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.

Myocardial dysfunction is an important manifestation of sepsis/septic shock. Activation of Phosphatidylinositol 3-kinase(PI3K)/protein kinase B (Akt) signaling pathway has been shown to improve cardiac performance during sepsis/septic shock. We have reported previously that α-lipoic acid (LA) activates PI3K/Akt pathway in neuronal cells. It is possible, therefore, that treatment with LA will attenuate cardiac dysfunction during sepsis/septic shock through a PI3K/Akt-dependent mechanism. To test this possibility, we treated mice with LA prior to lipopolysaccharide (LPS) challenge. Cardiac function was analyzed by echocardiography 6 h after LPS challenge. LPS significantly suppressed cardiac function as evidenced by decreases in EF% and FS% in mice. However, LA pretreatment significantly attenuated cardiac dysfunction following LPS challenge. LA pretreatment also improved survival in LPS-challenged mice. Furthermore, LA markedly attenuated the LPS-induced inflammatory response in myocardium, as evidenced by decreases in the upregulation of VCAM-1, ICAM-1 and iNOS, as well as myocardial leucocytes infiltration. Moreover, LPS challenge significantly decreased the phosphorylation levels of Akt and Gsk-3β, which was prevented by LA pretreatment. More importantly, inhibition of PI3K/Akt signaling by Wortmannin (WM) completely abrogated the LA-induced protection in cardiac dysfunction following LPS challenge. Collectively, our results demonstrated that LA improved cardiac function during endotoxemia. The mechanism was through, at least in part, preserved activation of the PI3K/Akt signaling.

Myocardial dysfunction is an important manifestation of sepsis/septic shock. Activation of Phosphatidylinositol 3-kinase(PI3K)/protein kinase B (Akt) signaling pathway has been shown to improve cardiac performance during sepsis/septic shock. We have reported previously that α-lipoic acid (LA) activates PI3K/Akt pathway in neuronal cells. It is possible, therefore, that treatment with LA will attenuate cardiac dysfunction during sepsis/septic shock through a PI3K/Akt-dependent mechanism. To test this possibility, we treated mice with LA prior to lipopolysaccharide (LPS) challenge. Cardiac function was analyzed by echocardiography 6 h after LPS challenge. LPS significantly suppressed cardiac function as evidenced by decreases in EF% and FS% in mice. However, LA pretreatment significantly attenuated cardiac dysfunction following LPS challenge. LA pretreatment also improved survival in LPS-challenged mice. Furthermore, LA markedly attenuated the LPS-induced inflammatory response in myocardium, as evidenced by decreases in the upregulation of VCAM-1, ICAM-1 and iNOS, as well as myocardial leucocytes infiltration. Moreover, LPS challenge significantly decreased the phosphorylation levels of Akt and Gsk-3β, which was prevented by LA pretreatment. More importantly, inhibition of PI3K/Akt signaling by Wortmannin (WM) completely abrogated the LA-induced protection in cardiac dysfunction following LPS challenge. Collectively, our results demonstrated that LA improved cardiac function during endotoxemia. The mechanism was through, at least in part, preserved activation of the PI3K/Akt signaling.

It has been long noted that cardiac cell apoptosis provoked by excessive production of nitric oxide (NO) plays important roles in the pathogenesis of variant cardiac diseases. Attenuation of NO-induced injury would be an alternative therapeutic approach for the development of cardiac disorders. This study investigated the effects of α-lipoic acid (LA) on the injury induced by sodium nitroprusside (SNP), a widely used NO donor, in rat cardiomyoblast H9c2 cells. SNP challenge significantly decreased cell viability and increased apoptosis, as evidenced by morphological abnormalities, nuclear condensation and decline of mitochondrial potential (δ. Ψm). These changes induced by SNP were significantly attenuated by LA pretreatment. Furthermore, LA pretreatment prevented the SNP-triggered suppression of Akt and Gsk-3β activation. Blockade of Akt activation with triciribin (API) completely abolished the cytoprotection of LA against SNP challenge. In addition, LA moderately increased intracellular ROS production. Interestingly, inhibition of ROS with N-acetylcysteine abrogated Akt/Gsk-3β activation and the LA-induced cytoprotection following SNP stimulation. Taken together, the results indicate that LA protected the SNP-induced injury in cardiac H9c2 cells through, at least in part, the activation of Akt/Gsk-3β signaling in a ROS-dependent mechanism.

α-Lipoic acid (LA) has been shown to improve the diabetic cardiac symptoms. However, the underlying mechanisms have not been elucidated precisely. We have reported recently that LA potentially protected neurons from substance-induced apoptosis. We hypothesized that LA could attenuate cardiac cells death induced by oxidative stress derived from high glucose. To test this possibility, we examined the effects of LA on . d-glucose/glucose oxidase (DG/GO, 30. mM/5. mU)-induced injury in rat cardiomyoblast H9c2 cells. We observed that LA pretreatment significantly increased cell viability in DG/GO-challenged cells. LA pretreatment also attenuated DG/GO-induced apoptosis as evidenced by decreases in both nuclear condensation and loss of mitochondrial potential. In addition, LA activated ERK1/2 and moderately increased ROS production. Blockade of ERK1/2 activation by PD98059 completely abolished LA-induced protection against DG/GO challenge. Inhibition of ROS by . N-acetylcysteine abrogated LA-induced ERK1/2 activation and cytoprotection. Furthermore, we observed that the ROS production induced by LA was significantly slower and milder than that by DG/GO. Our results suggest that pretreatment with LA moderately increased ROS production to induce a preconditioning-like effect by ERK1/2 activation thereby increased tolerance of H9c2 cells to DG/GO challenge.

Background: Bupivacaine is an amide type local anesthetic which is widely used for epidural anesthesia and nerve blockade in patients. However, local administration of bupivacaine could cause neuron injury showing transient neurologic symptoms. α-Lipoic acid (LA) was shown to protect nerve cells from substance-induced injury. We hypothesized that LA administration could attenuate bupivacaine-induced neurotoxicity. Methods: To evaluate our hypothesis, we treated mouse neuroblastoma N2a cells with LA 30 min before the cells were exposed to bupivacaine. We evaluated cellular injury by examination of cell viability, morphology changes, nuclear condensation, and Annexin V staining. We also examined the levels of intracellular reactive oxygen species (ROS) and activation of PI3K/Akt signaling pathway. In a separate experiment, we determined the effect of Akt inhibition on cell viability in the presence of LA and bupivacaine. Results: Bupivacaine treatment significantly induced cell injury as evidenced by decreased cell viability, increased nuclear condensation and Annexin V staining. Administration of LA significantly attenuated bupivacaine-induced cell injury. In addition, LA treatment increased the levels of phospho-Akt and phospho-GSK3β and attenuated bupivacaine decreased the levels of ROS. More significantly, pharmacological inhibition of Akt abolished the LA-induced protection from bupivacaine-caused cell injury. Conclusions: Our findings suggest that pretreatment of neuroblastoma cells with LA protected neural cells from bupivacaine-induced injury. The mechanisms involve activation of the PI3K/Akt signaling pathway.