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.

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.

No description available.

Isoflavones have antioxidant activities in vivo, however, their antioxidative potential against oxidative stress initiated by exercise was not explored. The first study investigated the effect of high-genistin isoflavone (HGI) supplementation on erythrocyte antioxidant enzymes and tissues' thiobarbituric reactive substances (TBARS) in acutely exercised one-year old rats. All tissue genistein concentrations increased after exercise. Ingestion of HGI seemingly enhanced running time to exhaustion, and maintained glutathione peroxidase (GPx) and catalase (CAT) activities decreased due to exercise. The second study investigated the dose effect of HGI supplementation. Genistein concentrations were significantly higher (P<0.05) in tissues of rats fed the 1045 PPM HGI diet than in rats fed 522 or 209 PPM HGI diets and increased the glutathione (GSH)/total glutathione (TGSH) ratio (P<0.03). Reductions of the in vivo MDA concentrations (P<0.05) were observed only in the plasma of rats fed 522 and 1045 PPM HGI diets compared to those fed 0 PPM (-1.08, -0.82, and 0.03 mM, respectively). Therefore, isoflavones at 522-1045 PPM HGI diet have antioxidative effects in rats. The last two studies investigated the effect of isoflavone supplementation on the modulation of erythrocyte antioxidant enzyme activities, glutathione homeostasis, and other oxidative biomolecules in healthy young men undergoing 80%VO2pk exercise. In Study 3 exercise at 80%VO2pk increased oxidative stress which was best demonstrated by increased superoxide dismutase (SOD) activity (16.5%), GSH/TGSH ratio, in vivo MDA (12.6%), plasma uric acid (4.9%) and ferric reducing/antioxidant ability (FRAP) ( 7.8%). Therefore, 30 minutes 80% VO2pk exercise induced oxidative stress in moderately active college men. In study 4, four-week HGI supplementation produced plasma genistein and daidzein concentrations of 499 and 415 ng/ml, which were significantly increased to 633 and 539 ng/ml by exercise (P=0.04 and P=0.05). Isoflavones significantly decreased in vivo pre-exercise plasma MDA (P<0.05), increased pre-exercise blood TGSH (P=0.01) and pre-exercise erythrocyte SOD activity (P=0.0006), and maintained the decreased activities of GPx due to exercise at pre-exercise levels. Results demonstrated that isoflavones had antioxidant activity in vivo under normal physiological conditions in healthy young men. They also maintaining GPx activity which was decreased due to exercise, however, isoflavones may not overcome all oxidative stress initiated by intense exercise. / Ph. D.

Studies were conducted to investigate the effects of in ovo injection of selenium (Se) either as seleno-methionine (Se-Met) or sodium selenite (Na2SeO3) into the yolk of incubating eggs on tissue Se concentration, embryo livability, lipid peroxidation, immune response and growth performance. When white-shelled eggs were injected with 0.1ml of solutions providing 0, 2.5, 5, 10 or 20 μg Se/egg, no detrimental effects on embryo viability at 20 days of incubation were noted. The effects on tissue Se concentrations suggested that Se-Met and Na2SeO3 were metabolized differently by the chick embryo. In a subsequent study using injection doses up to 60 μg/egg, a greater linear response in tissue Se was obtained with Se-Met, compared with Na2SeO3 (P < 0.01). Minimal changes in heart and breast muscle Se concentrations were noted above the 40 μg dose when Na2SeO3 was used (P > 0.05). In a study with broiler eggs, injection doses of 0, 2.5, 5, 10, 20 and 40 μg Se/egg were used. Se-Met or Na2SeO3 at doses up to 40 μg Se/egg had little effect on embryo viability. Injecting Se-Met resulted in greater tissue Se accumulation than Na2SeO3 at 20 days of incubation. In another study with broiler eggs using injection doses up to 40 μg Se/egg, Se-Met injection resulted in higher hatchability, reduced lipid peroxidation in the lung and heart muscle of the embryos after 20 days incubation and higher Se concentrations in heart and breast muscle of hatched chicks through 7 days and in lung through 21 days of growth. In a feeding trial with broiler breeder hens, adding 0.3 mg/kg of Se as Se yeast or Na2SeO3 to the diet improved tissue Se status at hatching of progeny chicks. Taken together, these results indicate that injection of Se into the yolk of incubating eggs may be useful for enhancing Se status during embryonic and early post-hatch development. Therefore, the improvement in Se status using this method in conjunction with dietary Se supplementation of breeder hens would be much greater than with only using dietary supplementation.

As Espécies Reativas de Oxigênio (EROs) conferem proteção aos seres vivos contra os contínuos ataques de microrganismos, além de serem responsáveis por diversos eventos fisiológicos. No entanto um desequilíbrio entre a produção das EROs e os agentes antioxidantes resulta diversos processos patológicos nos mais variados sistemas em seres humanos e em animais. Durante a gestação ocorrem alterações do metabolismo que podem levar a um aumento de subprodutos da oxidação. Assim como a gestação, o nascimento também impõe um período com alta demanda energética, alta tensão de oxigênio e, por consequência, determina um momento crítico na vida do neonato, por ser exigida uma rápida adaptação da condição hipóxica (intra útero) para hiperóxica (extra útero). Desta forma, o objetivo desta dissertação foi verificar o estado oxidativo das éguas no terço final da gestação, no periparto e no pós-parto levando em consideração o efeito da paridade, bem como a condição oxidativa de neonatos durante os primeiros sete dias de vida. Como indicadores de oxidação foram mensurados os níveis de TBARS e oxidação de proteína. Foi quantificado o ferro total e como parâmetros antioxidantes, foram medidas as atividades das enzimas GPx e SOD, e as concentrações de bilirrubina. Nos potros verificamos que a SOD não apresentou diferença significativa no periodo analisado. As concentrações de bilirrubina foram mais baixas no primeiro tempo avaliado, e tanto a bilirrubina total quanto a indireta elevaram-se às 12 horas e então caíram entre as 72h e 168h. Já a GPx demonstrou aumento da sua atividade nos tempos 12 e 72h quando comparada ao tempo 0h. Verificou-se altos níveis de TBARS no primeiro momento pós-nascimento, uma conseguinte diminuição às 12h seguida de estabilização nos demais tempos. Já a avaliação do período gestacional das éguas indicou um efeito de interação entre paridade e tempo gestacional apenas para o ferro total. SOD e oxidação proteica não apresentaram alterações significantes no período estudado. Tanto a GPx quanto as TBARS apresentaram efeito de tempo, com evidente alteração entre o parto, apresentando aumento e o pós-parto apresentando diminuição de atividade e das concentrações dessas variáveis. Concluímos que em potros, a peroxidação lipídica ao nascimento apresentou-se alta sugerindo um balanço pró-oxidativo durante tal período, o que poderia caracterizar um aumento nos níveis de EROs com finalidade de completar importantes eventos fisiológicos. Quanto a bilirrubina indireta e a GPx podemos sugerir que frente aos altos níveis da peroxidação lipídica houve um estímulo para ativação dos sistemas antioxidantes que envolvem essas biomoléculas e que as duas tenham agido concomitantemente visando equilibrar os níveis de EROs. Com relação às éguas, apontamos que a paridade não tem influencia sobre o estabelecimento da homeostase oxidativa em éguas e que no momento do parto as mesmas passam por um desbalanço oxidativo transiente. Ou seja, o desbalanço oxidativo faz parte tanto do momento do parto quanto da primeira semana de vida dos potros, possivelmente desempenhando um papel fisiológico em abas categorias. / Reactive Oxygen Species (ROS) produced protect living beings against the continuous attacks of microorganisms, as well as being responsible for several physiological events, so oxidative homeostasis is a premise. However, an imbalance between the production of ROS and the antioxidant agents causes several pathological processes in the most varied systems in humans and animals. During pregnancy it is suggested that alterations of the metabolism take place, consequently the increase of by-products of the oxidation during the gestational period. Like gestation, the birth also imposes a period with high energy demand, high oxygen tension and, consequently, it determines a critical moment in the life of the newborn because it is required a rapid adaptation of the same to the change of hypoxic condition (intra uterus) To hyperoxic (extra uterus). The aim of this dissertation was to verify the oxidative status of mares in the final third of gestation, peripartum and postpartum, taking into account the parity effect, as well as the oxidative condition of neonates during the first seven days of life. As oxidation indicators, the levels of TBARS and protein oxidation were measured. Total iron was quantified and as antioxidant parameters, activities of GPx and SOD enzymes, and bilirubin concentrations were measured. In the foals we verified that the SOD showed no significant difference in the analyzed time. Bilirubin concentrations were lower in the first time evaluated, and both total and indirect bilirubin increased at 12 hours and then fell between 72h and 168h. On the other hand, GPx showed an increase in its activity in times 12 and 72h when compared to time 0h. There were high levels of TBARS at the first post-birth moment, a consequent decrease at 12h, followed by stabilization at the other times. The results with mares indicated interaction effect between parity and gestational time only for total iron. SOD and protein oxidation did not present significant alterations in the studied period. Both GPx and TBARS presented a time effect, with an evident alteration between childbirth and postpartum, and there was an increase and decrease, respectively, in the activity and concentrations of these variables. We conclude that in foals, lipid peroxidation at birth was high suggesting a pro-oxidative balance during such period, which could characterize an increase in the levels of ROS in order to complete important physiological events. Regarding indirect bilirubin and GPx, we can suggest that in the face of the high levels of lipid peroxidation there was a stimulus for the activation of the antioxidant systems that involve these biomolecules and that the two have acted concomitantly in order to maintain the high levels of EROs at non detrimental levels. Regarding mares, we pointed out that parity has no influence on the establishment of oxidative homeostasis in mares and that at the time of delivery they undergo transient oxidative imbalance. That is, oxidative imbalance is part of both the calving moment and the first week of life of foals, possibly playing a physiological role in categories.

O aumento da incidência do câncer de pele está associado à maior exposição à luz solar e a adoção de ações de proteção ao Sol é uma estratégia para minimizar os níveis cumulativos de danos à pele. Os raios ultravioletas (UV), ao alcançarem o tecido cutâneo, podem causar eritema, inflamação, fotoenvelhecimento, formação de rugas e imunossupressão, entre outros, devido à formação de espécies reativas de oxigênio (ERO´s). A formação de ERO´s, como o oxigênio singleto, radical ânion superóxido, peróxido de hidrogênio e radical hidroxil, elevam o risco dos danos foto-oxidativos. O desequilíbrio entre a formação de ERO´s e os mecanismos antioxidantes do organismo desencadeia o estresse oxidativo. Na pele, as ERO´s são as responsáveis pelo dano oxidativo no DNA, proteínas e lipídeos. Identificar e quantificar biomarcadores do estresse oxidativo cutâneo é essencial para a correlação entre os raios UV e seus efeitos. Deve-se isto, em parte, à limitação de métodos para quantificar os parâmetros que são diretamente afetados pela exposição aos raios UV, tais como a peroxidação lipídica. São necessários métodos complementares para avaliação da eficácia de fotoprotetores perante os danos causados por este tipo de estresse. Esta pesquisa projeto compreendeu a avaliação ex vivo da eficácia de filtros solares UVB por meio da quantificação da peroxidação lipídica proveniente do estrato córneo removido por tape stripping. Foram preparados sistemas emulsionados do tipo O/A com os filtros octocrileno, metoxicinamato de octila e salicilato de octila. A caracterização funcional da eficácia fotoprotetora in vitro demonstrou que o filtro octocrileno manteve-se estável, mesmo após exposição solar artificial. Os filtros octocrileno (10% p/p), metoxicinamato de octila (10% p/p) e salicilato de octila (5% p/p) alcançaram, após irradiância, respectivamente, os valores de FPS 5,7 &#177; 2,1; 4,7 &#177; 1,5 e 1,0&#177; 0,0. As formulações foram utilizadas na avaliação da eficácia fotoprotetora ex vivo. O método por CLAE, para quantificação da peroxidação lipídica no estrato córneo, possuiu linearidade e demonstrou exatidão e precisão satisfatórias. O estresse pela radiação UV desencadeou a peroxidação lipídica no estrato córneo. Em função do protocolo aplicado, não houve diferenças entre as amostras. A eficácia, com relação à inibição da peroxidação lipídica, foi similar em todas as amostras. / The increasing of incidence of skin cancer is associated with greater exposure to sunlight and the adoption of sun protection actions is a strategy to minimize cumulative levels of skin damage. Ultraviolet (UV) rays, when they reach the cutaneous tissue, can cause erythema, inflammation, photoaging, wrinkling and immunosuppression, among other things, due to the formation of reactive oxygen species (ROS). The formation of ROS, such as singlet oxygen, superoxide anion radical, hydrogen peroxide and hydroxyl radical, raise the risk of photooxidative damage. The variation between the formation of ROS and the antioxidant mechanisms of the organism triggers oxidative stress. In the skin, ROS are responsible for oxidative damage in DNA, proteins and lipids. Identifying and quantifying biomarkers of cutaneous oxidative stress is essential for the correlation between UV rays and their effects. This is partially due to the limitation of methods for quantifying parameters that are directly affected by exposure to UV rays, such as lipid peroxidation. Complementary methods are needed to evaluate the effectiveness of photoprotectors because of the damage caused by this type of stress. This research project had the ex vivo evaluation of the efficacy of UVB sunscreens by quantifying the lipid peroxidation from the stratum corneum removed by tape stripping. Emulsified O/A type systems were prepared with the octocrylene, octyl methoxycinnamate and octyl salicylate filters. The functional characterization of photoprotective efficacy in vitro revealed that the octocrylene filter remained stable even after artificial sun exposure. Octocrylene (10% w / w), octyl methoxycinnamate (10% w / w) and octyl salicylate (5% w / w) respectively reached the values of FPS 5.7 &#177; 2.1; 4.7 &#177; 1.5 and 1.0 &#177; 0.0. The formulations were used in the evaluation of ex vivo photoprotective efficacy. The method by HPLC, for quantification of the lipid peroxidation in the stratum corneum, had linearity and demonstrated satisfactory accuracy and precision. UV radiation stress triggered lipid peroxidation in the stratum corneum. Due to the protocol applied, there were no differences between the samples. The efficacy, compared to the inhibition of lipid peroxidation, was similar in all samples.