Muitas espécies de animais vivenciam situações nas quais há uma profunda depressão metabólica, como na anóxia, na hipóxia e na hibernação. Durante a reoxigenação ou o despertar, ocorre aumento da produção de espécies reativas de oxigênio (ROS), que tendem a causar danos oxidativos. Diferentes enzimas antioxidantes protegem o organismo contra as ROS, porém não se sabe qual a real importância de cada uma delas durante a reoxigenação. A hibernação é uma das formas de hipometabolismo menos estudadas, fazendo com que haja questionamentos sobre como os hibernantes se protegem das ROS durante o despertar. A análise dos dados existentes é complexa devido à existência de variáveis não controladas, como o efeito do jejum associado à hibernação. Nesta tese, foram desenvolvidos dois projetos. Em um, investigou-se a importância da catalase num ciclo de anóxia e reoxigenação em caramujos pulmonados. No segundo, investigou-se o efeito da hibernação e da privação alimentar no intestino de lagartos teiús. Com base nos resultados, foi possível concluir que a catalase exerce um papel complementar contra os danos oxidativos causados pelas ROS e em conjunto com os demais componentes do sistema antioxidante. Porém, sua função não parece ser essencial, sendo em grande parte compensada pela atividade de glutationa peroxidase. Também foi possível concluir que a hibernação, estudada sem a interferência de drásticas quedas da temperatura, causa nítidas alterações no metabolismo de radicais livres no intestino de lagartos, com queda de atividades enzimáticas e de concentração de glutationa. A ausência de grandes danos oxidativos durante o despertar dos animais mostra que eles têm um sistema antioxidante eficiente. A privação alimentar resultou em respostas semelhantes as da hibernação, mas parece ter causado um certo grau de estresse oxidativo. Os resultados apresentados nesta tese respondem dois questionamentos no estudo do metabolismo de radicais livres em situações que envolvem flutuações na disponibilidade e no consumo de oxigênio. / Many species of animals experience situations in which occurs a profound metabolic depression, like anoxia, hypoxia and hibernation. During reoxygenation or arousal, there is an increase of the production of reactive oxygen species (ROS), which tend to cause oxidative damage. Different antioxidant enzymes protect the organims against the ROS, however the real importance of each one of them during reoxygenation is not known. Hibernation is one of the types of hypometabolism less studied, and questions about how the hibernators protect themselves from ROS during the arousal have not yet been answered. The analysis of the existent data is complex due to the existence of uncontrolled variables. In this thesis were carried out two studies in which were investigated: the importance of catalase in a cycle of anoxia and reoxygenation in pulmonate snails, and the effect of hibernation and of food deprivation in the intestine of tegu lizards. Considering the results, it was possible to conclude that catalase plays an complementary role against the damages caused by ROS and in association with the other components of the antioxidant system. However, its function seems to be non-essential, being greatly compensated by the glutathione peroxidase activity. It was also possible to conclude that hibernation, studied without the interference of drastic falls in temperature, causes clear alterations in free radicals metabolism in the lizards intestine, with a reduction in enzymes activities and in glutathione concentration. The absence of big oxidative damage during the arousal of the animals shows that they have an efficient antioxidant system. Food deprivation resulted in similar responses of those from hibernation, but seemed to cause some degree of oxidative stress. The results presented in this thesis answer two questions in the study of the free radical metabolism in situations that involve fluctuations in oxygen availability and consumption.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Conjugated linoleic acids (CLA) are geometrical and positional isomers of n-6 octadecadenoic acid (linoleic acid, LA, 18:2n-6), which form part of a family of essential polyunsaturated fatty acids (PUFA). There are 28 identified CLA isomers that mostly found in the meat and milk from ruminant animals. CLA has shown to possess a number of health benefits including; reduction in body fat and increased lean body mass, prevention of atherosclerosis, hypertension, increased immune function and in particular the prevention of cancer. The effects of CLA on cancer cell lines will be evaluated to discover the mechanisms that are employed to achieve this great phenomenon on cell growth. The aim of this study was to determine the effect of CLA on various parameters that are essential in the development of cancer cell phenotype. The objectives were to evaluate the effect of CLA on iron-induced lipid peroxidation of microsomes isolated from rat liver cells and in vitro cytotoxicity, cell proliferation and apoptosis in HepG2 hepatocarcinoma cells. The Fatty acid incorporation in HepG2 cells was also assessed.

This thesis studies the interaction between free radical species and supported noble metal nanoparticles (silver and gold) in the context of oxidation reactions. The peroxidation of cumene is the first reaction to be discussed and the difference in peroxidation product distribution using silver nanoparticles (AgNP) versus gold nanoparticles (AuNP) is examined. Specifically, cumyl alcohol is obtained as the major product obtained when using supported AuNP, whereas cumene hydroperoxide is favoured for AgNP. Such variations in product distribution are partially explained by the differences in the nanoparticle Fenton activity, where the TiO2 support was proposed to enhance such activity due to possible electron shuttling capabilities with the nanoparticle surface. Use of hydrotalcite as a support was found to minimize this characteristic, due to its insulator properties. The stability of hydroperoxide was tested in the presence of various others supports (activated carbon, Al2O3, ZnO, SiO2 and clays) with little success, with hydroperoxide exhibiting stability in the presence of HT. Using an oxygen uptake apparatus, the interaction of the cumyl peroxyl radical with the AuNP surface was demonstrated. Furthermore, this interaction promotes decomposition leading to the corresponding alkoxyl radical and subsequent hydrogen abstraction to form the observed cumyl alcohol product. The radical interaction with supported nanoparticles, and its reversibility appear different for gold and silver and accounts for a large part of the product distribution differences observed between AuNP and AgNP, as illustrated below. The peroxidation of ethylbenzene and propylbenzene was studied and revealed the participation of a reactive surface oxygen species due to the decomposition of peroxyl radicals on the nanoparticle surface. The reactive oxygen species was found to be transient in nature in the case of AuNP . Furthermore, this surface species was found to be an important participant in hydrogen abstraction leading to peroxide product formation. Finally, supported nanoparticle catalyzed tetralin peroxidation was investigated to determine the influence of temperature on the peroxidation product distribution and how changes in the reaction temperature can effect the radical-nanoparticle surface interactions.

Lipid peroxidation has been implicated in the onset and progression of many degenerative diseases, including cardiovascular disease, Alzheimer’s disease and cancer. Accordingly, for more than 50 years, considerable effort has been devoted to the design of synthetic compounds or the discovery of natural products that can slow lipid peroxidation. Despite the enormous investments made to date, no clear antioxidant strategies have emerged for the treatment and/or prevention of degenerative disease. We argue that this is because of a lack of fundamental understanding of the chemical reactivity of these compounds in relevant contexts. Herein, we describe studies of our optimized synthetic radical-trapping antioxidant (RTA) – the tetrahydronaphthyridinols (THNs). We first present the synthesis of a series of THN analogs of α-tocopherol (Nature’s premier lipid-soluble radical-trapping antioxidant) with varying sidechain substitution and then demonstrate how systematic changes in the lipophilicity of these potent antioxidants impact their peroxyl radical-trapping activities in lipid bilayers and mammalian cell culture. Their regenerability by water-soluble reductants in lipid bilayers, binding to human tocopherol transport protein (hTTP), and cytotoxicity were also evaluated to provide insight on whether this type of antioxidant can be potentially pushed toward animal studies. We also describe analogous studies of natural products such as the garlic-derived thiosulfinate allicin and the grape-derived polyphenol resveratrol. These compounds have attracted significant attention in the past 20 years due to their purported health benefits, which are often ascribed to their purported radical-trapping activities. To date, systematic studies on their radical-trapping activities in solution, lipid bilayers and mammalian cells have been lacking. We have determined that allicin and petivericin, while effective RTAs in solution, are not so in lipid bilayers. Moreover, the compounds are not antioxidants in cell culture, but instead kill the cells. Similarly, resveratrol and its dimers pallidol and quadrangularin A, are found to be inefficient RTAs in lipid bilayers. Our studies to date rather suggest that they autoxidize readily to produce hydrogen peroxide, which may induce expression of phase 2 antioxidant enzymes, affording cytoprotection. Our insights underscore the need for systematic studies of antioxidant activity in multiple contexts.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants that have been associated with various adverse health effects in humans and wildlife. Dioxin-like PCBs elicit a broad spectrum of biochemical and toxic changes including cardiovascular disorders. Paraoxonase 1 (PON1), an antioxidant enzyme, prevents oxidative stress and plays key roles in the pathogenesis of atherosclerosis. The overall goal of this dissertation is to investigate the mechanism and role of PON1 in the antioxidant defense to the exposure of 3, 3', 4, 4', 5-pentachlorobiphenyl (PCB 126), the most potent congener in PCB family. My overall hypothesis is that: The up-regulation of PON1 is an antioxidant response to PCB 126 exposure, which involves the Ah receptor (AhR) and results in changes in the PON1 protein level and activity which in turn has an influence on the oxidative stress status. First, the responses of PON1 gene expression and activities in serum and liver upon the PCB126 treatment were evaluated in the rat model. I found that PCB 126 up-regulated PON1, gene expression and activities, in a time and dose dependent manner. Next, I investigated the molecular mechanism of this response. My results show that the up-regulation of PON1 by PCB 126 involves the AhR and the XRE-like sequence in the gene promoter region. The up-regulation of PON1 was tissue specific, and this response probably protected liver and serum from lipid oxidation to some extent. The structure-activity relationship studies with PCB congeners indicate that the up-regulation of PON1 was specific to dioxin-like PCB 126. Other different AhR ligands displayed different PON1 induction capabilities. Also, reduction of PON1 activity was found in male rats dosed with non-dioxin-like PCBs. Finally I investigated the interaction, and possible protection, of other antioxidants (Se and NAC) on the response to PCB 126. I found that these antioxidants reduced the magnitude of the response of PON1 to the PCB 126 exposure in liver. Both the increase of PON1 activities and addition of antioxidants may be the reason for the lack in increase of lipid peroxidation. In total, these findings support my hypothesis and suggest that up-regulation of PON1 by PCB 126 may be an adaptive antioxidant mechanism that is involved in the body's antioxidant system.

Effects of Fe and Se status on GSHPx activity and lipid peroxidation in liver and intestinal mucosa were studied. Rats were Se and Fe supplemented ( +Se+Fe), Sedeficient and Fe-supplemented (-Se+Fe), Se supplemented and Fe overloaded (+Se++Fe) by intramuscular injection, or Se deficient and Fe overloaded (-Se++Fe) for 20d. Fe overloaded tissues had more Fe, but hemoglobin was unaffected. Liver and mucosal GSHPx activity was low in Se-deficient rats. Thiobarbituric acid reactive substances (TBARS) were higher in Fe overloaded and -Se++Fe vs +Se++Fe tissues. Mucosal TBARS was higher in -Se++Fe rats gavaged with CBrCl3. In experiment 2, Fe overload was induced by a 2% carbonyl iron, low-Se diet fed for 2mo, the +Se++Fe and -Se++Fe groups. Low Se-, low Fe-diet was fed to rats supplemented with Fe or Fe and Se in the water, the -Se+Fe and +Se+Fe controls. Iron overloaded tissues had more Fe. Liver and mucosal GSHPx activity was lower in Se-deficient and +Se++Fe vs +Se+Fe rats. TBARS was higher in Fe overloaded, -Se++Fe vs +Se++Fe, and CBrCl3 tissues. Hemoglobin and serum Fe were lower in the -Se++Fe group. In experiment 3, low-Se, low-Fe diet was fed for 20d, the -Se-Fe, +Se-Fe, -Se+Fe and +Se+Fe groups. Mucosal Fe was lower in the Fe-deficient rats. Cytochrome P-450 and GSHPx activities were lower and TBARS was higher in Se-deficient tissues. In experiment 4, the +Se+Fe, +SeFe, -Se+Fe, -Se-Fe, +Se++Fe, and -Se++Fe groups treated as in experiment 3. Iron overloaded tissues had more Fe and TBARS, but hemoglobin and serum Fe were unaffected. GSHPx and Cytochrome P-450 activities were lower in Se-deficient and in +Se++Fe vs +Se+Fe rats. CBrCl3 did not affect TBARS. High TBARS occurred in liver and mucosa of Fe overloaded rats. Chronic Fe overload was required to reduce liver and mucosal GSHPx activity. The combination of Se deficiency and Fe overload caused very high TBARS. Low oral CBrCl3 doses elevated mucosal TBARS, a first report of extrahepatic action of CBrCl3 in vivo. Iron deficiency did not affect GSHPx activity or CBrCl3 induced lipid peroxidation.

Obesity is linked to nonalcoholic steatohepatitis. Peroxisome proliferator-activated receptor-a (PPARa) regulates lipid metabolism. Cytochrome P-450 2A5 (CYP2A5) is a potential antioxidant and CYP2A5 induction by ethanol is CYP2E1 dependent. High-fat diet (HFD)-induced obesity and steatosis are more severe in CYP2A5 knockout (cyp2a5 -/- ) mice than in wild-type mice although PPARa is elevated in cyp2a5 -/- mice. To examine why the upregulated PPARa failed to prevent the enhanced steatosis in cyp2a5 -/- mice, we abrogate the upregulated PPARa in cyp2a5 -/- mice by cross-breeding cyp2a5 -/- mice with PPARa knockout (ppara-/- ) mice to create ppara-/- /cyp2a5 -/- mice. The ppara-/- /cyp2a5 -/- mice, ppara-/- mice, and cyp2a5 -/- mice were fed HFD to induce steatosis. After HFD feeding, more severe steatosis was developed in ppara-/- /cyp2a5 -/- mice than in ppara-/- mice and cyp2a5 -/- mice. The ppara-/- /cyp2a5 -/- mice and ppara-/- mice exhibited comparable and impaired lipid metabolism. Elevated serum alanine transaminase and liver interleukin-1β, liver inflammatory cell infiltration, and foci of hepatocellular ballooning were observed in ppara-/- /cyp2a5 -/- mice but not in ppara-/- mice and cyp2a5 -/- mice. In ppara-/- /cyp2a5 -/- mice, although redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 and its target antioxidant genes were upregulated as a compensation, thioredoxin was suppressed, and phosphorylation of JNK and formation of nitrotyrosine adduct were increased. Liver glutathione was decreased, and lipid peroxidation was increased. Interestingly, inflammation and fibrosis were all observed within the clusters of lipid droplets, and these lipid droplet clusters were all located inside the area with CYP2E1-positive staining. These results suggest that HFD-induced fibrosis in ppara-/- /cyp2a5 -/- mice is associated with steatosis, and CYP2A5 interacts with PPARa to participate in regulating steatohepatitis-associated fibrosis.

Ferroptosis is an iron-dependent form of cell death driven by peroxidation of phospholipids with polyunsaturated fatty acyl (PUFA) tails. Dietary factors, such as fatty acids and iron, regulate ferroptosis. Moreover, the incidence and progression of several cancers is correlated with diet; models of lymphoma have shown sensitivity to ferroptosis. We investigated the effects of altering dietary factors linked to ferroptosis on diffuse large B cell lymphomas (DLBCL). We found that DLBCL cells undergo ferroptosis in response to iron and PUFA treatments in vitro, and that their growth in xenograft models is substantially reduced. We observed that monounsaturated fatty acids (MUFAs), in contrast, suppress ferroptosis and promote growth in DLBCL cell and animal models. The inhibitory effect of the ferroptosis inducer imidazole ketone erastin (IKE) on DLBCL xenograft growth was lessened by dietary MUFA. Ferroptosis linked fatty acids and iron thus impact the growth and response to ferroptosis treatment of DLBCL tumors.

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