The studies described in this dissertation were performed with the goal of understandingthe function of antioxidant compounds delivered in vivo to rodents and the implication of theresults towards oxidative stress (OS)-related neurodegenerative disorders with particularemphasis on Alzheimer's disease (AD). OS has been implicated in AD and is characterized byextensive oxidative damage to protein, lipids and DNA. A major thrust of this dissertation workwas to gain insight into antioxidant properties of compounds used in the following studies andtheir efficacy as potential therapeutics for treatment of OS-related disorders.D609, a glutathione (GSH) mimetic is known to trap OH. Radicals, scavenge H2O2 andreduce the A?? (1-42)-induced OS and cytotoxicity in neurons. The present dissertation studyshowed in vivo protective effect of D609 in synaptosomes and mitochondria isolated fromgerbils against OS mediated by Fe2+/H2O2, AAPH, and A?? (1-42). Upon intraperitonial (i.p.)injection of gerbils, D609 showed protection of subsequently isolated brain moieties against OS.In vivo administration of D609 also modulates brain GSH levels and increases the activity of keyGSH-related enzymes, thereby likely provides a protection against OS.Adriamycin (ADR), a quinone-containing chemotherapeutic, is known to produce ROS inheart. Patients under treatment with ADR often show persistent changes in cognitive function(effect called as chemobrain by patients). Upon i.p. injection, ADR causes OS, increasesexpression of multidrug resistant protein-1 (MRP-1) in brain and alters GSH levels and itsrelated enzyme activities. ??-Glutamyl cysteinyl ethyl ester (GCEE) is known to increase GSHlevels in brain, in vivo. Research reported in this dissertation shows that in vivo GCEE reversesthe ADR-mediated OS in mice brain.N-acetylcysteine (NAC), a GSH precursor provides the limiting substrate cysteine inGSH synthesis. Previously, our laboratory showed increased GSH levels post i.p. injection ofNAC and reduces OS in synaptosomes treated with acrolein. The present study showed thatNAC given in drinking water to APP/PS-1 mice, a model of AD can significantly reduce OS.These results provide a potential therapeutic intervention by antioxidants that can modulateGSH in OS-mediated neurodegenerative disorders.

Pawpaw (Asimina triloba (L.) Dunal) has significant potential as a new fruit crop. During ripening, loss of firmness is extremely rapid, and this trait may be the biggest obstacle to the development of a broader market as handling without injury is difficult. Cold storage of pawpaw seems limited to 4 weeks at 4 C. A study of several cultivars with commercial appeal showed that ripening traits such as ethylene production, respiration and loss of firmness were similar in all genotypes, and that no cultivar showed superior responses to cold storage. Cold storage for longer than 4 weeks caused the development of cold injury symptoms such as black discoloration, rapid loss of firmness, impaired respiration, tissue acidification, decrease in antioxidant content, decrease in volatile ester production and development of off-flavor volatile compounds. Overall cold storage injury symptoms observed in pawpaw may be due to oxidative damage linked to the failure of the two major antioxidant systems that could protect against such damage: phenolics and the ascorbateglutamate system. With the aim of enhancing pawpaw low temperature tolerance and prolonging cold storage length, different techniques such as hot air exposure and hot water dips of fruit prior to beginning cold storage, and intermittent warming periods during cold storage, were evaluated. Despite positive results with these techniques for other commodities, all the strategies failed to appreciably alter fruit ripening, loss of firmness or maintain fruit quality during and/or after cold storage.

The studies presented in this work were completed with the goal ofgaining greater insight into the roles of protein oxidation in brain aging and age-relatedcognitive impairment. Aging is associated with the impairment of physiological systemssuch as the central nervous system (CNS), homeostatic system, immune system, etc.Functional impairments of the CNS is associated with increased susceptibility to developmany neurodegenerative diseases such as Alzheimer's diseases (AD), Parkinson's disease(PD), and amyotrophic lateral sclerosis (ALS). One of the most noticeable functionalimpairments of the CNS is manifested by cognitive decline. In the past three decades, thefree radical theory of aging has gained relatively strong support in this area. Excessiveproduction reactive oxygen species (ROS) was demonstrated as a contributing factor inage-related memory and synaptic plasticity dysfunction. This dissertation use proteomicsto identify the proteins that are oxidatively modified and post-translationally altered inaged brain with cognitive impairment and normal aging brain.Ongoing research is being pursued for development of regime to preventoxidative damage by age-related oxidative stress. Among which are those that scavengefree radicals by antioxidants, i.e. ??-lipoic acid (LA), and protecting the brains byreducing production of neurotoxic substance, i.e. reducing production of amyloid ??(A??).Therefore, proteomics were also used to identify the alteration of specific proteins in agedbrain treated with LA and antisense oligonucleotides again amyloid protein precursor.This dissertation provides evidences that certain proteins are less oxidatively modifiedand post-translationally altered in cognitively impaired aged brain treated with LA andantisense oligonucleotides against the A?? region of amyloid precursor protein (APP)(AO).Together, the studies in this dissertation demonstrated that increased oxidativestress in brain play a significant role in age-related cognitive impairment. Moreover, suchincreased oxidative stress leads to specific protein oxidation in the brain of cognitiveimpaired subject, thereby leading to cognitive function impairment. Moreover, thefunctional alterations of the proteins identified by proteomics in this dissertation mayleads to impaired metabolism, decline antioxidant system, and damaged synapticcommunication. Ultimately, impairment of these processes lead to neuronal damages andcognitive decline. This dissertation also show that several of the up-regulated andoxidized proteins in the brains of normal aging mice identified are known to be oxidizedin neurodegenerative diseases as well, suggesting that the expression levels of certainproteins may increase as a compensatory response to oxidative stress. This compensationwould allow for the maintenance of proper molecular functions in normal aging brainsand protection against neurodegeneration.

The studies presented in this dissertation were conducted with the objective ofgaining greater understanding into the mechanisms of successful aging, the role ofmitochondria dysfunction in traumatic brain injury, and also on the mechanisms ofimproved learning and cognitive function in the aging.Aging is usually characterized by impairments in physiological functionsincreasing its susceptibility to dementia and neurodegenerative disorders. In thisdissertation, the mechanisms of dementia-free aging were investigated. The use of anantioxidant fortified diet and a program of behavioral enrichment in the canine model ofhuman aging was shown to result in a significant decrease in the levels of oxidativestress. A proteomic analysis of these brains also demonstrated a significant decrease inthe oxidative modification of key brain proteins and an increase in the expression levelsof other key brain proteins associated with energy metabolism and antioxidant systemswhich correlated with improved learning and memory.We show that following TBI key mitochondrial-related proteins undergoextensive oxidative modification, possibly contributing to the severe loss ofmitochondrial energetics and neuronal cell death previously observed in experimentalTBI.Taken together, these findings support the role of oxidative stress in thepathophysiology of aging and age-related neurodegenerative disorders and in CNS injury.These studies also show that antioxidants and a program of behavioral enrichmentprovide protection against oxidative stress-mediated cognitive impairments.

No description available.

The introduction of high fructose corn syrup into the diet has been proposed to be the cause of many illnesses related to the metabolic syndrome. Fructose and its metabolites can be metabolized into cytotoxic reactive dicarbonyls that can cause damage to macromolecules leading to deleterious consequences. Dihydroxyacetone, a fructose metabolite, was studied in this thesis. Its ability to autoxidize and cause protein carbonylation under standard (pH 7.4, 37°C) and oxidative stress conditions (Fentons reagent) was investigated. Dihydroxyacetone was able to form significant amounts of dicarbonyls and protein carbonylation. Several selected natural polyphenols were chosen for an in vitro toxicological study involving rat hepatocytes. The chosen dietary polyphenols were rutin, gallic acid, methylgallate, ethylgallate, propylgallate and curcumin. In this thesis, the polyphenols were found to be able to significantly protect against the deleterious effects of glyoxal and methylglyoxal. In summary, these polyphenols could be candidates for future in vivo studies.

The introduction of high fructose corn syrup into the diet has been proposed to be the cause of many illnesses related to the metabolic syndrome. Fructose and its metabolites can be metabolized into cytotoxic reactive dicarbonyls that can cause damage to macromolecules leading to deleterious consequences. Dihydroxyacetone, a fructose metabolite, was studied in this thesis. Its ability to autoxidize and cause protein carbonylation under standard (pH 7.4, 37°C) and oxidative stress conditions (Fentons reagent) was investigated. Dihydroxyacetone was able to form significant amounts of dicarbonyls and protein carbonylation. Several selected natural polyphenols were chosen for an in vitro toxicological study involving rat hepatocytes. The chosen dietary polyphenols were rutin, gallic acid, methylgallate, ethylgallate, propylgallate and curcumin. In this thesis, the polyphenols were found to be able to significantly protect against the deleterious effects of glyoxal and methylglyoxal. In summary, these polyphenols could be candidates for future in vivo studies.

Ozone therapy is an alternative form of therapy that has gained attention in the last couple of years. It is believed that O3 may exert a stimulatory effect on the antioxidant defence and immune systems and may therefore be effective in the treatment of ischemic disorders. diabetes mellitus. AIDS and other diseases. On the other hand. it is well known that O3 is a reactive molecule that is toxic to the pulmonary system. Therefore. there remains scepticism regarding its use as a form of therapy. In order to shed some light on this. the effects of ozone autohemotherapy (O3-AHT) on the energy homeostasis of white blood cells were investigated. The possible protective effects of the plasma antioxidant defence system during O3-AHT, were also investigated. Venous blood from six apparently healthy human donors was collected in heparin. In one aliquot a precise volume of blood was mixed with an equal volume of O2/O3 gas mixture containing 20 or 80 μg/ml O3 for 20 minutes. In the other aliquot, the plasma was washed out and the cells resuspended in a buffered phosphate solution. The buffered blood cells were treated with the same concentrations of O3. Control samples was either not treated or treated with a corresponding volume of O2 . Various biochemical analyses were done on the whole blood and buffered cells to determine the oxidant/antioxidant status, cell viability, apoptosis and mitochondrial function. The higher concentration of O3 increased oxidative stress and caused death of white blood cells. Antioxidant enzyme (catalase, glutathione reductase and glutathione peroxidase) activity and the plasma antioxidant capacity decreased, whereas superoxide dismutase levels increased slightly. Exposure to O3 also increased caspase 3/7 activity. A decrease in mitochondrial function was measured by a decrease in ATP levels and an increase in NADH/NAD+ ratio. Complex IV of the respiratory chain was almost completely inhibited by both O3 concentrations. These results indicated that the death of white blood cells was probably through apoptosis. These effects were more evident in the absence of plasma antioxidants. Therefore. high concentrations of O3 were damaging to the cells, but this effect was lessened by antioxidants present in plasma. In view of the results, the use of O3 as a therapy needs to be reconsidered. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2007.

Hyperhomocysteinemia, a condition of elevated blood homocysteine level, is an independent risk factor for cardiovascular diseases. Folic acid can effectively reduce blood homocysteine levels. Recent studies have shown that hyperhomocysteinemia is also associated with liver disorders. However, the underlying mechanisms remain unclear. The general objective of my study was to investigate the biochemical and molecular mechanisms of homocysteine-induced liver injury and abnormal lipid metabolism. Hyperhomocysteinemia was induced in Sprague-Dawley rats by feeding a high-methionine diet for 4 weeks. An elevation of serum aminotransferases activities (indicator for liver injury) and an increase in hepatic lipid peroxidation were observed in hyperhomocysteinemic rats. Hyperhomocysteinemia-induced superoxide anion production led to oxidative stress in the liver. Reduction of oxidative stress by inhibiting superoxide anion production ameliorated hyperhomocysteinemia-induced liver injury. A significant elevation of hepatic and serum cholesterol concentrations in hyperhomocysteinemic rats was observed, exclusively due to increased expression of HMG-CoA reductase in hepatocytes. The molecular mechanisms of homocysteine-induced adverse effects were further investigated in isolated rat hepatocytes and in human hepatoma cells (HepG2). Hcy stimulated HMG-CoA reductase expression in hepatocytes via activation of transcription factors, namely, sterol regulatory element-binding protein-2 (SREBP-2), cAMP response element binding protein (CREB) and nuclear factor Y (NF-Y). Activation of these 3 transcription factors was detected in hyperhomocysteinemic rat liver and in homocysteine-treated hepatocytes. Pretreatment of hepatocytes with inhibitors for individual transcription factors effectively attenuated Hcy-induced HMG-CoA reductase mRNA expression. Supplementation of folic acid in diet significantly reduced serum homocysteine level and effectively inhibited hyperhomocysteinemia-induced superoxide anion production, resulting in amelioration of oxidative stress-mediated liver injury in hyperhomocysteinemic rats. These results reflected a protective role of folic acid in hyperhomocysteinemia-induced liver injury. In conclusion, the present study demonstrates that (1) hyperhomocysteinemia can cause oxidative stress and liver injury; (2) homocysteine stimulates cholesterol biosynthesis in hepatocytes via transcriptional regulation of HMG-CoA reductase expression; (3) supplementation of folic acid offers a hepatoprotective effect during hyperhomocysteinemia. Oxidative stress and accumulation of cholesterol in the liver contribute to liver injury associated with hyperhomocysteinemia. The role of folic acid in maintaining good health may extend beyond the cardiovascular system to encompass hyperhomocysteinemia-associated liver disorders.

Many consumer and medical products contain engineered nanomaterials (ENMs) due to their unique properties arising from their small size of <100 nm in at least one dimension. Although ENMs could greatly improve the quality of daily life, concerns for their health and environmental safety emerged in recent years because the same properties that make ENMs beneficial may also render them toxic. The small size allows ENMs’ entrance into the cell where they may attach to biological molecules and membranes, disrupting their function and/or leading to oxidative stress and/or damage. This thesis focused on silver nanoparticles (AgNPs). Several articles demonstrated that during washing AgNPs are released from the AgNP-impregnated fabrics and could pose a risk to aquatic species. Given that the toxicity mechanisms of AgNPs are yet to be clearly understood this thesis investigated the effects of AgNPs from ‘oxidative stress’ and ‘endocrine disruption’ points of view, using both in vivo and in vitro model fish systems. A 4 d exposure of zebrafish (Danio rerio) embryos to AgNPs increased mortality, delayed hatching, and increased oxidative stress. The silver ion (Ag+) was more effective in eliciting these effects at equivalent silver concentrations. Moreover, the Ag-chelator cysteine reduced the toxicity of both Ag-types. Despite these effects AgNPs or Ag+ did not affect the ability of zebrafish larvae or adults (raised to adulthood in Ag-free water) to increase cortisol levels, but there were differential effects on the expression of corticotropin-releasing factor (CRF)-related genes, suggesting that other physiological processes regulated by CRF may be impacted. Furthermore, a 48 h exposure of rainbow trout (Oncorhynchus mykiss) erythrocytes and hepatocytes to AgNPs or Ag+ increased oxidative stress, but Ag+ was more potent. Moreover, AgNPs elevated lipid peroxidation, while Ag+ increased DNA damage, suggesting different modes of action for the two Ag-types. Cysteine treatment reduced the toxicity of Ag+ and AgNPs, while buthionine sulfoximine, which inhibits glutathione synthesis, increased it, suggesting the importance of glutathione in silver toxicity. Finally, AgNPs increased glycogenolysis in trout hepatocytes independently of the beta-adrenoreceptor or the glucocorticoid receptor.