Red raspberries, as an excellent source of dietary antioxidants, were investigated for their effect on oxidative stress in healthy adults. Study 1 measured effects of chronic exposure in a parallel, multi-dose intervention. Subjects consumed one-cup red raspberries (1cR) daily for two-weeks, then were randomized to consume 1cR, 2cR or 4cR for additional two-weeks (n=8, by group). There was a reduction in TBARS, indicating a decrease in lipid peroxidation, after two-weeks of intervention in the 1cR group, but effects were not significant at week 4, or for other treatment groups. Study 2 measured effects of acute exposure using a cross-over design. Subjects (n=8) consumed single treatments of 1cR, 2cR, 4cR, bread and bread plus vitamin C. Post-prandial oxidative stress responses were complex and appeared related to calorie and antioxidant load. Overall there was no clear relationship between red raspberry consumption and protection against oxidative stress.

Reactive oxygen species (ROS) are a by-product of aerobic metabolism and have been implicated in cancer, arthrosclerosis, diabetes and aging. Antioxidant enzymes, such as superoxide dismutase (SOD), work to neutralize ROS and oxidative stress occurs when the antioxidant capacity of the cell is overwhelmed. Using a Drosophila mutant with defective cytoplasmic SOD function (cSODn108), we are able to study the consequences of excess ROS on gene expression. Microarray experiments indicate gene expression changes associated with immune response, heat shock, detoxification, proteolysis, carbohydrate metabolism, lipid metabolism and behaviour. Behavioural and physiological assays investigated possible phenotypes predicted by changes in gene expression. We found that cSODn108 mutants feed less yet demonstrate a remarkable resistance to starvation. In addition, cSODn108 mutants show a reduced response to sucrose, odorants and decreased locomotor activity. These phenotypes correlate with observed gene expression changes and suggest a potentially altered energy metabolism in response to chronic oxidative stress.

Reactive oxygen species (ROS) are a by-product of aerobic metabolism and have been implicated in cancer, arthrosclerosis, diabetes and aging. Antioxidant enzymes, such as superoxide dismutase (SOD), work to neutralize ROS and oxidative stress occurs when the antioxidant capacity of the cell is overwhelmed. Using a Drosophila mutant with defective cytoplasmic SOD function (cSODn108), we are able to study the consequences of excess ROS on gene expression. Microarray experiments indicate gene expression changes associated with immune response, heat shock, detoxification, proteolysis, carbohydrate metabolism, lipid metabolism and behaviour. Behavioural and physiological assays investigated possible phenotypes predicted by changes in gene expression. We found that cSODn108 mutants feed less yet demonstrate a remarkable resistance to starvation. In addition, cSODn108 mutants show a reduced response to sucrose, odorants and decreased locomotor activity. These phenotypes correlate with observed gene expression changes and suggest a potentially altered energy metabolism in response to chronic oxidative stress.

Spinal cord injury occurs at a rate of 11.5 - 53.4 per million in developed countries with great emotional and financial consequences. The damage caused by the initial injury is followed by secondary damage, a complex cascade of mechanisms including ischemia, oxidative stress, inflammation and apoptosis. Although nothing can be done to reverse the initial damage to the spinal cord once it occurs, the secondary damage can be targeted by therapeutics to improve recovery. Following injury, concentrations of the potent antioxidant glutathione (GSH) are decreased in the spinal cord which potentiates mechanisms of secondary damage. In an attempt to maintain the GSH concentrations, the non-essential amino acid glutamine was tested as it was shown to increase GSH concentrations both in vivo and in vitro. Glutamine is being used extensively in clinical research in an expansive number of physiological and pathological conditions including brain trauma. To examine the therapeutic potential of glutamine after spinal cord trauma, two compression injury models, the modified aneurysm clip and the modified forceps, were used to induce an injury in male Wistar rats. We have demonstrated the ability of glutamine treatment (1 mmol/kg), given 1 hour after a 30 g aneurysm clip injury to increase GSH not only in whole blood samples but within the spinal tissue at the site of injury. Increasing GSH in this way also resulted in improved locomotor scores and maintenance of white matter tissue at the injury epicenter. Experiments using the forceps model were then performed to determine if the potency of glutamine treatment would be carried over to a different model and at a variety of severities. Glutamine, again, demonstrated the ability to improve maintenance of whole blood GSH, locomotor scores and tissue histology. In our experiments, glutamine has proven to be a potent therapeutic for spinal cord injury with an effect that is matched by few compounds currently being studied and well exceeding the standard therapeutic, methylprednisolone. Given the breadth of knowledge regarding the effects of glutamine clinically in numerous paradigms and the potency of the therapeutic effect seen in these studies, we believe that glutamine is fit for clinical trial and has a high potential for success.

<p>The present research was designed to investigate the mechanisms of cellular transport, metabolism and toxicity of selenium [inorganic (selenite) and organic (selenomethionine)] in a model teleost, rainbow trout (<i>Oncorhynchus mykiss</i>), using both <i>in vitro</i> and <i>in vivo</i> experimental approaches. The transport properties of selenite and its thiol (glutathione and cysteine) reduced forms were examined in isolated enterocytes and hepatocytes. The kinetics of selenite uptake revealed a linear profile in both cell types, suggesting a low affinity transport process. However, the uptake kinetics was different between the two cell types in the presence of extracellular glutathione, since a concentration-dependent Hill uptake kinetics was recorded in enterocytes, while a linear kinetics persisted in hepatocytes. Both cysteine and glutathione augmented cellular selenium accumulation in these cells. The selenium transport was found to be energy independent, but sensitive to the extracellular pH and inorganic mercury. The pharmacological examination suggested that the cellular transport of selenite is primarily mediated by anion transport systems (e.g., sulphite transporters and/or bicarbonate transporters), although cell-specific differences in transport efficiency was apparent. The metabolism of selenite, selenate and selenomethionine in hepatocytes was examined using X-ray absorption near edge structure spectroscopy (XANES). Inorganic and organic forms of selenium appeared to be metabolized <i>via</i> different cellular pathways, as both selenite and selenate were found to be metabolized into elemental selenium, whereas selenocystine constituted the primary metabolite of selenomethionine. My findings also suggested direct enzymatic transformation of selenomethionine into methylselenol at high exposure level, a process that leads to enhanced intracellular reactive oxygen species generation because of the redox-reactive properties of methylselenol. To validate the metabolite profile of selenium observed in <i>in vitro</i> studies, the tissue-specific differences in selenium metabolism <i>in vivo</i> was analyzed in fish exposed to elevated dietary selenomethionine for two weeks. Similar to the observation in hepatocytes, selenocystine and selenomethionine were found to be the major selenium species across tissues, although there were differences in their relative proportion in different tissues. In addition, a good correlation between the total selenium burden and selenocystine fraction was recorded among all the major tissues except gonads. To understand the role of oxidative stress in cellular toxicity of selenium, isolated trout hepatocytes were exposed to increasing dosage of selenite and selenomethionine over a period of 24h. Selenite was found to be 10 times more toxic than selenomethionine to the hepatocytes. Both selenite and selenomethionine induced rapid generation of reactive oxygen species, which subsequently triggered an upregulation of enzymatic antioxidants. Interestingly, a sharp dose-dependent decrease in intracellular thiol redox (reduced to oxidized glutathione ratio) was recorded with exposure to both selenite and selenomethionine, indicating that glutathione plays an important role in mediating selenium toxicity. At the high exposure dosage, both selenium compounds compromised membrane and DNA integrity, disrupted intracellular calcium homeostasis, and induced enzymatic apoptosis pathway, ultimately leading to cell death <i>via</i> aponecrosis. These findings suggested that high selenium exposure causes cellular toxicity by inducing a rapid loss of the intracellular reducing milieu. Overall, the findings from the present study provided novel information on the transport, metabolism and toxicity of selenium in fish. This fundamental information will be useful in understanding the chemical species-specific toxicity of selenium in fish, and may help in identifying cellular biomarkers for assessing the health of selenium-impacted natural fish populations.

Apoptosis is essential not only to the normal development of a multicellular organism but also for the maintenance of tissue homeostasis. This proposal seeks to investigate, in part, the role of oligodendrocyte (OL) apoptosis in myelination. We used an OL-specific conditional knockout animal to study caspase-8 function in OL development; analyzing histological differences in myelination at postnatal day 10 and alterations to OL proliferation, differentiation, and cell death in culture. Our preliminary data suggests that deletion of caspase-8 did not alter OL proliferation or differentiation in culture, but reduced the percentage of apoptotic cells following nutrient deprivation. In vivo, we found an increase in myelinated axons in the spinal cord of caspase-8 deficient mice, indicating a role for caspase-8 in the myelination process. This study also seeks to investigate mechanisms of cell death in OLs, astrocytes, and neurons following oxidative injury. Exposure of primary OLs, astrocytes, and neurons to arachidonic acid (AA) resulted in oxidative stress and cell death. Necrostation-1, the specific inhibitor of receptor interacting protein kinase 1 (RIP-1), markedly prevented AA-induced oxidative death in OLs and astrocytes, but not in neurons. Similarly, we found that blockade of 12-lipoxygenase (LOX) and c-Jun N-terminal kinase (JNK) protected OLs and astrocytes but not neurons against AA toxicity. Consistent with the inability of necrostatin-1 to rescue neurons, we found very low expression of RIP-1 as well as RIP-3 in neurons. Finally, the zinc chelator TPEN effectively abolished AA-induced oxidative death in all three cell types, suggesting zinc release as a common mechanism. Taken together, our findings indicate differences in cell death mechanisms following oxidative injury in astrocytes, OLs, and neurons.

Oxidative stress and mutations in DJ-1, a redox sensitive protein, are linked to Parkinson's Disease. The protective mechanism of DJ-1 is unclear. I hypothesized that: 1) DJ-1 mediates protection by translocating to mitochondria after oxidative stress and, 2) when DJ-1 is downregulated, apoptotic pathways regulated by calcineurin are also downregulated. In PC12 cells and rat cortical neurons, oxidative stress resulted in the upregulation of DJ-1 and increased DJ-1 in the nucleus, but did not increase mitochondrial translocation of DJ-1. In cortical neurons and wildtype mouse embryonic fibroblasts, H2O2 induced cleavage of CnA into an inactive fragment. DJ-1 knockout fibroblasts had less nuclear localization of the transcription factor NFATc4, a substrate of calcineurin involved in apoptosis. H2O2 increased CnA cleavage in DJ-1 knockout fibroblasts, but NFATc4 localization was unchanged. These results suggest that the downregulation of apoptotic pathways regulated by calcineurin may be a compensatory response to the downregulation of DJ-1.

<p>The present research was designed to investigate the mechanisms of cellular transport, metabolism and toxicity of selenium [inorganic (selenite) and organic (selenomethionine)] in a model teleost, rainbow trout (<i>Oncorhynchus mykiss</i>), using both <i>in vitro</i> and <i>in vivo</i> experimental approaches. The transport properties of selenite and its thiol (glutathione and cysteine) reduced forms were examined in isolated enterocytes and hepatocytes. The kinetics of selenite uptake revealed a linear profile in both cell types, suggesting a low affinity transport process. However, the uptake kinetics was different between the two cell types in the presence of extracellular glutathione, since a concentration-dependent Hill uptake kinetics was recorded in enterocytes, while a linear kinetics persisted in hepatocytes. Both cysteine and glutathione augmented cellular selenium accumulation in these cells. The selenium transport was found to be energy independent, but sensitive to the extracellular pH and inorganic mercury. The pharmacological examination suggested that the cellular transport of selenite is primarily mediated by anion transport systems (e.g., sulphite transporters and/or bicarbonate transporters), although cell-specific differences in transport efficiency was apparent. The metabolism of selenite, selenate and selenomethionine in hepatocytes was examined using X-ray absorption near edge structure spectroscopy (XANES). Inorganic and organic forms of selenium appeared to be metabolized <i>via</i> different cellular pathways, as both selenite and selenate were found to be metabolized into elemental selenium, whereas selenocystine constituted the primary metabolite of selenomethionine. My findings also suggested direct enzymatic transformation of selenomethionine into methylselenol at high exposure level, a process that leads to enhanced intracellular reactive oxygen species generation because of the redox-reactive properties of methylselenol. To validate the metabolite profile of selenium observed in <i>in vitro</i> studies, the tissue-specific differences in selenium metabolism <i>in vivo</i> was analyzed in fish exposed to elevated dietary selenomethionine for two weeks. Similar to the observation in hepatocytes, selenocystine and selenomethionine were found to be the major selenium species across tissues, although there were differences in their relative proportion in different tissues. In addition, a good correlation between the total selenium burden and selenocystine fraction was recorded among all the major tissues except gonads. To understand the role of oxidative stress in cellular toxicity of selenium, isolated trout hepatocytes were exposed to increasing dosage of selenite and selenomethionine over a period of 24h. Selenite was found to be 10 times more toxic than selenomethionine to the hepatocytes. Both selenite and selenomethionine induced rapid generation of reactive oxygen species, which subsequently triggered an upregulation of enzymatic antioxidants. Interestingly, a sharp dose-dependent decrease in intracellular thiol redox (reduced to oxidized glutathione ratio) was recorded with exposure to both selenite and selenomethionine, indicating that glutathione plays an important role in mediating selenium toxicity. At the high exposure dosage, both selenium compounds compromised membrane and DNA integrity, disrupted intracellular calcium homeostasis, and induced enzymatic apoptosis pathway, ultimately leading to cell death <i>via</i> aponecrosis. These findings suggested that high selenium exposure causes cellular toxicity by inducing a rapid loss of the intracellular reducing milieu. Overall, the findings from the present study provided novel information on the transport, metabolism and toxicity of selenium in fish. This fundamental information will be useful in understanding the chemical species-specific toxicity of selenium in fish, and may help in identifying cellular biomarkers for assessing the health of selenium-impacted natural fish populations.

Restenosis is one of the major adverse outcomes of Percutaneous Coronary Intervention (PCI). Previous studies have shown conflicting reports for homocysteine as a predictor of restenosis following PCI. The conflicting reports may be due to oxidative factors (stimulation of polymorphonuclear leukocyte [PMNL]-induced reactive oxygen species generation, xanthine- xanthine oxidase, and arachidonic acid metabolism) other than homocysteine which could cause endothelial cell dysfunction leading to restenosis. Malondialdehyde (MDA), a lipid peroxidation product, is a marker for oxidative stress and is related to all oxidative factors. Therefore, it is possible that serum MDA may be a better predictor of restenosis than plasma homocysteine. The purpose of this study is to determine whether or not the pre-procedural serum MDA and plasma homocysteine levels are elevated in patients who develop restenosis post PCI. <p>The study included fifty-one patients undergoing elective PCI who consented to participate in a protocol that was approved by the Ethics Committee of the University of Saskatchewan. Homocysteine and malondialdehyde were measured in the plasma and serum respectively. Blood samples were collected pre-procedural, 0 time, 8 hours, 24 hours, and 6 months post-procedure. Exercise tolerance tests were performed at two weeks, and six months post-procedure to determine if there was any evidence of restenosis. <p>The results of the study showed that pre-procedural values of plasma homocysteine in the restenosis and non-restenosis groups were 10.37 ± 0.46 and 10.73 ± 0.49 respectively. These values were not significantly different (p=0.60) between the groups. The pre-procedural levels of plasma homocysteine were not significantly different (p=0.08) from the post-PCI values of those patients who did not develop restenosis at the 6-month time interval. However, the pre-procedural levels of plasma homocysteine were significantly different from the post-PCI values of those patients in the restenosis group at the 24hr (p=0.04) and 6-month (p=0.002) time intervals. In the restenosis group there was a significant increase (24%) after six months in the values of homocysteine from the pre-procedural levels. Thus, this indicates that restenosis is associated with higher post-PCI levels of homocysteine. <p>The pre-procedural levels of serum MDA in the restenosis and non-restenosis groups were 0.124± 0.16 and 0.147± 0.02 respectively. There was no significant difference (p=0.60) between the two groups. There was also no significant difference (p=0.053) between the pre-procedural values and the 6-month post-PCI values in those patients who did not develop restenosis. However, there was a significant difference (p=0.001) between the pre-procedural values and the 6-month post-PCI values in those patients who developed restenosis. The levels of serum MDA in patients with restenosis at 6-months increased by 109% and were significantly different (p=0.001) in the restenosis group. <p>The results suggest that pre-procedural levels of plasma homocysteine and serum MDA were not predictors of restenosis following PCI. However, the post-PCI six-month levels of both homocysteine and MDA are predictors of restenosis. Moreover, the post-PCI levels of MDA were better predictors of restenosis than the post-PCI levels of homocysteine because the increase in MDA levels were greater at six months than the rise in homocysteine levels at the same time interval.

Spinal cord injury occurs at a rate of 11.5 - 53.4 per million in developed countries with great emotional and financial consequences. The damage caused by the initial injury is followed by secondary damage, a complex cascade of mechanisms including ischemia, oxidative stress, inflammation and apoptosis. Although nothing can be done to reverse the initial damage to the spinal cord once it occurs, the secondary damage can be targeted by therapeutics to improve recovery. Following injury, concentrations of the potent antioxidant glutathione (GSH) are decreased in the spinal cord which potentiates mechanisms of secondary damage. In an attempt to maintain the GSH concentrations, the non-essential amino acid glutamine was tested as it was shown to increase GSH concentrations both in vivo and in vitro. Glutamine is being used extensively in clinical research in an expansive number of physiological and pathological conditions including brain trauma. To examine the therapeutic potential of glutamine after spinal cord trauma, two compression injury models, the modified aneurysm clip and the modified forceps, were used to induce an injury in male Wistar rats. We have demonstrated the ability of glutamine treatment (1 mmol/kg), given 1 hour after a 30 g aneurysm clip injury to increase GSH not only in whole blood samples but within the spinal tissue at the site of injury. Increasing GSH in this way also resulted in improved locomotor scores and maintenance of white matter tissue at the injury epicenter. Experiments using the forceps model were then performed to determine if the potency of glutamine treatment would be carried over to a different model and at a variety of severities. Glutamine, again, demonstrated the ability to improve maintenance of whole blood GSH, locomotor scores and tissue histology. In our experiments, glutamine has proven to be a potent therapeutic for spinal cord injury with an effect that is matched by few compounds currently being studied and well exceeding the standard therapeutic, methylprednisolone. Given the breadth of knowledge regarding the effects of glutamine clinically in numerous paradigms and the potency of the therapeutic effect seen in these studies, we believe that glutamine is fit for clinical trial and has a high potential for success.