Numerous diseases are associated with reduced antioxidant defence and oxidative stress. The antioxidant defence includes dietary and endogenous antioxidants and involves complex interactions between them. The effects of dietary factors on antioxidant status and oxidative stress of healthy humans were investigated in the studies described in this thesis. Assays of plasma antioxidant capacity encompass interactions between various antioxidants. Although uric acid has an unclear function as an antioxidant, it is a major determinant of antioxidant capacity. We measured antioxidant capacity in the presence and absence of uric acid to provide more information on the application of measures of antioxidant capacity. Individuals with high dietary intakes of various antioxidants and antioxidant rich foods, especially when combined, had higher plasma antioxidant capacities than those with lower antioxidant intakes. However, there were no associations between dietary intake of antioxidants or antioxidant rich foods and the plasma concentration of F2-isoprostanes, which is considered a reliable biomarker for oxidative stress. Intakes of various doses of a mixture of bilberry juice and black tea, rich in flavonoids for four weeks, increased antioxidant capacity in some groups, but urine levels of F2-isoprostanes were not affected. There were substantial individual variations in responses to the drinks related to baseline antioxidant capacity. Supplementation with eicosapentaenoic acid and docosahexaenoic acid decreased the plasma levels of F2-isoprostanes, but not prostaglandin F2α formation or antioxidant capacity. It was concluded that a high intake of foods rich in antioxidants is related to improved antioxidant status. After intake of foods rich in antioxidants, the antioxidant status may increase, but with considerable individual variation in the responses, which warrants further investigation. Lipid peroxidation in vivo is not easily affected by dietary antioxidants in healthy humans. Although n-3 fatty acids are highly unsaturated, they reduce nonenzymatic free radical-catalyzed lipid peroxidation, but not enzymatic lipid peroxidation.

Exposure of cells to hydrogen peroxide can cause oxidative damage to cellular constituents including lipids, protein, and DNA. At elevated concentrations, hydrogen peroxide can trigger cell death by apoptosis or necrosis. Apoptotic cell death can be prevented by overexpression of the oncoprotein Bcl-2. The exact mechanism by which Bcl-2 blocks cell death is controversial. Some researchers believe that Bcl-2 possesses antioxidant properties that protect cells from apoptosis. The purpose of this thesis was to assess oxidative stress and apoptosis following hydrogen peroxide exposure in Jurkat T cells overexpressing Bcl-2. One of the major objectives was to ascertain whether or not Bcl-2 overexpression elevated the antioxidant capacity of Jurkat T cells to provide protection from oxidant-induced cell death. Hydrogen peroxide treated Jurkat cells became apoptotic at moderate levels of oxidant (25-100 uM H2O2), and necrotic at higher doses (greater than 200 uM H2O2). Bcl-2 overexpression prevented caspase activation and cell death at the apoptotic doses of H2O2, but not the necrotic doses. Caspase inhibition studies demonstrated that Bcl-2 overexpression provided a greater level of resistance from H2O2-induced cell death than the broad-spectrum caspase inhibitor z-VAD.fmk. A systematic study was carried out examining the antioxidant status of Jurkat cells overexpressing Bcl-2. Several Bcl-2 transfectants were utilised for the study, so that any differences seen could be correlated to the level of Bcl-2 expression. Surprisingly, there were no statistically significant differences among the Bcl-2 transfectants for any of the antioxidant enzymes. Jurkat cells overexpressing Bcl-2 exhibited the same level of oxidative damage to lipids and protein in response to H2O2 exposure as the parental Jurkat cells. Interestingly, Jurkat cells overexpressing Bcl-2 continued to grow in culture after H2O2 exposure, despite harboring damage to cellular constituents. Consistent with these results, H2O2 treated Jurkat cells overexpressing Bcl-2, which failed to undergo apoptosis, were more prone to genomic instability. Together, these findings suggest that Bcl-2 overexpression protects Jurkat cells from H2O2-induced cell death by blocking apoptosis. Jurkat cells overexpressing Bcl-2 were no better at detoxifying oxidants and showed the same level of oxidative damage following H2O2 exposure. As a result, the overexpression of Bcl-2 considerably enhanced the mutagenicity of H2O2.

NF-E2-related factor 2 (Nrf2) is a transcription factor that is responsible for maintaining cellular homeostasis by controlling the fate of cells through transcriptional upregulation of antioxidant response element-bearing genes critical for eliminating toxicants and carcinogens. Under quiescent conditions, basal levels of Nrf2 are relatively low due to tight regulation by Keap1, a substrate adaptor protein for a Cullin 3 (Cul3)-E3 ubiquitin ligase complex that facilitates the ubiquitination and degradation of Nrf2. It is thought that when cells are exposed to oxidative stress, naturally-occurring compounds, or synthetic chemicals, cysteine residues in Keap1, particularly cysteine 151 (C151), are modified causing a conformational change that compromises the ability of the Keap1-Cul3-E3 ubiquitin ligase complex to properly ubiquitinate Nrf2. It is then stabilized and allowed to translocate into the nucleus to transcriptionally activate downstream genes. Interestingly, recent emerging data has revealed the "dark side" of Nrf2. Epigentic alterations and somatic mutations in either Nrf2 or Keap1 disrupting the Nrf2-Keap1 axis and causing constitutive activation of Nrf2 have been found in many human cancer cell lines and tumors. Thus, Nrf2 provides mutated cells a protective advantage against cytotoxic chemotherapeutics, allowing for further cell survival and growth. It is well known that arsenic is a human carcinogen and can activate the Nrf2 pathway through a Keap1-C151 independent mechanism. It has also been shown that arsenic can activate autophagy, a bulk-lysosomal degradation pathway. In this dissertation, we establish the cross-talk between the Nrf2-Keap1 pathway and autophagy by elucidating a novel non-canonical mechanism of Nrf2 activation. We found that deregulation of autophagy causes accumulation of p62, a substrate adaptor protein, which sequesters Keap1 into autophagosomes and activates the Nrf2 pathway. Moreover, we also demonstrate how arsenic blocks autophagic flux and prolongs Nrf2 activation through this novel mechanism. Additionally, activation of the Nrf2 pathway has been shown to confer protection against arsenic-induced toxicity and carcinogenicity. We demonstrate that co-treatment with sulforaphane alleviates arsenic-mediated autophagy. These studies suggest that the Keap1-C151 dependent mechanism triggers the chemopreventive role of Nrf2 while activation through p62 elicits the dark side. Therefore, the use of Keap1-C151-dependent compounds to counteract environmental insults continuous to be a promising strategy for cancer prevention.

Motivation: Metabolic syndrome is a constellation of risk factors predisposing to coronary heart disease (CHD) and is classified as a "disease of modern civilization". Characteristics of the metabolic syndrome include abdominal obesity, increased triacylglycerol (TG) concentrations, increased small dense low-density lipoprotein(LDL) particles, decreased high-density lipoprotein cholesterol (HDL-C), hypertension, insulin resistance, inflammation, glucose intolerance and/or type 2 diabetes mellitus. Subjects with metabolic syndrome may be susceptible to oxidative stress due to their prolonged exposure to elevated glucose levels. A variety of natural antioxidants exists (e.g. glutathione, l3-carotene, vitamin C, polyphenols) that may prevent oxidative damage to biological structures. Nuts are rich sources of unsaturated fatty acids, protein, fibre, .micronutrients, phytochemicals and antioxidants. Duet o their high antioxidant content, it can, therefore, be speculated that nuts may play a role in the prevention of oxidative stress in subjects with the metabolic syndrome. Objective: - To investigate the effect of a high walnut and a high unsalted cashew nut diet on the antioxidant status of subjects with metabolic syndrome. Methods: Sixty eight subjects with diagnosed metabolic syndrome (according to the ATP III criteria) were recruited to take part in this parallel, randomized, controlled feeding trial. Subjects were mainly recruited from the North-West University, Potchefstroom Campus and surrounding areas. After a run-in period of three weeks during which the participants followed a prudent diet, subjects were randomly divided into three groups receiving either walnuts or cashew nuts (63- 108g/day)as part of a prudent diet, or continued with the prudent control diet. The intervention was followed for eight weeks. Fasting blood samples were taken at the beginning(after the three week run-in period) and at the end of the intervention. Antioxidant variables including oxygen radical absorbance capacity (ORAC), reduced glutathione (GSH)/oxidized glutathione (GSSG), diacron reactive oxygen metabolites (dRom) were measured at the beginning and the end of the intervention. C-reactive protein (CRP), fibrinogen and plasminogen activator-inhibitor activity (PAI-1a) were also measured as markers of inflammation. The antioxidant capacity and the polyphenol content of the diets and the walnuts and cashew nuts were determined at the end of the intervention. Results: A significant decrease in dRom and significant increases in GSSG, the redox status of glutathione (GSH/GSSG) and ORAC were observed in all three groups from baseline to end. GSH remained unchanged from baseline to end in all three groups. No significant differences in changes in dRom (p = 0.92), GSSG (p = 0.99), GSH/GSSG (p = 0.86), antioxidant capacity (p = 0.10) and GSH (p = 0.34) were observed from baseline to end between groups. The total polyphenol content of the walnut and control diets were similar and significantly higher than the cashew nut diet. The antioxidant capacity of the walnut and cashew nut diets showed a tendency to be higher than the control diet (p = 0.07 and p = 0.06 respectively). CRP, fibrinogen and PAI-1a concentrations did not differ significantly between groups. Conclusion No significant differences between the groups receiving walnuts, cashew nuts or no nuts were observed in GSH, GSSG, GSH/GSSG, dRom or ORAC. Therefore, there seems to be no beneficial effect of the inclusion of walnuts and cashew nuts in the diet on the antioxidant status of the participants. / Thesis (M.Sc. (Dietetics))--North-West University, Potchefstroom Campus, 2006.

ABSTRACT ASSOCIATION OF ANTIOXIDANT INTAKE AND BODY MASS INDEX IN PRE-TO-EARLY ADOLESCENT CHILDREN by E. Kelly Imboden Background: The prevalence of overweight (Body Mass Index [BMI]85-<95th percentile) and obesity (BMI>95thpercentile) for individuals aged 2-19 years in the United States in 2009-2010 was estimated to be 31.8%. Excessive body fat increases the risk for chronic conditions such as hypertension and type 2 diabetes mellitus. Studies have established an association between obesity and oxidative stress and inflammation in children and adolescents. Antioxidants have been shown to have protective effects against inflammation and oxidative stress. However, the effect of dietary antioxidant intake on obesity is not fully understood. Objective: To examine dietary antioxidant intake by BMI classification in a population of normal, overweight and obese children. Methods: The study population included 296 healthy pre-to-early adolescent (age 6-15 years) African American and Caucasian children residing in Pittsburgh, PA. Demographic characteristics, anthropometric measures and nutrient intake were assessed at baseline and six months. A food frequency questionnaire was used to assess antioxidant intake (vitamin C, carotene, total vitamin A, zinc and vitamin E). Frequency analysis was used to describe demographic, anthropometric and nutrient data. The Kruskal Wallis test was used to evaluate difference in median antioxidant and kilocalorie intake by BMI classification at baseline. A Kendall’s tau correlation was performed to test for a linear relationship between BMI and antioxidant intake at baseline. Results: The median age of the population was 10 years (range, 8 to 11 years). The majority of the population was male (53%) and African American (60%). Weight and BMI (p = 0.028 and 0.000, respectively) were the only demographic and anthropometric characteristics that differed by gender. For the total cohort, median nutrient intake by BMI classification was significantly different for vitamin C (p = 0.015), zinc (p = 0.019), vitamin E (p = 0.022) and kilocalories (p = 0.015). When divided by gender, zinc intake in males (p = 0.047) and kilocalorie intake in females (p = 0.017) were the only nutrients found to be statistically different by weight classification. No linear relationship was observed between antioxidant intake and BMI for the total cohort and for each gender. Conclusion: Our results do not support a linear relationship between antioxidant intake and BMI. In contrast to our hypothesis, antioxidant intake was found to be highest in children who were overweight. Future studies should include a serum measure of inflammation and antioxidant levels in addition to antioxidant intake to better understand the impact, if any, of antioxidants in overweight and obese children and adolescents.

Oxidative stress is a feature of many chronic neurodegenerative diseases as well as a contributing factor in acute disorders including stroke. Fork head class of transcription factors (Foxos) play a key role in promoting oxidative stress-induced apoptosis in neurons through the upregulation of a number of pro-apoptotic genes. Here I demonstrate that synaptic NMDA receptor activity not only promotes Foxos nuclear exclusion but also suppresses the expression of Foxo1 in a PI3K-dependent fashion. I also found that Foxo1 is in fact, a Foxo target gene and that it is subject to a feed-forward inhibition by synaptic activity, which is thought to result in longerterm suppression of Foxo downstream gene expression than previously thought. The nuclear factor (erythroid 2-related) factor 2 (Nrf2) is another transcription factor involved in oxidative stress and the key regulator of many genes, whose products form important intrinsic antioxidant systems. In the CNS, artificial activation of Nrf2 in astrocytes has been shown to protect nearby neurons from oxidative insults. However, the extent to which Nrf2 in astrocytes could respond to endogenous signals such as mild oxidative stress is less clear. The data presented herein, demonstrate for the first time that endogenous Nrf2 could be activated by mild oxidative stress and that this activation is restricted to astrocytes. Contrary to the established dogma, I found that mild oxidative stress induces the astrocytic Nrf2 pathway in a manner distinct from the classical Keap1 antagonism employed by prototypical Nrf2 inducers. The mechanism was found to involve direct regulation of Nrf2's transactivation properties. Overall these results advance our knowledge of the molecular mechanism(s) associated with the control of endogenous antioxidant defences by physiological signals.

Oxidative stress is caused when there are more reactive oxygen species (ROS), than antioxidants to scavenge them, resulting in damage to cellular components. It has been implicated as a major player at multiple points in the disease process of Parkinson’s disease (PD) and many other conditions. For example, evidence suggests oxidative damage to the α-synuclein protein may affect its aggregation propensity. In addition, α-synuclein may increase ROS production. However, how this oxidative stress relates to neurodegeneration is not known. Therefore, there is a need for models of α-synucleinopathies and tools to assess the involvement of oxidative stress in the disease process. In order to model α-synucleinopathies, overexpression of the α-synuclein protein was used. A BacMam viral expression system containing human α-synuclein was generated and used to assess toxicity. α-Synuclein overexpression in undifferentiated or differentiated SH-SY5Y cells failed to show toxicity. However, the stability of α-synuclein protein expression and the cell line used may have influenced in the lack of toxicity. The current work provides important guidance for future experimental design. Flavonoids are found in plants and have antioxidant capability. AO-1-530 is a synthetic compound with a flavonoid head group and a long hydrocarbon tail. It is highly cell permeable and localises to the mitochondria. In order to investigate its protective properties, toxin-induced oxidative stress cell assays were established. AO-1-530, in the low micromolar range, was protective against high doses of tert-butyl hydroperoxide (tBHP), whereas natural antioxidants, such as myricetin and quercetin, showed limited protection or required at least 10-fold higher concentrations to achieve similar protection. The ability of AO-1-530 to directly scavenge radicals was assessed cell-free in solution and in a cell-based assay. In solution the mechanism of action was investigated by electron paramagnetic resonance (EPR) spectroscopy. AO-1-530 had similar scavenging ability to myricetin, but was a slightly stronger scavenger than quercetin. The intracellular scavenging ability was quantified by CellROX® Deep Red live imaging. Although the compounds had similar cell-free scavenging abilities, AO-1-530 significantly out-performed both myricetin and quercetin in the intracellular assay, suggesting the mitochondrial localisation is critical to its highly protective properties. AO-1-530 is a powerful, novel tool to study the involvement of oxidative stress in diverse disease models.

Master of Science / Food Science / J. Scott Smith / The objective of this study was to observe the effect of natural cocoa powder versus 200 ppm of tocopherols on delaying the onset of oxidative rancidity in peanuts, peanut butter, and peanut oil. The samples were obtained from a single lot of blended Runner peanuts after roasting, grinding, and pressing. The samples were treated within a week of initial roasting with either 200 ppm of mixed tocopherols or 2.5% cocoa powder. The development of oxidation was monitored by peroxide value (PV) and gas chromatography monitoring of hexanal development. The peanut butter samples were assessed by a professional sensory panel using descriptive analysis for the development of rancidity. The data was analyzed using JMP SAS software. In peanuts, the cocoa powder sample developed significantly lower levels of oxidation identifiers than the tocopherol or control samples. In peanut oil, there was no significant difference in levels of oxidation identifiers between the treatments. In peanut butter, the PV was significantly higher in the tocopherol sample than the cocoa powder or control samples, but no significant difference was observed in hexanal. The results of the sensory analysis indicated that the cocoa powder depressed the perception of both positive and negative attributes compared to the tocopherol and control samples. This study showed that cocoa powder may be a more effective preservative than an untreated sample or a sample treated with 200 ppm of tocopherols in peanuts and peanut butter; however, cocoa powder at 2.5% w/w basis did not perform as a significant antioxidant in peanut oil.

Inhibition of protein synthesis serves as a general measure of cellular consequences of chemical stress. A few proteins are translated selectively and influence cell fate. How these proteins can bypass the general control of translation remains unknown. We found that low to mild doses of oxidants induce de novo translation of the NRF2 protein. Here we demonstrate the presence of a G-quadruplex structure in the 5' untranslated region (UTR) of NRF2 mRNA, as measured by circular dichroism, nuclear magnetic resonance, and dimethylsulfate footprinting analyses. Such a structure is important for 5'-UTR activity, since its removal by sequence mutation eliminated H2O2-induced activation of the NRF2 5' UTR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics revealed elongation factor 1 alpha (EF1a) as a protein binding to the G-quadruplex sequence. Cells responded to H2O2 treatment by increasing the EF1a protein association with NRF2 mRNA, as measured by RNA-protein interaction assays. The EF1a interaction with small and large subunits of ribosomes did not appear to change due to H2O2 treatment, nor did post translational modifications, as measured by two-dimensional (2-D) Western blot analysis. Since NRF2 encodes a transcription factor essential for protection against tissue injury, our data have revealed a novel mechanism of cellular defense involving de novo NRF2 protein translation governed by the EF1a interaction with the G-quadruplex in the NRF2 5' UTR during oxidative stress.

>Magister Scientiae - MSc / Maize is one of the most abundantly produced cereals and contributes to about 40% of the global cereal production. This figure will have to increase in order to feed the ever-growing human population. One of the major environmental constraints that impact maize production is drought. Plants use antioxidant defences to cope with drought stress. Understanding and improving these defence mechanisms will be important to improve overall drought tolerance. A previous study done by Gokul and authors in 2016 showed that 3,3’-diindolylmethane (DIM) improves both seed germination and seedling shoot growth in Brassica napus. Plants belonging to the Brassicaceae family have the metabolic machinery to synthesize glucosinolates such as DIM, which play vital roles in physiological and stress responses. These responses have not been investigated in plants such as maize, which lack the machinery to produce DIM. Therefore, this study investigated the effects of exogenously applied DIM on the physiological and biochemical responses of maize under drought stress. Physiological parameters such as relative water content, chlorophyll content and lipid peroxidation, were determined in order to understand how drought and DIM , as separate or combined treatments, affected the plants. Additionally, proline accumulation was also assessed because free proline plays a role as an osmoprotectant during stress. The accumulation of ROS, namely hydrogen peroxide, was measured using spectrophotometric assays to determine how the above treatments affect ROS accumulation in maize. As a result of changes in the ROS content in due to the treatments, it would only be natural to investigate the changes in antioxidants as well. Given that hydrogen peroxide was the ROS to be measured, we therefore investigated the antioxidant enzymatic activities responsible for hydrogen peroxide scavenging. Therefore, changes in Ascorbate peroxidase (APX) and catalase (CAT) were assessed. An improved drought response was observed in maize plants treated with DIM as these plants had better ability to maintain their water status than when no DIM was applied. This is indicated by water-deprived plants treated with DIM having a higher RWC than water-deprived plant without DIM.