Oxidative and nitrosative stress are deleterious physiological processes caused by an imbalance between reactants such as reactive oxygen and nitrogen species and antioxidants. Due to the links between oxidative and nitrosative stress and disease, there is much interest in accurately quantifying these in biological and physiological samples. There are numerous methods to quantify the in vivo oxidative and nitrosative damage to lipids, DNA and proteins however they are generally time-consuming, expensive and difficult. Furthermore, due to the complex nature of oxidative and nitrosative stress it would be appropriate to measure a number of different biomarkers, however this is rarely done. The first section of this thesis contains research aimed at developing a bioassay to simultaneously detect markers of oxidative and nitrosative stress. This includes; 1) a review of the studies investigating the ability of these biomarkers to predict the onset of disease, 2) a description of the attempts to develop the bioassay, 3) a study designed to test the sensitivity of the bioassay to detect changes in oxidative stress. Unfortunately, the attempts to develop the bioassay were not as successful as hoped and, in the interests of completing the PhD in the time allowed, the PhD changed focus to look at the effects of oxidant:antioxidant balance on mitochondrial biogenesis. The second section of the thesis contains a review of the literature on this topic and two original investigations. It is well documented that oxidative and nitrosative stress contributes to the progression of many diseases including; cardiovascular disease, type 2 diabetes, Alzheimer’s disease, kidney disease and cancer. To determine which biomarkers would have the greatest efficacy in the bioassay, a comprehensive review was undertaken. The aim of the review was to investigate studies which have measured oxidative and nitrosative biomarkers to determine whether they are independent predictors of cardiovascular events (Chapter two). From the review, fifty-one studies were identified with twenty-six of these measuring oxidised (Ox)-LDL, fifteen assessing myeloperoxidase (MPO), seven using lipid peroxidation measures and three quantifying protein oxidation in plasma/serum. The recommendation of the review was that all areas require further investigation, however, it was determined that Ox-LDL and MPO would be beneficial for inclusion in the bio-assay. Other biomarkers considered for the bio-assay were nitrotyrosine, superoxide dismutase and glutathione peroxidase. Chapter three outlines method development used to measure the oxidative and nitrosative markers simultaneously. Recent technology allows multiple analytes to be detected simultaneously from the one sample. The Mulit-plex system is used to detect analytes that have been sandwiched between primary capture and secondary biotinylated detection antibodies. The secondary antibody attaches to streptavidin-phycoerythrin and is used by the Mulit-plex analyser to quantify the analyte. During development of the bio-assay, clumping of microspheres, high background, no detection of standard curve or samples, matrix effects, mislabeling of antibodies by manufacturers and lack of commercial available antibodies were obstacles that limited the success of this method. MPO was the only biomarker that was successful. Chapter four contains a study that investigated the sensitivity of the MPO mulitplex bio-assay. Nine highly trained cyclists underwent an extensive exercise protocol designed to induce dehydration by 4 % body mass, rehydration of 150 % fluid loss and a performance time-trial. Plasma samples were taken at five time points; baseline, post dehydration, post rehydration, pre time-trial and post time-trial and analysed using the mulitplex bio-assay. The results showed that there was a significant increase in MPO post dehydration and post time-trial compared with all other time points (P<0.05), thereby demonstrating that the mulitplex bio-assay is sensitive to detect changes in exercise and appropriate rehydration reduces oxidative stress. The MPO mulitplex bio-assay requires further testing on patients with diseases to further validate its future applications. As mentioned above, due to time constraints it was decided to stop the attempts to create a multi-analyte bioassay and focus on another important area of cellular oxidative stress. Currently, there is much interest in the involvement of oxidant:antioxidant balance in mitochondrial biogenesis. The increase of mitochondrial content within the skeletal muscle, termed mitochondrial biogenesis, provides an increased capacity to generate ATP during exercise and is recognized as one of major cellular adaptations to exercise. Reactive oxygen species are produced during exercise and have been shown to induce mitochondrial biogenesis. One of the key instigators of mitochondrial biogenesis is peroxisome proliferator activated receptor gamma coactivator-1α (PGC-1α). PGC-1α is central to the transcription of mitochondrial and nuclear encoded genes, which regulate downstream pathways such as oxidative phosphorylation and fatty acid oxidation. Antioxidant supplementation is common among athletes and healthy individuals; however, antioxidant supplements suppress reactive oxygen species and could therefore could hinder mitochondrial biogenesis and the positive adaptations associated with exercise. To establish whether antioxidant supplementation reduced mitochondrial biogenesis in skeletal muscle, male Wistar rats were supplemented with α-tocopherol and α-lipoic acid for fourteen weeks (Chapter six). Animals were separated into four groups: 1) sedentary control diet, 2) sedentary antioxidant diet, 3) exercise control diet and 4) exercise antioxidant diet. The exercise animals were trained 5 days/week for 14 weeks. Consistent with increased mitochondrial biogenesis and antioxidant defences following training, there were significant increases in PGC-1α mRNA and protein, COX IV and Cyt C protein abundance, citrate synthase activity, Nfe2l2 and SOD2 protein (P<0.05). Antioxidant supplementation reduced PGC-1α mRNA, PGC-1α and COX IV protein, and citrate synthase enzyme activity (P<0.05) in both sedentary and exercise-trained rats. In summary, antioxidants α-tocopherol and -lipoic acid supplementation suppresses beneficial adaptations in skeletal muscle such as markers of mitochondrial biogenesis and mitochondrial proteins, regardless of training status. The reduction in mitochondrial biogenesis may affect exercise training adaptations and reduce the ability of healthy individuals to attain optimal exercise adaptations. The last investigation (Chapter seven) studied the effect of reduced glutathione, through diethyl maleate (DEM) administration, on upstream regulators of mitochondrial biogenesis, markers of mitochondrial biogenesis and downstream signalling. Glutathione is a key antioxidant that reduces the amount of hydrogen peroxide. Male Wistar rats were divided into six groups 1) sedentary control, 2) sedentary DEM, 3) post-exercise control, 4) post-exercise DEM, 5) exercise-recovery and 6) exercise-recovery DEM. After an exercise bout to fatigue, animals were euthanized directly after exercise (post-exercise) or four hours post exercise (exercise-recovery). Exercising animals given DEM had significantly (P<0.05) decreased glutathione in skeletal muscle and had a significantly (P<0.05) greater increase in PGC-1α gene expression. There were also main interaction effects between exercise and DEM administration on SOD2 activity. Exercise altered the gene expression of GPx and the phosphorylation of p38 MAPK. Glutathione depletion decreased GPX activity and oxidised glutathione levels. These novel findings represent important in vivo evidence of the involvement of glutathione and oxidant:antioxidant balance in mitochondrial biogenisis. Overall this thesis has provided 1) the first comprehensive review on the prognostic ability of oxidative stress biomarkers to predict the onset of cardiovascular disease, 2) detailed information to assist in the further development of a multi-analyte bioassay to quantify oxidative and nitrosative stress, 3) data indicating that the MPO Mulit-plex bioassay is sensitive to detect physiological perturbations to oxidative stress, 4) evidence that antioxidant supplementation suppresses mitochondrial biogenesis and 5) proof that glutathione is important in the regulation of exercise-induced mitochondrial biogenesis.

Drug induced liver injury is a leading cause of human illness and a major cause of drug withdrawals from the market. A systems biology approach has the potential to aid toxicology research since toxicological responses are a consequence of multiple non-linear and interdependent biological responses. Here such an approach is developed.The glutathione pathway is a key hepatic defence mechanism and deactivates reactive metabolites before they have the chance to damage cellular proteins. However, glutathione availability is limited and can vary between individuals. As hepatic glutathione levels cannot be measured directly, two serum-based biomarkers, i.e. 5-oxoproline and ophthalmic acid, have been proposed in literature as a means of tracking glutathione depletion. This thesis aims to test the reliability of the correlation between biomarker concentration and decreasing glutathione concentration.In this study a spiral between experiments, model predictions and falsifications, model improvement, and experimental design is described. Using this approach a kinetic model of the hepatic glutathione pathway and biomarker metabolism was constructed and subsequently expanded by adding physiologically based pharmacokinetic (PBPK) models of paracetamol and the proposed biomarkers. These models have increased the understanding of the glutathione pathway. For example, the model predicted that Glutamyl-Cysteine Synthetase induction should be a highly effective way to increase the robustness of the liver to a paracetamol challenge.In addition, it was possible to qualify with increasing precision, the correlation between biomarkers and hepatic glutathione depletion. 5-Oxoproline and ophthalmic acid provide different information about the status of the glutathione pathway. 5-Oxoproline is correlated with paracetamol-glutathione conjugate formation, but not with extreme toxicity. Ophthalmic acid is a biomarker of a more advanced stage of toxicity, where the cell is unable to protect against glutathione depletion. However, care must be taken when inferring hepatic glutathione concentration. Both models demonstrate that the sensitivity of biomarkers to exposure of paracetamol, depends on the dynamics of exposure as well as on the concentrations of intracellular metabolites, such as methionine.I discuss how the methodology of biomarker assessment could be personalised with regards to individual patients and how systems toxicology could be further developed towards reliable tools for the pharmaceutical industry.

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no / Glutathione depletion is a consistent feature of the progression of mild to severe acute pancreatitis. In this study, we examined the temporal relationship between cysteine, homocysteine, and cysteinyl-glycine levels; total reduced erythrocyte glutathione; gamma-glutamyl transpeptidase activity; and disease severity. Initially, cysteine concentration was low, at levels similar to those of healthy controls. However, glutathione was reduced whilst cysteinyl glycine and gamma-glutamyl transpeptidase activity were increased in both mild and severe attacks. As the disease progressed, glutathione and cysteinyl glycine were further increased in mild attacks and cysteine levels correlated with homocysteine (r = 0.8, P < 0.001) and gamma-glutamyl transpeptidase activity (r = 0.75, P < 0.001). The progress of severe attacks was associated with glutathione depletion, reduced gamma-glutamyl transpeptidase activity, and increased cysteinyl glycine that correlated with glutathione depletion (r = 0.99, P = 0.01). These results show that glutathione depletion associated with severe acute pancreatitis occurs despite an adequate cysteine supply and could be attributed to heightened oxidative stress coupled to impaired downstream biosynthesis.