Liver fatty acid binding protein has been reported to possess antioxidant properties in the liver. The aim of this study was to investigate the effect of this protein in a nonalcoholic fatty liver disease (NAFLD) cell culture model. Rat hepatoma cells were treated with an oleate:palmitate (2:1) mixture for either 1 and 2 days, or further treated with 500 µM clofibrate to induce L-FABP expression. Intracellular lipid accumulation was quantitated by Nile Red. Lipotoxicity was determined using the WST-1 assay. Dichlorofluorescein (DCF) was utilized to assess intracellular reactive oxidative species (ROS) level. Measurement of lipotoxicity showed statistical decreases in cell viability as lipid concentrations increased in a dose-dependent manner. NAFLD cell cultures showed characteristic cellular damage from increased ROS levels in fatty acid treated cells. All groups treated with clofibrate showed statistically increased intracellular L-FABP levels and reduced ROS levels. The results lead to the conclusion that clofibrate induces L-FABP expression and in this manner suppresses hepatocellular ROS generation.

Understanding the molecular basis of doxorubicin (Dox)-induced oxidative stress leading to cardiomyopathy is crucial to finding cardioprotective strategies to manage this important clinical problem. Improving the antioxidant defenses of cardiac cells could be one strategy for cardioprotection. The role of oxidative stress in Dox-induced cardiotoxicity as well as testing the efficacy of antioxidant Vitamin C (Vit C) in offering protection to cardiomyocytes was investigated. As stem cells have been suggested to play a role in this cardiotoxicity, Dox-mediated oxidative stress effects, with and without Vit C, on the stem cell antigen-1 (Sca) positive cells from heart as well as bone marrow were also examined. Our time-course studies of the effects of Dox on the isolated cardiomyocytes showed that the phosphorylation of mitogen-activated protein kinases and p53 followed the rise in reactive oxygen species (ROS) production. Dox also downregulated the Sodium-dependent Vit C Transporter-2 (SVCT-2) and this may have enhanced Dox-induced increase in oxidative stress. Pro-apoptotic markers Bax/Bcl-xL ratio and caspase 3 cleavage were higher after the activation of stress-induced pathways and viability of cells was decreased. Dox-induced increase in apoptosis and decrease in cell viability depended in part on the activation of p38/JNK and p53 proteins, but not on the ERK protein. Exposure to Dox, increased membrane leakage, autophagy and lipid peroxidation. On the other hand, Dox decreased overall antioxidant capacity as well as expression of the endogenous antioxidant enzymes glutathione peroxidase, Cu/Zn superoxide dismutase and catalase. Dox affected Sca-1 positive cells in a prominent manner which was marked by a dose-dependent increase in cell loss, cell leakage and ROS levels as well as decrease in cellular ATP levels. Vit C pre-treatment prior to the addition of Dox delayed and reduced Dox-induced injury to cardiomyocytes, preserving viability. Vit C was able to blunt the decrease in SVCT-2 as well as Dox-induced oxidative stress. Vit C also offered protection to Sca-1 positive cells by partially preventing Dox-induced changes to these cells. The data presented in this thesis improves our knowledge of the molecular mechanisms leading to Dox-induced cardiotoxicity as well as suggest cardioprotection by Vit C.

Infection with challenge virus standard-11 (CVS) strain, a laboratory fixed rabies virus strain, induces neuronal process degeneration in both in vivo and in vitro models. CVS-induced axonal swellings of primary rodent dorsal root ganglion neurons are associated with 4-hydroxy-2-nonenal staining indicating a critical role of oxidative stress. Mitochondrial dysfunction is one of the most important causes of oxidative stress. We hypothesized that CVS infection induces mitochondrial dysfunction leading to oxidative stress. We investigated the effects of CVS infection on several mitochondrial parameters in different cell types. CVS infection increased electron transport chain capacity, Complex I and IV activities, but did not affect Complex II-III, citrate synthase, and malate dehydrogenase activities. CVS maintained normal oxidative phosphorylation capacity and proton leak, indicating a tight mitochondrial coupling. Possibly as a result of enhanced Complex activity and efficient coupling, a high mitochondrial membrane potential was generated. CVS infection reduced the intracellular ATP level and altered the cellular redox state as indicated by high NADH/NAD+ ratio. CVS infection was associated with a higher rate of hydrogen peroxide production. We conclude that CVS infection induces mitochondrial dysfunction leading to ROS overgeneration, oxidative stress and neuronal process degeneration.

Pregestational and gestational diabetes (GDM) places the mother and her offspring at an increased risk for later development of insulin resistance and type 2 diabetes. Oxidative stress may mediate long-term disturbances in glucose homeostasis associated with type 2 diabetes and the metabolic syndrome. This thesis describes a cross-sectional study examining serum concentrations of free radical generated oxysterols as markers of oxidative stress in a cohort of teenage daughters from pregnancies with and without GDM. Daughters of GDM-pregnancies had a tendency of higher levels of serum oxysterols (7beta-hydroxycholesterol); however, this difference was not statistically significant after adjustment for total cholesterol. Serum oxysterols were significantly correlated with obesity measures such as waist circumference and BMI, which likely accounted for the tendency for higher measures of oxysterol concentrations in the GDM daughters. Oxysterols represent potentially important biomarkers for oxidative stress in adolescent girls as their levels track with the metabolic syndrome risk factors. / Le diabète pré-gestationnel et le diabète de gestation (DG) augmentent le risque dedéveloppement d'une future résistance à l'insuline et de diabète de type 2 autant pourla mère que pour l'enfant. Le stress oxydatif est un facteur potentiel impliqué dans ledéséquilibre du glucose sanguin associé au diabète de type 2 et au syndromemétabolique. La présente thèse est une étude sectionnelle croisée, ayant pour but demesurer des marqueurs du stress oxidatif, notamment la concentration des oxystérolsgénérés par les radicaux libres dans le sérum d'adolescentes, nées de mères ayantprésenté ou non un diabète de gestation. Nos résultats montrent des concentrationsd'oxystérols (7P-hydroxycholesterol) plus élevées dans le sérum de filles issues degestations diabétiques à comparer aux filles de mères n'ayant pas eu de DG.Cependant, la différence entre les deux groupes n'était pas statistiquementsignificative après un ajustement au cholestérol total. La concentration d'oxystérolsétait significativement corrélée aux marqueurs d'obésité, notamment la circonférencede la taille et l'index de masse corporelle, possiblement à l'origine de la tendance desoxystérols à être plus élevés dans le cas des adolescentes issues de gestationsdiabétiques.

High chronic fructose consumption has been linked to many diseases. Sugar metabolites, especially glyoxal and methylglyoxal can form advanced glycation products, which contribute to the pathology of diabetic complications. Our objective was to study the metabolism of these metabolites and the associated protein carbonyation and cytotoxicity in isolated hepatocytes. In addition, the effect of oxidative stress on the metabolism of these toxins was also investigated. Methylglyoxal and glyoxal can induce protein carbonylation, which contributes to hepatocyte toxicity. Methylglyoxal, but not glyoxal, was detoxified mainly by the glyoxalase system. Both toxins can be metabolized by mitochondrial aldehyde dehydrogenase. The detoxification of glyoxal was impaired under oxidative stress conditions (i.e. increased hydrogen peroxide level). Glyoxal was found to be a common autoxidation product from glyceraldehyde, hydroxypyruvate and glycolaldehyde. Glyoxal and the reactive oxygen species formation during the autoxidation process contributed to the hepatocyte toxicity of glyceraldehyde, hydroxypyruvate and glycolaldehyde.

High chronic fructose consumption has been linked to many diseases. Sugar metabolites, especially glyoxal and methylglyoxal can form advanced glycation products, which contribute to the pathology of diabetic complications. Our objective was to study the metabolism of these metabolites and the associated protein carbonyation and cytotoxicity in isolated hepatocytes. In addition, the effect of oxidative stress on the metabolism of these toxins was also investigated. Methylglyoxal and glyoxal can induce protein carbonylation, which contributes to hepatocyte toxicity. Methylglyoxal, but not glyoxal, was detoxified mainly by the glyoxalase system. Both toxins can be metabolized by mitochondrial aldehyde dehydrogenase. The detoxification of glyoxal was impaired under oxidative stress conditions (i.e. increased hydrogen peroxide level). Glyoxal was found to be a common autoxidation product from glyceraldehyde, hydroxypyruvate and glycolaldehyde. Glyoxal and the reactive oxygen species formation during the autoxidation process contributed to the hepatocyte toxicity of glyceraldehyde, hydroxypyruvate and glycolaldehyde.

In the Ottawa Hospital Weight Management Clinic, we have previously identified subpopulations of patients in the upper and lower quintiles for rate of weight loss, and characterized them as ‘obese diet sensitive’ (ODS) and ‘obese diet resistant’ (ODR) patient groups, respectively. Skeletal muscle is a major contributor to basal metabolic rate and mitochondrial proton leak in skeletal muscle can account for up to 50 % of resting oxygen consumption. The overall aim of this research is to explore differences in mitochondrial function in human primary myotubes from ODS and ODR subjects. Subsets of ODS and ODR subjects (n = 9/group) who followed a hypocaloric clinical weight loss program at the Ottawa Weight Management Clinic consented to a muscle (vastus lateralis) biopsy. Human primary myoblasts obtained from biopsies were immunopurified and differentiated into myotubes. Mitochondrial function and distribution were compared in intact myotubes from ODS and ODR subjects. Mitochondrial proton leak was significantly lower (p< 0.05) in ODR myotubes compared to ODS myotubes, independent of whether cells were differentiated in low or high glucose medium. In addition, in low glucose medium, ODR myotubes had higher MnSOD protein levels compared to ODS myotubes (p< 0.05). However, there were no significant differences in mitochondrial content, mitochondrial membrane potential, cellular ROS levels or ATP content between ODS and ODR myotubes. Overall, our in vitro mitochondrial proton leak results are consistent with our previous ex vivo results. Future research should examine the possibility that differences in proton leak between ODS and ODR groups may be related to mechanisms of cellular ROS regulation.

We hypothesized that with exercise training and the subsequent attenuation of hyperglycemia, β-cell adaptation to worsening insulin resistance would be maintained. Also, because classical stress-activated systems and oxidative stress are involved in hepatic insulin resistance we examined if exercise would be associated with improvements in hepatic markers of oxidative stress and inflammation. Exercise maintained fasted hyperinsulinemia and preserved normoglycemia in male Zucker diabetic fatty (ZDF) rats. β-cell function calculations indicate prolonged β-cell adaptation in exercised animals. Such improved β-cell function was associated with increased β-cell mass. Hypertrophy and replication contributed to expansion of β-cell mass; exercised animals had increased β-cell size and bromodeoxyuridine (BrdU) incorporation rates versus controls. Furthermore, we observed augmented β-cell-specific immunohistochemical staining of GLUT2 and Akt/PKB in exercised versus sedentary controls. We also observed large cytoplasmic ubiquitinated structures which form in response to oxidative stress in pancreatic tissue samples from hyperglycemic ZDF rats. In the exercised groups such aggregate numbers were reduced to numbers compared to those seen in younger non-diabetic basal ZDF animals and age-matched lean Zucker rats. With respect to the liver we investigated whether exercise alters kinases such as c-Jun NH2-terminal kinase (JNK) and IKKβ (as evidenced by IκBα levels) and related insulin receptor substrate-1 (IRS-1) serine phosphorylation which are associated with hepatic insulin resistance in obesity. On average, exercised animals ran 5250m/day which improved insulin sensitivity based on the homeostasis model assessment for insulin resistance (HOMA-IR) calculations, and maintained fed and fasted glucoregulation and glucose tolerance. Ten weeks of running decreased whole-body markers of inflammation and oxidative stress in the blood and in the liver. Exercise lowered circulating interleukin-6 (IL-6), haptoglobin, malondialdehyde (MDA) levels, and protein oxidation in the liver. Exercise reduced phosphorylated JNK (pJNK) indicating decreased JNK activity; in accordance serine phosphorylated IRS-1 was reduced in exercised rats. In conclusion, improvements in glucoregulation were associated with increased β-cell compensation at least in part due to a reduction in oxidative stress. Furthermore, we show exercise attenuates development of hyperglycemia in ZDF rats in association with decreases in plasma and hepatic markers of inflammation, oxidative stress, JNK activation, and serine phosphorylation of IRS-1.

Phosphine, hydrogen phosphide (PH3), gas is a fumigant that is used worldwide to protect stored grain from infestation by insect pests. Despite a long history of phosphine use, little is known about either the mode of action of this compound or the mechanisms whereby insect pests have become resistant. To better understand phosphine toxicity and resistance mechanisms, a genetically well-characterised model organism, Caenorhabditis elegans, was used in my PhD project. Three previously created phosphine resistant C. elegans mutants (pre-1, pre-7 and pre-33) developed from the wild type N2 strain were used in this study, though analysis of pre-33 was the primary focus. The three mutants were determined to be 2, 5 and 9 times more resistant toward phosphine than was the parental N2 strain by comparison of LC50 values. Molecular oxygen was shown to be an extremely effective synergist with phosphine as, under hyperoxic conditions, 100% mortality was observed in wild-type nematodes exposed to 0.1 mg/l phosphine, a non-lethal concentration in air. All three mutants were resistant to the synergistic effects of oxygen in proportion to their resistance to phosphine with one mutant, pre-33, showing complete resistance to this synergism. I take the proportionality of cross-resistance between phosphine and the synergistic effect of oxygen to imply that all three mutants circumvent a mechanism of phosphine toxicity that is directly coupled to oxygen metabolism. Compared with the wild-type strain, each of the three mutants has an extended average life expectancy of 12.5 to 25.3%. This is consistent with the proposed involvement of oxidative stress in both phosphine toxicity and ageing. Indeed, a correlation between phosphine resistance and resistance to other stressors (e.g. heavy metal, heat and UV) was also detected. On the other hand, no significant difference in methyl viologen sensitivity was found between pre-33 and N2 strains, suggesting that pre-33 mutant does not seem to provide resistance to phosphine via protection against oxidative damage. Additionally, to test for possible involvement of the DAF-2/DAF-16 signalling pathway in the phosphine response, the levels of phosphine sensitivity of mutants in this pathway were tested. Phosphine resistance levels were increased in daf-2 and age-1 mutants but decreased in daf-16 nematodes, which mirrors the longevity phenotypes of these mutants, suggesting some congruence in glucose signalling between the phosphine resistance and longevity traits. In contrast, no congruence is observed between phosphine resistance and oxidative metabolism as the clk-mutation, which disrupts oxidative metabolism does not cause phosphine resistance and neither do the phosphine resistant mutants cause the severe developmental delay of the clk-1 mutation. The phosphine induced time-dependent mortality was assessed in both N2 and pre-33 nematodes at two fixed phosphine concentrations (0.3 and 3.0 mg/l), allowing the determination of minimum exposure periods required for any mortality as well as the exposure time required to achieve 50% mortality. As a result, it was determined that 15 hours of exposure was needed for significant mortality in N2 and pre-33 strain when exposed to 0.3 and 3.0 mg/l of phosphine, respectively; whereas this period is 5 hours for N2 when treated with 3.0 mg/l phosphine. The fact that the LT50 value for N2 at 0.3 mg/l phosphine is indistinguishable from that of pre-33 at 3.0 mg/l (24.6 and 24.5 respectively) suggests that 0.3 and 3.0 mg/l of phosphine have the same toxic effects on N2 and pre-33 nematodes respectively. This result is consistent with the finding that pre-33 is ~9 fold more resistant to phosphine than is the N2 strain. Moreover, the LT50 was determined to be 8.4 hours for N2 when treated with 3.0 mg/l of phosphine, which is only three times faster than pre-33 when exposed to the same level of phosphine. In contrast to the differential toxicity of phosphine between the N2 and pre-33 lines, the delay in reaching reproductive maturity caused by phosphine exposure is indistinguishable between WT and pre-33 nematodes. This indicates that the phosphine induced delay in maturation is independent of the toxic effects of phosphine. Since the inhibition of complex IV (cytochrome c oxidase) in the mitochondrial electron transport chain has been proposed as a mechanism of phosphine toxicity, the phosphine effects on cellular ATP metabolism, presented as ATP+ADP content and ATP/ADP ratio, were also assessed. Phosphine exposure (0.3 mg/l, 25 hours) led to a significant decrease in ATP+ADP levels as well as the ATP/ADP ratio in N2 nematodes. Similar results were also detected in pre-33 nematodes when exposed to 3.0 mg/l phosphine for 25 hours. These observations indicate that phosphine can interrupt cellular ATP metabolism, which is associated with phosphine induced mortality. Additionally, the fact that mutant pre-33 can maintain its ATP levels under phosphine exposure at 0.3 mg/l suggests it has a greater ability to maintain mitochondrial function than does the N2 strain. To better understand the mechanism of phosphine toxicity in the wild type N2 strain, gene expression profiling by DNA microarray analysis was employed. A significant overlap between phosphine and DAF-16 regulated genes was detected, supporting the previous finding that the DAF-2/DAF-16 pathway can contribute to phosphine resistance. Phosphine exposure also strongly induced xenobiotic detoxification and stress responses, indicating nematodes are able to sense phosphine induced toxic effects and protect themselves by switching on native detoxification mechanisms. Furthermore, glycolysis and gluconeogenesis were also up-regulated by phosphine, possibly due to an increase in energy demand caused by increased xenobiotic detoxification activities. Consistent with the previous findings that phosphine delays median reproductive age and reduces fertility, expressions of a large number of genes involved in growth, embryonic development and reproduction were suppressed by phosphine. Moreover, the microarray results of seven genes whose expression levels were significantly altered by phosphine were validated using RT-PCR, confirming the robustness of the microarray results. The most direct way to determine the phosphine resistance mechanism in mutant pre-33 is to identify and characterise the mutation itself. Using a classic F1 test, the resistance mutation in pre-33 was determined to be incompletely recessive. Additionally, using three mapping strategies, the resistance mutation was mapped to Chromosome IV between 12,591,683 and 12,879,637 bp with 45 genes located in this small region. In an attempt to identify the resistance gene, the effect of suppressing each of 28 of the 45 genes in the interval was determined using a commercially available gene suppression library. It was observed that only knockdown of gene vha-7 resulted in a slight decrease in phosphine sensitivity (84.6%) compared to N2 (97.6%). However, this result does not clearly implicate vha-7 as the resistance gene in pre-33. The microarray results indicated that linoleate and arachidonate signalling pathways might be activated by phosphine. This was observed as induction of a phospholipase A2 gene that regulates the release of arachidonic acid from the C-2 position of membrane phospholipids, as well as several CYP genes predicted to catalyse the oxidation of linoleate and arachidonate. Therefore, phosphine effects on the linoleate and arachidonate dependent signalling pathways were assessed. It was found that, in the presence of phosphine, the pre-33 mutant has a greater ability to transform linoleate and arachidonate epoxides to diols than does N2. This activity may help pre-33 to better maintain mitochondrial function and, therefore, ATP metabolism than N2 during phosphine exposure. The microarray results also showed that phosphine exposure caused up-regulation of glycolysis and gluconeogenesis, indicating phosphine regulation of carbohydrate metabolism. As expected, a preliminary metabonomic analysis by 1H nuclear magnetic resonance (NMR) into the effect of phosphine exposure on metabolism in N2 nematodes revealed significant alteration of the metabonomic profile.

Cardiovascular disease is the leading cause of death globally. Chronic hypertension can lead to a gradual deterioration in the structure and performance of the cardiovascular system in a process described as cardiovascular remodelling. The ultimate response to this remodelling is heart failure. While cardiovascular remodelling is characterised by features such as cardiac and vascular hypertrophy, endothelial dysfunction and fibrosis; mechanisms leading to such pathologies are still unclear. However, oxidative stress, the damage caused by increased production of reactive oxygen species such as free radicals, or their reduced removal by anti-oxidants, appears to have a significant role. The major aim of this thesis is to investigate the involvement of reactive oxygen species in cardiovascular remodelling and to further investigate the potentials of three anti-oxidant approaches (a combination of alpha-lipoic acid and vitamin E, apocynin and a red-wine component, resveratrol) in preventing or reversing cardiovascular remodelling. These studies were conducted in two well-established rat models of cardiovascular remodelling including the deoxycorticosterone acetate (DOCA)-salt hypertensive rat and the ageing spontaneously hypertensive rat (SHR). Additionally, this thesis also characterises the effects of ovariectomy on the blood pressure and survival rate of female SHR. The cardiovascular structure and function in the animals have been defined using in vivo echocardiography, ex vivo isolated Langendorff heart perfusion, isolated thoracic aortic rings, histological analysis of the myocardial extracellular matrix and inflammation along with terminal organ weight measurements. Reactive oxygen species were assessed through the measurement of plasma malondialdehyde (MDA) while the anti-oxidant capacities have been assessed through measurements of plasma total anti-oxidant status (TAS) and plasma glutathione peroxidase (GPx) activity. DOCA-salt treated rats exhibited hypertension, oxidative stress and cardiovascular remodelling, evidenced by their increased left ventricular weights, excess collagen deposition in the heart and increased values for diastolic stiffness, increased plasma MDA concentrations along with impaired contraction and relaxation of the vessels. Treatment with a combination of alpha-lipoic acid and vitamin E or apocynin significantly inhibited the increases in blood pressure, left ventricular weights, cardiac stiffness, interstitial collagen deposition along with improvements in the vascular responses. The treatment of ageing male SHRs with resveratrol, although without any decrease in blood pressure, was shown to reduce left ventricular hypertrophy, fibrosis, diastolic stiffness and improve functional performance of the heart. The level of oxidative stress was also lowered in the male SHRs treated with resveratrol as evidenced by decreased plasma MDA and increased TAS. Female SHRs had a higher blood pressure and diastolic stiffness compared to their age-matched WKYs. Similar to male SHRs, treatment with resveratrol did not affect blood pressure, but attenuated the increased diastolic stiffness, in female SHRs. Furthermore, the ovariectomised SHRs were proven to be extremely hypertensive and had a significantly poorer survival rate. Overall, these studies demonstrated great potential for the alpha-lipoic acid and vitamin E combination and apocynin in the treatment of cardiovascular remodelling. However, further clinical and experimental research is essential to confirm the complete cardiovascular health benefits of resveratrol or red wine intake.