energy production, immune defense and volemic reserve. The human being is exposed occupationally and in the environment to a variety of hepatotoxic compounds, such as the use of paints and their derivatives (2-nitropropane, 2-NP), chemical reagents (carbon tetrachloride, CCl4) and exposure to cigarette (2-NP). Therefore, it is interesting the study of therapies to prevent or even reverse the poisoning caused by these compounds. Considering that reactive oxygen species (ROS) have an important role in various diseases, especially in liver diseases, the use of antioxidant therapies should be considered. In this context, the heterocyclic compounds containing selenium in their structures have attracted the attention of researchers. Thus, this study investigated the antioxidant activity of 3-alkynyl selenophenes in models of oxidative damage in vitro and ex vivo in rats (Wistar, male, weighing 200-300g). A class of 3-alkynyl selenophene compounds with different substitutions was tested, with the objective to assess their antioxidant profile and their possible toxic effect in vitro. As a result, 3-alkynyl selenophenes had antioxidant activity, but this activity was dependent on the presence of terminal alkynes in the molecule or easy conversion to it. The possible toxic effect of 3-alkynyl selenophenes was evaluated through the activity of the enzyme δ-aminolevulinate dehydratase (δ-ALA-D) in vitro. The results showed that none of 3-alkynyl selenophenes inhibited the activity of this enzyme, suggesting that this class of compound did not present toxicity on this enzyme. From these results, selenophene h (compound that had the best antioxidant activity in vitro) was selected for the evaluation of its protective effect against oxidative damage induced by 2-NP and CCl4 (ex vivo). Selenophene h (25 mg/kg) protected against the increase of markers of liver damage (aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities) and oxidative stress induced by administration of 2-NP in rats. 2-NP induced microscopic changes, evaluated by histopathological inspections, that were protected by this compound. Selenophene h showed a protective effect against the increase of lipid peroxidation and inhibition of activity of δ-ALA-D in animals treated with 2-NP. Selenophene h protected against oxidative damage induced by CCl4 in rats. A single dose of CCl4 caused significant hepatotoxicity, evidenced by elevated plasma enzyme activity of AST and ALT, increased incidence of histopathological lesions, increased lipid peroxidation levels and the activity of Glutathione-S-transferase (GST), decreased levels of ascorbic acid and the activity of catalase and δ-ALA-D. In conclusion, 3-alkynyl selenophene protected from all these changes, confirming its hepatoprotective effect. Considering the results, we suggest that 3-alkynyl selenophene, an antioxidant, may be a useful therapy for the oxidative damage induced by 2-NP or CCl4 . / O fígado apresenta extraordinária pluralidade funcional, destacando-se no controle de produção de energia, defesa imunológica e reserva volêmica. No meio ambiente e ocupacionalmente, o ser humano está exposto a uma variedade de compostos hepatotóxicos, como por exemplo, no uso de tintas e seus derivados (2-nitropropano, 2- NP), reagentes químicos (tetracloreto de carbono, CCl4) e na exposição ao cigarro (2-NP). Portanto, é interessante o estudo de terapias que previnam ou até mesmo revertam a intoxicação causada por estes compostos. Considerando que as espécies reativas de oxigênio (EROs) apresentam importante papel sobre diversas patologias, em especial nas doenças hepáticas, o uso de terapias antioxidantes deve ser considerada. Neste contexto, destacam-se os compostos heterocíclicos contendo selênio em sua estrutura. Deste modo, neste estudo investigou-se a atividade antioxidante de 3-alquinil selenofenos em modelos de dano oxidativo in vitro e ex vivo em ratos (Wistar, machos, pesando entre 200 300 g). Para esse fim, testou-se uma classe de compostos 3-alquinil selenofeno, com diferentes substituições na estrutura química, com o objetivo de avaliar o perfil antioxidante e seu possível efeito tóxico in vitro em ratos. Como resultado, 3- alquinil selenofenos tiveram atividade antioxidante, porém esta atividade foi dependente da presença de um alquino terminal na molécula ou da fácil conversão da molécula a um alquino terminal. Além disso, o possível efeito tóxico dos 3-alquinil selenofenos foi avaliado através da atividade da enzima δ-aminolevulinato desidratase (δ-ALA-D) in vitro. Os resultados obtidos demonstraram que nenhum dos 3-alquinil selenofenos testados inibiu a atividade desta enzima, sugerindo que esta classe de compostos não apresentou toxicidade sobre a atividade da δ-ALA-D. A partir destes resultados, selecionou-se o selenofeno h (que obteve melhor atividade antioxidante in vitro) para a avaliação do seu efeito protetor contra o dano oxidativo induzido por 2-NP e CCl4 em ratos (ex vivo). O selenofeno h (25 mg/kg) protegeu contra o aumento dos marcadores de dano hepático (aspartato aminotranferase (AST) e alanina aminotransferase (ALT)) e de estresse oxidativo induzidos pela administração do 2-NP. O 2-NP induziu alterações microscópicas avaliadas por inspeções histopatológicas as quais foram protegidas pelo composto. O selenofeno h demonstrou efeito protetor contra o aumento da peroxidação lipídica e inibição da atividade da δ-ALA-D nos animais tratados com 2-NP. Além disso, o selenofeno h protegeu contra o dano oxidativo induzido pelo CCl4 em ratos. Uma única dose de CCl4 causou significante hepatotoxicidade, evidenciada por elevação da atividade plasmática das enzimas AST e ALT, aumento da incidência de lesões histopatológicas, aumento dos níveis de peroxidação lipídica e da atividade da enzima glutationa-S-transferase (GST), bem como diminuição dos níveis de ácido ascórbico e da atividade das enzimas catalase e δ-ALA-D. A partir dos resultados demonstrados, verificou-se que o selenofeno h protegeu contra todas estas alterações, confirmando o seu efeito hepatoprotetor. Considerando os resultados obtidos, pode-se sugerir que o selenofeno h, uma molécula com atividade antioxidante, pode ser uma útil terapia contra o dano oxidativo induzido pelos hepatotoxicantes: 2-NP e CCl4.

Submitted by Adriana Martinez (amartinez@unoeste.br) on 2016-12-16T13:59:28Z No. of bitstreams: 1 Fernanda Serra.pdf: 916933 bytes, checksum: 7854d6fb1afaa501232fb8cc37be07f7 (MD5) / Made available in DSpace on 2016-12-16T13:59:28Z (GMT). No. of bitstreams: 1 Fernanda Serra.pdf: 916933 bytes, checksum: 7854d6fb1afaa501232fb8cc37be07f7 (MD5) Previous issue date: 2016-10-24 / Bidens pilosa is a medicinal plant made in abundance by flavonoids and polyacetylene, which act in the body with anti-tumor, antioxidant and antimicrobial effects. It is an herb widely used as tea to combat jaundice and hepatitis. The hypothesis is that the Bidens pilosa can be used as a hepatoprotective in cases of exposure to carbon tetrachloride and also may confer protection against mutagenicity caused by this chemical agent. Objective: To evaluate whether the topical treatment and oral with Bidens pilosa may have anti-mutagenic effect on bone marrow cells in animals exposed to carbon tetrachloride (CCl4). Material and methods: 64 Wistar rats, adults, males, were divided into seven groups, treated for 10 weeks (except the positive control group). The dose of CCl4 solution was 1 ml / 100g, intraperitoneally, twice a week; the aqueous extract of Bidens pilosa was admistrated by oral gavage daily (0.5 ml / 100g); topical Bidens pilosa was by daily bath for 1 minute; the negative control group received 1ml olive oil / kg of body weight intraperitoneally twice a week and the positive control received subcutaneous single dose of cyclophosphamide (50 mg / kg) on the first day of the experiment. Groups: A - CCl4 group (n = 10); B - CCl4 Group + Bidens oral hairy, (n = 10); C - + CCl4 Group Bidens pilosa topical (n = 10); D - + CCl4 Group Bidens pilosa oral and topical (n = 10); E - Bidens pilosa orally (n = 8); F - negative control group (n = 8); G - Group positive control (n = 8). The animals of groups A, B, C, D, E and F were euthanized 10 weeks after the start of the experiment and the G group 24 hours after the start. He retired the femur to collect the bone marrow cells and realization of the micronucleus test. Results: The mean of micronuclei in the group exposed only to CCl4 was 4.00. In groups exposed to CCl4 and Bidens pilosa (oral and / or topical) and exposed only to Bidens pilosa and not exposed to CCl4 group was 0.00. In the group exposed to cyclophosphamide was 9.00. There was no statistically significant difference between groups A and C (p> 0.05), but they differed significantly from the other groups (p <0.05). Conclusion: The Bidens pilosa in oral and topical given anti-mutagenic effect of exposure to carbon tetrachloride. / A Bidens pilosa é uma planta medicinal composta em abundancia por flavonoides e poliacetileno, que atuam no organismo com efeitos antitumorais, antioxidante e antimicrobiano. É uma erva muito utilizada na forma de chá para combater icterícia e hepatite. A hipótese deste estudo é que se a Bidens pilosa podendo ser usada como hepatoprotetora em casos de exposição ao tetracloreto de carbono, também, possa conferir proteção contra mutagenicidade deste agente químico. Objetivo: Avaliar se o tratamento tópico e por via oral com Bidens pilosa pode ter efeito anti-mutagênico sobre as células da medula óssea em animais expostos ao tetracloreto de carbono (CCl4). Material e Métodos: Foram utilizados 64 ratos Wistar albinos, adultos, machos, divididos em sete grupos, tratados por 10 semanas (exceto o grupo controle positivo). A dose administrada da solução de CCl4 foi de 1 ml/100g de peso, via intraperitoneal, duas vezes por semana; o extrato aquoso de Bidens pilosa oral foi admistrado por gavagem diariamente, (0,5ml/100g de peso); a Bidens pilosa tópica foi mediante banho diário durante 1 minuto; o grupo do controle negativo recebeu 1ml de óleo de oliva por cada quilo de peso via intraperitoneal, duas vezes por semana e o controle positivo recebeu ciclofosfamida em dose única subcutânea (50 mg/kg) no primeiro dia do experimento. Grupos: A – Grupo CCI4, (n=10); B – Grupo CCI4 + Bidens pilosa oral, (n=10); C – Grupo CCI4 + Bidens pilosa tópica, (n=10); D – Grupo CCI4 + Bidens pilosa oral e tópica, (n=10); E – Bidens pilosa oral, (n=8); F – Grupo controle negativo, (n=8); G – Grupo controle positivo (n=8). Os animais dos grupos A, B, C, D, E e F foram eutanasiados 10 semanas após o início do experimento e os do grupo G, 24 horas após o início. Retirou-se o fêmur para coleta das células da medula óssea e realização do teste do micronúcleo. Resultados: A mediana de micronúcleos no grupo exposto somente ao CCl4 foi de 4,00. Nos grupos expostos ao CCl4 e Bidens pilosa (oral e/ou tópica) e exposto apenas a Bidens pilosa e no grupo não exposto ao CCl4 foi de 0,00. No grupo exposto a ciclofosfamida foi de 9,00. Não houve diferença estatisticamente significante entre os grupos A e G (p>0,05), porém estes diferiram estatisticamente dos demais grupos (p<0,05). Conclusão: A Bidens pilosa em uso oral e tópico conferiu efeito anti-mutagênico a exposição ao tetracloreto de carbono.