Free radicals have been implicated in the progression of various diseases, such as cancers and cardiomyopathies. When the body is overburdened with free radicals and endogenous antioxidants become depleted, oxidative stress ensues with resultant damage to biomolecules. During oxidative stress high levels of reactive oxygen species are generated, cellular viability decreases, and apoptosis and lipid peroxidation are induced. Supplementation with exogenous supplements rich in antioxidants, such as herbal remedies containing polyphenols, could result in increased protection against oxidative stress. The aim of the study was to assess the effect of Burkea africana and Syzygium cordatum in a cellular oxidative stress model for the potential development of an antioxidant supplement. Crude aqueous and methanolic extracts were prepared by solvent maceration, while a polyphenolic-rich extract was created through liquid-liquid extraction. Polyphenolic content and antioxidant activity was assessed in cell-free systems. Polyphenolic content was determined through the Folin-Ciocalteau and aluminium trichloride methods, while antioxidant activity was assessed by the Trolox Equivalence Antioxidant Capacity and 1,1-diphenyl-2-picrylhydrazyl radical assays. Identification of phytochemical classes was done through thin layer chromatography and biochemical reactions. Inherent cytotoxicity of samples was determined in four cell cultures (3T3-L1 pre-adipocytes, C2C12 myoblasts, normal human dermal fibroblasts and U937 macrophage-like cells) using the neutral red uptake assay. The effect on oxidative stress was assessed in 2,2`-azobis-(2-methylpropionamidine) dihydrochloride-exposed U937 macrophage-like cells with regards to reactive oxygen species generation, cytotoxicity, apoptosis, lipid peroxidation and GSH depletion. Both B. africana and S. cordatum showed enrichment of polyphenols from the aqueous extract, to methanolic extract, to polyphenolic-rich extract. Antioxidant activity showed the same trend, which correlated well with the increased concentration of polyphenols, such as catechin, gallic acid and myricetin. Samples indicated toxicity in the 3T3-L1 and C2C12 cell lines, though no toxicity was noted in the U937 cell line and normal human dermal fibroblast cultures. Free radical-induced generation of reactive oxygen species, cytotoxicity, lipid peroxidation and apoptosis was successfully reduced by crude extracts of B. africana and the polyphenolic-rich extracts of both plants between concentrations of 10 and 20 ìg/ml. The crude extracts of S. cordatum were mostly ineffective in reducing these parameters, even though cell viability was increased. B. africana pre-treatment decreased reduced glutathione concentrations significantly in a dose-dependent manner, while the methanolic and polyphenolic-rich extract of S. cordatum increased concentrations moderately. Polyphenolic-rich extracts of B. africana and S. cordatum had the most potent decrease in oxidative stress-related parameters in the present study, which could be attributed to the polyphenolic content and antioxidant activity. Limited cytotoxicity was apparent in two of the four cell lines tested; further isolation and purification needs to be carried out to assess the bioactive constituents which do not elicit a toxic response. Further investigation through the use of quantitative structure–activity relationship modeling could give more insight on conformational and chemical changes that need to be brought about to modify the bioactive phytochemicals for reduced cytotoxicity, but increased antioxidant activity. Copyright / Dissertation (MSc)--University of Pretoria, 2013. / Pharmacology / unrestricted

O objetivo deste trabalho foi investigar o efeito da castração, tempo de confinamento, metabolismo celular e regiões do musculo sobre a oxidação proteica e lipídica e outras características de qualidade da carne bovina. Oitenta e quatro bovinos (castrados e inteiros) Nelore, confinados por diferentes períodos, foram usados para conduzir estudos em músculos Longissimus dorsi e Biceps femoris. O segundo musculo foi dividido em duas porções: origem (PO) e inserção (PI). No estudo com L. dorsi, bifes foram embalados sob condições de aerobiose (PVC) e anaerobiose (vácuo) e maturados por 1, 3, 5, 7 e 9, e 1, 7, 14 e 21 dias, respectivamente. Para este músculo, nenhuma diferença na estabilidade oxidativa [tióis, carbonilas e Substancias Reativas ao Ácido Tiobarbiturico (TBARS)] e cor entre as carnes dos animais inteiros e castrados foram encontradas. Isto poderia ser explicado pela falta de diferença no status oxidativo inicial, mensurados através da atividade de enzimas antioxidantes, conteúdo de glutationa total e composição de ácidos graxos, entre as condições sexuais. Os resultados também indicaram que a oxidação dos bifes embalados a vácuo leva o dobro de dias para iniciar, em comparação aos bifes em aerobiose. No estudo com o Bíceps femoris, os animais foram abatidos com 59 e 129 dias de confinamento e os bifes da PO e PI foram maturados por 1, 30, 60 e 100 dias. Os resultados da atividade das enzimas lactato desidrogenase e citrato sintase mostraram que a PO tem metabolismo mais oxidativo (aeróbio) e a PI glicolítico (anaeróbio). A carne dos animais inteiros tiveram menor TBARS e maior luminosidade (L*), perda de peso por cocção (PPC) e força de cisalhamento (FC) em comparação aos animais castrados. A PO foi mais susceptível a oxidação proteica (menor tióis) em comparação a PI. A carne dos animais confinados por 129 dias tiveram maiores PPC e oxidação proteica (menores tióis) em comparação a carne dos animais confinados por 59 dias. Diferenças de estabilidade oxidativa entre a carne de animais castrados e inteiros confinados por menor período desapareceram quando os animais foram confinados por maior período. Valores de pH e tióis na carne dos animais castrados e inteiros foram afetados pelo tempo de maturação. Ambas as condições sexuais tiveram carne com maior valores de pH no dia 30 de maturação e este, se manteve ao longo do tempo. A PO teve maiores valores de TBARS no dia 60, PPC no dia 100 e FC nos dias 30 e 60 de maturação em comparação a PI. Foi observada uma interação entre tempo de confinamento e tempo de maturação para tióis, TBARS, metamioglobina, pH, L* e FC. Quando comparado aos animais confinados por 59 dias, os animais confinados por 129 dias tiveram: maior oxidação (maior TBARS e menor tióis) nos dias 60 e 100 de maturação; oxidação da mioglobina (metamioglobina) mais tardia, sendo o maior valor obtido no dia 100; menor luminosidade (L*) em todos os tempos de maturação; maior maciez (menor FC) aos 100 dias de maturação. Os animais confinados por 59 dias tiveram: maior oxidação proteica (menor tióis) e maciez (menor FC) aos 30 dias de maturação. De forma geral, todos os efeitos testados tais como castração, tempo de confinamento, metabolismo celular e regiões do musculo pareceram influenciar sobre a oxidação proteica e lipídica e outras características de qualidade da carne bovina. / The objective of this work was to investigate the effect of the castration, time on confinement, cellular metabolism and muscle region on the protein and lipid oxidation, and other traits of beef quality. Eight-four Nellore cattle (steers and bulls), confined for different periods, were used to conduct studies in Longissimus dorsi and Biceps femoris muscles. The latter muscle was divided in two portions: origin (OP) and insertion (IP). In the study of L. dorsi muscle, steaks were packaged under aerobiosis (PVC) and anaerobiosis (vacuum) conditions and aged for 1, 3, 5, 7 and 9, and 1, 7, 14 and 21 days, respectively. For this muscle, no differences in oxidative stability [thiols, carbonyls and Thiobarbituric Acid Reactive Substances (TBARS)] and color between the meat from bulls and steers were found. This could be explained by the lack of differences in initial oxidative status, measured through the activity of the antioxidants enzymes, content of total glutathione and composition of fatty acids, between the sexual conditions. The results also indicated that the oxidation of the steaks vacuum-packaged took about twice more days to start than the steaks under aerobiosis. In the study of Biceps femoris muscle, the animals were slaughtered after 59 and 129 days on confinement and the steaks from OP and IP were aged for 1, 30, 60 and 100 days. The results of the lactate dehydrogenase and citrate synthase enzymes activity showed that the OP has a more oxidative metabolism and the IP has a more glycolytic metabolism. The meat from bulls had lower TBARS and higher lightness (L*), cooking loss (CL) and shear force (SF) in comparison with steers. The OP was more susceptible to protein oxidation (lower thiols) than the IP. Animals confined for 129 days had meat with higher CL when compared to those ones confined for 59 days. The meat from animals confined for 129 days had higher CL and protein oxidation (lower thiols) in regard to the meat from animals confined for 59 days. Differences in oxidative stability between the meat from steers and bulls confined for shorter period disappeared when the animals were confined for larger period. Values of pH and thiols in meat from steers and bulls were affected by the time of aging. Both the sexual conditions had meat with higher pH values at the day 30 of aging and this was kept across the time. The OP had higher values of TBARS at the day 60, CL at the day 100 and SF at the days 30 and 60 of aging when compared to the IP. It was observed an interaction between confinement time and aging time for thiols, TBARS, metmyoglobin, pH, L* and SF. When compared to the animals confined for 59 days, the animals confined for 129 days had: higher oxidation (higher TBARS and lower thiols) at the days 60 and 100 of aging; oxidation of myoglobin (metmyoglobin) slower, since the higher value was obtained at the day 100; lower lightness (L*) in all the times of aging; tender meat (lower SF) at 100 days of aging. The animals confined for 59 days had: higher protein oxidation (lower thiols) and tender meat (lower SF) at 30 days of aging. Overall, all the effects tested such as castration, time on confinement, cellular metabolism and muscle region seemed to influence on the protein and lipid oxidation and other traits of beef quality.

The effect of coffee and cocoa on oxidative damage to macromolecules has been investigated in several studies, often with controversial results. This study aimed to investigate the effect of one-month consumption of different doses of coffee or cocoa-based products containing coffee on markers of DNA damage and lipid peroxidation in young healthy volunteers. Twenty-one volunteers were randomly assigned into a three-arm, crossover, randomized trial. Subjects were assigned to consume one of the three following treatments: one cup of espresso coffee/day (1C), three cups of espresso coffee/day (3C), and one cup of espresso coffee plus two cocoa-based products containing coffee (PC) twice per day for 1 month. At the end of each treatment, blood samples were collected for the analysis of endogenous and H2O2-induced DNA damage and DNA oxidation catabolites, while urines were used for the analysis of oxylipins. On the whole, four DNA catabolites (cyclic guanosine monophosphate (cGMP), 8-OH-2′-deoxy-guanosine, 8-OH-guanine, and 8-NO2-cGMP) were detected in plasma samples following the one-month intervention. No significant modulation of DNA and lipid damage markers was documented among groups, apart from an effect of time for DNA strand breaks and some markers of lipid peroxidation. In conclusion, the consumption of coffee and cocoa-based confectionery containing coffee was apparently not able to affect oxidative stress markers. More studies are encouraged to better explain the findings obtained and to understand the impact of different dosages of these products on specific target groups.

Free radical driven lipid peroxidation is a chain reaction which can lead to oxidative degradation of biological membranes. Propagation vs. termination rates of peroxidation in biological membranes are determined by a variety of factors including fatty acyl chain composition, presence of antioxidants, as well as biophysical properties of mono- or bilayers. Sphingomyelins (SMs), a class of sphingophospholipids, were previously described to inhibit lipid oxidation most probably via the formation of H-bond network within membranes. To address the “antioxidant” potential of SMs, we performed LC-MS/MS analysis of model SM/glycerophosphatidylcholine (PC) liposomes with different SM fraction after induction of radical driven lipid peroxidation. Increasing SM fraction led to a strong suppression of lipid peroxidation. Electrochemical oxidation of non-liposomal SMs eliminated the observed effect, indicating the importance of membrane structure for inhibition of peroxidation propagation. High resolution MS analysis of lipid peroxidation products (LPPs) observed in in vitro oxidized SM/PC liposomes allowed to identify and relatively quantify SM- and PC-derived LPPs. Moreover, mapping quantified LPPs to the known pathways of lipid peroxidation allowed to demonstrate significant decrease in mono-hydroxy(epoxy) LPPs relative to mono-keto derivatives in SM-rich liposomes. The results presented here illustrate an important property of SMs in biological membranes, acting as “biophysical antioxidant”. Furthermore, a ratio between mono-keto/mono-hydroxy(epoxy) oxidized species can be used as a marker of lipid peroxidation propagation in the presence of different antioxidants.

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L'acide docosahexaénoïque (DHA, C22 :6 w3), l'acide icosapentaénoïque (EPA, C20 : 5, w3) et l'acide adrénique (AdA, C22 :4 w6) sont présents en quantités importantes dans les membranes neuronales. Lors d'un stress oxydant, l'oxydation radicalaire de ces acides gras polyinsaturés conduit à la formation de métabolites nommés neuroprostanes (NeuroPs). Souhaitant avoir accès à de nouveaux biomarqueurs du stress oxydant neuronal, nous sommes intéréssés à la synthèse de NeuroPs de type F, issues du DHA, de l'EPA et de l'AdA. / Docosahexaenoïc acid (DHA, C22 :6 w3), eicosapentaenoïc acid (EPA, C20 : 5, w3) and adrenic acid (AdA, C22 :4 w6) are the major polyunstaurated acids in neuronal membrane. During an oxidative stress, their lipidic peroxidation led to oxygenated metabolites called neuroprostanes (NeuroPs). In order to access to new neuronal oxidtive stress biomarkers, we were interested in the syntheses of F-type NeuroPs derived from DHA, EPA and AdA.

Introduction : Les enfants prématurés ont la particularité de naître alors que leur développement est souvent incomplet et nécessite la mise en œuvre de soins intensifs visant à poursuivre leur croissance en dehors de l’environnement utérin. Souvent cependant, le stade développemental de l’enfant ne lui permet pas d’assimiler une alimentation entérale du fait de l’immaturité de son système digestif. Le recours à une voie centrale délivrant les nutriments assurant le développement devient alors une nécessité. Ce type de nutrition, appelée nutrition parentérale (NP, ou total parenteral nutrition TPN), permet l’administration de molécules simples, directement dans le sang du prématuré. Il n’est toutefois pas exempt de risques puisqu’exposée à la lumière, la NP peut s’oxyder et générer des molécules oxydantes telles que des hydroperoxydes lipidiques susceptibles de se fragmenter par la suite en hydroxy-alkénals. Ceci devient problématique au vu de l’immaturité des systèmes de défenses antioxydants du nouveau-né prématuré. L’utilisation prolongée de la NP est d’ailleurs à l’origine de maladie hépatiques dans lesquelles le stress oxydant et la nécro-inflammation sont des composantes majeures. Nous avons émis l’hypothèse que l’infusion chez les enfants prématurés, d’aldéhydes d’origine lipidique est en relation avec le développement du stress oxydant et de l’inflammation hépatique. Objectif : Notre étude a consisté à évaluer la relation entre les quantités d’hydroxy-alkénals dans la NP et les effets hépatiques engendrés sur les marqueurs de stress oxydant et les voies de signalisation responsables d’une induction de processus inflammatoire. Dans ce but, nous avons cherché à mesurer la peroxydation lipidique dans l’émulsion lipidique de la NP et la conséquence de l’infusion en continue d’hydroxy-alkénals sur les marqueurs de stress oxydant, sur la voie de signalisation médiée par le Nuclear Factor κB et sur le déclenchement du processus inflammatoire hépatique. A la suite de ce travail, nous avons également travaillé sur des alternatives à la photoprotection, qui est la seule méthode réellement optimale pour réduire la peroxydation des lipides de la NP, mais cliniquement difficilement praticable. Résultats : Nos résultats ont mis en évidence la génération de 4-hydroxynonenal in vitro dans la NP, ce phénomène est augmenté par une exposition lumineuse. Dans ce cadre, nous avons montré l’inefficacité de l’ajout de multivitamines dans l’émulsion lipidique comme alternative à la photoprotection. Dans la validation biologique qui a suivi sur un modèle animal, nos résultats ont permis de démontrer que l’augmentation des adduits glutathion-hydroxynonenal était imputable à l’augmentation de 4-hydroxynonenal (4-HNE) dans la NP, et non à une peroxydation endogène. Nos données indiquent que la probable augmentation hépatique des niveaux de 4-HNE a conduit à une activation du NFκB responsable de l’activation de la transcription des gènes pro-inflammatoires du Tumour Necrosis Factor-α (TNF-α) et de l’interleukine-1 (IL-1). Nous avons alors évalué la capacité d’une émulsion lipidique enrichie en acides gras polyinsaturés (AGPI) n-3 à baisser les concentrations de 4-HNE dans la NP, mais également à moduler le stress oxydant et les marqueurs pro-inflammatoires. Enfin, nous avons démontré, en collaboration avec l’équipe du Dr Friel, que certains peptides isolés du lait humain (par un processus mimant la digestion) permettent également une modulation du stress oxydant et du processus inflammatoire. Conclusion : Le stress oxydant exogène issu de la NP a conduit par activation de facteurs de transcription intra-hépatiques au déclenchement d’un processus inflammatoire potentiellement responsable du développement de maladies hépatiques reliées à la NP telle que la cholestase. Dans ce sens, les AGPI n-3 et les peptides antioxydants peuvent se poser en tant qu’alternatives crédibles à la photoprotection. / Introduction: Premature infants usually born before full term require intensive care to continue to grow up outside the uterine environment. Premature newborns are born with gastrointestinal systems that are too immature to absorb nutrients safely. Therefore they receive their initial nutrients through intravenous feeding, called total parenteral nutrition which delivers simple nutrients directly into bloodstream. However, light exposed-TPN can generate oxidant molecules such as lipid hydroperoxides, which can potently break up into hydroxy-alkenals. Prolonged use of TPN is also a cause of liver disease in which oxidative stress and necro-inflammation are major components. Thus, we hypothesize that lipid aldehydes contained in TPN are associated with oxidative stress and hepatic inflammation developments. Objectives: The aim of our study is to assess the relationship between quantities of hydroxyl-alkenals generated in TPN and effects on oxidative stress biomarkers and cell-signalling pathways molecules implicated in hepatic inflammation induction. To this end, we measure lipid peroxidation in the TPN lipid emulsion in and the consequence of continuous infusion of hydroxy-alkenals on markers of oxidative stress, on cell-signaling pathway mediated by the NFkB, and on liver inflammation induction. Following these data, we also worked on alternatives of photoprotection, which is the only optimal method for preventing lipid peroxidation, but unfortunately clinically impractical. Results: In vitro studies have highlighted the generation of 4-HNE in the TPN, increased under light exposure. In this context, we have demonstrated that the addition of multivitamins in the lipid emulsion cannot be a valuable alternative to photoprotection. Concerning the biological validation in our guinea pig animal model, our results demonstrated that the increase of GS-HNE adducts was due to increased 4-HNE in the TPN, and does not provide from endogenous peroxidation. Our data also indicate that the increase of hepatic 4-HNE led to an activation of NFkB, responsible for the activation of the transcription of proinflammatory genes TNF-α, IL-1. In the next study, we have evaluated the ability of a lipid emulsion enriched with n-3 polyunsaturated fatty acids (PUFA) to reduce 4-HNE concentrations generated in TPN, and to modulate oxidative stress markers and pro-inflammatory process on the same animal model. We also have demonstrated, in collaboration with Dr Friel’s team, that two antioxidant peptides (derived from a process mimicking digestion process of human milk) allow also a modulation of oxidative stress and inflammatory process in the liver. Conclusion: This form of exogenous oxidative stress from the TPN led to an inflammatory process resulting from the activation of intrahepatic transcription, which is potentially responsible of liver disease development such as cholestasis. In this sense, the n-3 PUFA and antioxidant peptides may arise as a valuable alternative of photoprotection.

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