The process of autophagy is defined as the degradation of cellular cytoplasmic constituents via a lysosomal pathway. Herein I sought to examine the regulation of autophagy in the placental pathologies preeclampsia (PE) and intrauterine growth restriction (IUGR). I hypothesized that the Bcl-2 family proteins Mcl-1L and MtdL regulate placental autophagy and contribute towards dysregulated autophagy in PE. My results demonstrate that Mcl-1L acts to repress autophagy via a Beclin 1 interaction, while MtdL induces autophagy when it interacts with Mcl-1L. My data indicate that while autophagy is elevated in PE, a pathology characterized by oxidative stress, it is decreased in IUGR, a hypoxic pathology. Treatment with sodium nitroprusside to mimic PE caused a decrease in Mcl-1L and an increase in MtdL levels in response to oxidative stress, thereby inducing autophagy. Overall, my data provide insight into the molecular mechanisms contributing to the pathogenesis of preeclampsia.

Subarachnoid hemorrhage (SAH) comprises 7% of all stroke cases, and is associated with a disproportionately high morbidity and mortality with few therapeutic options available. The goal of this project was to understand the mechanism of neurological deterioration after experimental SAH, with a focus on cerebral vasospasm and brain injury after SAH. We tested the hypothesis that endothelial nitric oxide synthase (eNOS) is upregulated and uncoupled after SA, resulting in exacerbated neurological injury in a mouse model of SAH. The project entailed the investigation of eNOS-dimer uncoupling, its association with oxidative and nitrosative stress in the brain parenchyma and finally its association with secondary complications after SAH. In our studies we demonstrated the crucial role eNOS plays in anti-microthromboembolism, anti-apoptosis and maintenance of physiological superoxide (O2-)/NO balance. This study suggests that SAH up-regulates and disrupts eNOS, producing peroxynitrite (OONO-) and other radicals that further exacerbate the oxidative insult and neurological injury.

Type 1 and type 2 diabetes are characterized by an excessive loss of insulin producing β-cells. β-cells are particularly susceptible to increased oxidative stress induced apoptosis due to low expression of major antioxidants. Peroxiredoxin-2 (PRDX2) belongs to a group of antioxidants with antiapoptotic roles. Preliminary data indicate PRDX2 is expressed in the β-cells. Endogenous PRDX2 in the β-cell line MIN6 is found to decrease under oxidative stress conditions. I hypothesize that PRDX2 has a role in protecting β-cells against oxidative stress induced apoptosis. Overexpression or knockdown strategies were used to examine the role of PRDX2 in insulin-secreting MIN6 cells treated with various stimuli (cytokines, palmitate, streptozotocin) to induce apoptosis. Results showed that PRDX2 overexpression decreased oxidative stress induced apoptosis markers and cell death indicators, whereas knockdown of PRDX2 exaggerated oxidative stress induced toxicity. These findings suggest that PRDX2 plays a protective role in pancreatic β-cells under oxidative stress conditions.

The etiology of bipolar disorder (BD) is multidimensional and thought to involve several factors that increase neuronal oxidative stress and disrupt intracellular calcium homeostasis. As calcium-permeable canonical transient receptor potential channels (TRPC) have been linked to bipolar pathophysiology, I sought to determine whether oxidative stress affects TRPC3/TRPC5/TRPC6 expression and/or function. Chronic (4-day) but not acute (24-hour) rotenone-induced oxidative stress dose-dependently reduced TRPC5 and TRPC6 protein levels in primary rat cortical neurons. A decrease in TRPC5 mRNA levels was only found following acute but not chronic rotenone whereas TRPC6 mRNA levels did not change significantly with either treatment. Reduced TRPC3 function was seen after chronic stress when stimulated by TRPC3/6 activator, 1-oleoyl-2-acetyl-sn-glycerol. Lithium pre-treatment attenuated the rotenone-induced reduction in TRPC3 but not TRPC6 protein levels. These results suggest TRPC subtypes are differentially regulated by oxidative stress and support a potential mechanistic link between oxidative stress and calcium dyshomeostasis in BD.

Heavy metals, such as copper and cadmium have been linked to a number of cellular dysfunctions in single and multicellular organisms that are associated with apoptosis. The yeast, Saccharomyces cerevisiae, provides a valuable model for elucidating apoptosis mechanisms, and this study extends that capability to Cu and Cd-induced apoptosis. We demonstrate that S. cerevisiae undergoes a glucose-dependent, programmed cell death in response to low cadmium concentrations, which is initiated within the first hour of Cd exposure. The response was associated with induction of the yeast caspase, Yca1p, and was abolished in YCA1∆ mutant. Other apoptotic markers, including sub-G1 DNA fragmentation and hyper-polarization of mitochondrial membranes, were also evident among Cd-exposed cells. We also show that low levels of copper can induce a similar apoptotic response in yeast within the first hour of exposure. Such cellular responses were verified by analyzing mitochondrial perturbation, generation of superoxide ions, activation of the yeast caspase1, and the eventual fragmentation of nuclear DNA (through TUNEL). In analyzing the response of yeast to the different metals, we also demonstrated that the metal-induced PCD is instigated through the sequential activity of at least two caspase-like proteins (i.e., Yca1 and Atg4), both of which appear to be in involved in the process of inducing mitochondrial stress. The additional caspase-like activity is shown to be derived from an enzyme involved in the latter stages of autophagy (Atg4), and provides an intriguing association of apoptosis with autophagy. Here we also demonstrate that metals such as copper and cadmium causes oxidative damage to mitochondrial proteins. Such oxidative attack is targeted and we show that oxidation of certain crucial proteins is required for apoptosis upon metal exposure. By showing that such targeted protein oxidation is dependent on YCA1 and ATG, we also confirm the finding that in yeast that have been exposed to a heavy metal, YCA1 and ATG are essential for damaging mitochondria and to initiate apoptosis. These novel findings highlight several new perspectives about the mechanism of metal-dependent apoptosis, while opening up future analyses to the power of the yeast model system.

Saccharomyces cerevisiae exhibits an apoptotic response upon exposure to toxic metals such as cadmium (Cd) and copper (Cu). Preliminary findings indicate that this response is dependent –to some extent- on the presence of a fermentable carbon source, glucose. To investigate this dependency we monitored the apoptotic response to both metals in the presence and absence of glucose and have shown that glucose is absolutely necessary in order to induce apoptosis in yeast at least during the exposure to metal. We have also looked at the biochemical changes that are taking place in yeast when treated with Cd using Fourier Transform Infra-Red (FTIR) Spectroscopy. Our results suggest that there are definitive changes in cellular activities that are discernable at 1660-1640cm-1 (amide I), 1540-1510cm-1 (amide II) and 1140-1080cm-1(DNA absorption bands).

Primary brain injury in stroke is followed by oxidative stress and further neural damage. Glutathione (GSH) is critical in antioxidant defense. Since cysteine is limiting in GSH synthesis, Phase 1 of this study investigated the effect of a dietary sulphur amino acid deficiency (-SAA) on neural damage in global hemispheric hypoxia-ischemia (GHHI). Rats were fed a -SAA or control diet for 6 days, and subjected to GHHI after 3 days. Histologically evaluated neural damage at 7 days post hypoxia-ischemia was greater in -SAA rats. Brain GSH concentration was decreased in -SAA rats 3 days after ischemia. A cysteine precursor, L-2-oxothiazolidine-4-carboxylic acid (OTC) administered to -SAA rats did not ameliorate neural damage. GSH is decreased by protein-energy malnutrition (PEM) in some tissues. Phase 2 investigated the effect of PEM on brain oxidative stress, neural damage and behaviour after global ischemia in adult male gerbils. In a 2x2 factorial design, gerbils were fed an adequate protein (12%; C) or low protein (2%; PEM) diet for 4 weeks, then subjected to transient ischemia (I) or sham surgery (S). After 12 hours of reperfusion, brain from half the gerbils was collected for biochemical analyses. Remaining gerbils were fed pre-surgery diets for 10 more days. To assess functional consequences of ischemia, gerbils were placed in an open field on Days 3, 7 and 10 after surgery. On Day 10, viable hippocampal CA1 neurons were counted. C-I gerbils did not habituate as readily in the open field on day 3 as C-S, but normalized by day 7. PEM-I gerbils failed to habituate by day 10, traveled greater distance than other gerbils and 7 of 12 displayed thigmotaxis, a <i>wall-hugging</i> preference for the outer perimeter of the open field. CA1 neuron loss in I was 61.5% of S, but unaffected by PEM. Four of 12 PEM-I gerbils had marked increases in hippocampal glia. Hippocampus protein thiols were reduced by PEM and by ischemia, consistent with oxidative stress. GSH concentration, glutathione reductase activity and thiobarbituric acid reactive substances were not significantly affected by PEM or ischemia. Findings from these two studies suggest well-nourished but not nutritionally-deficient rodents tolerate a mild brain insult. This is clinically relevant because many elderly stroke victims suffer from PEM at the time of ischemia, which may compromise recovery.

Primary brain injury in stroke is followed by oxidative stress and further neural damage. Glutathione (GSH) is critical in antioxidant defense. Since cysteine is limiting in GSH synthesis, Phase 1 of this study investigated the effect of a dietary sulphur amino acid deficiency (-SAA) on neural damage in global hemispheric hypoxia-ischemia (GHHI). Rats were fed a -SAA or control diet for 6 days, and subjected to GHHI after 3 days. Histologically evaluated neural damage at 7 days post hypoxia-ischemia was greater in -SAA rats. Brain GSH concentration was decreased in -SAA rats 3 days after ischemia. A cysteine precursor, L-2-oxothiazolidine-4-carboxylic acid (OTC) administered to -SAA rats did not ameliorate neural damage. GSH is decreased by protein-energy malnutrition (PEM) in some tissues. Phase 2 investigated the effect of PEM on brain oxidative stress, neural damage and behaviour after global ischemia in adult male gerbils. In a 2x2 factorial design, gerbils were fed an adequate protein (12%; C) or low protein (2%; PEM) diet for 4 weeks, then subjected to transient ischemia (I) or sham surgery (S). After 12 hours of reperfusion, brain from half the gerbils was collected for biochemical analyses. Remaining gerbils were fed pre-surgery diets for 10 more days. To assess functional consequences of ischemia, gerbils were placed in an open field on Days 3, 7 and 10 after surgery. On Day 10, viable hippocampal CA1 neurons were counted. C-I gerbils did not habituate as readily in the open field on day 3 as C-S, but normalized by day 7. PEM-I gerbils failed to habituate by day 10, traveled greater distance than other gerbils and 7 of 12 displayed thigmotaxis, a <i>wall-hugging</i> preference for the outer perimeter of the open field. CA1 neuron loss in I was 61.5% of S, but unaffected by PEM. Four of 12 PEM-I gerbils had marked increases in hippocampal glia. Hippocampus protein thiols were reduced by PEM and by ischemia, consistent with oxidative stress. GSH concentration, glutathione reductase activity and thiobarbituric acid reactive substances were not significantly affected by PEM or ischemia. Findings from these two studies suggest well-nourished but not nutritionally-deficient rodents tolerate a mild brain insult. This is clinically relevant because many elderly stroke victims suffer from PEM at the time of ischemia, which may compromise recovery.

Inflammatory diseases such as hypertension are associated with high levels of oxidative stress. Characteristic of oxidative stress is the inflammatory acute phase protein response. Oxidative stress and its accompanied inflammation can be reduced via phase 2 enzyme induction. Broccoli sprouts, a rich source of phase 2 enzyme inducers such as isothiocyanates, can be incorporated into the diet to increase phase 2 enzymes. <p> The hypothesis of this study is that, the dietary intake of dried broccoli sprouts, by inducing liver phase 2 enzymes, will decrease oxidative stress and the acute phase response in the blood of spontaneously hypertensive stroke-prone rats. <p> Spontaneously hypertensive stroke-prone rats (SHRsp) and Sprague Dawley (SD) rats were placed either on a control diet of modified AIN-93G or an experimental diet of modified AIN-93G supplemented with dried broccoli sprouts. The following parameters were examined: 1. Isothiocyanate absorption (an increased level of dithiocarbamates is reflective of ITC absorption), 2. Oxidative stress (a reduction in oxidative stress is evidenced by an increase in plasma protein thiols and blood glutathione (GSH)), 3. Acute phase proteins (a decreased APR is reflected by an increase in plasma albumin and a decrease in ceruloplasmin), 4. Activity of phase 2 enzymes (increased phase 2 enzyme induction results in higher activities of liver quinone reductase (QR), glutathione-S-transferase (GST) and glutathione reductase (GR)). <p> My experimental results demonstrated that broccoli sprout feeding results in higher protein thiol levels in female SHRsp and higher blood GSH levels in males but no acute phase protein changes were observed in either male or female SHRsp. Broccoli sprout feeding caused higher QR and lower GST activities in female SHRsp but did not affect the activities of phase 2 enzymes in male SHRsp. The activities of GST and QR were higher in SD rats than in SHRsp. Levels of dithiocarbamates were higher in the broccoli fed group than in the control fed group. <p> The results from this study do not present a clear pattern to support the hypothesis that dietary intake of broccoli sprouts by inducing phase 2 enzymes will decrease parameters of oxidative stress and the acute phase response. <p> In conclusion, there is an interactive role played by animal gender and the induction of phase 2 enzymes by dried broccoli sprouts.

Recent epidemiological evidence suggests that many chronic health disorders in the developed world are associated with endogenous toxins formed from the Western diet. The Western diet, which encompasses calorie dense foods, processed foods and increased quantities of red meat, can cause intracellular oxidative stress through increased formation of reactive oxygen species(ROS) and reactive carbonyl species (RCS). A number of micronutrients have been investigated for their protective capacity in in vitro and in vivo models of oxidative stress. This thesis investigated the cytotoxic targets of Fenton-mediated ROS and RCS and the subsequent protective mechanisms of vitamins B1 (thiamin) or B6 (pyridoxal, pyridoxamine or pyridoxine) in an isolated rat hepatocyte model. The approach was to use an “accelerated cytotoxicity mechanism screening” technique (ACMS) to develop an in vitro cell system that mimicked in vivo tissue cytotoxicity. Using this technique, we investigated the protective mechanisms of vitamins B1 and/or B6 against the cytotoxic effects of two endogenous toxins associated with the Western diet: 1) RCS, as exemplified by glyoxal, a glucose/fructose autoxidation product and 2) biological ROS induced by exogenous iron. Firstly, we developed an understanding of the sequence of events contributing to glyoxal-induced oxidative stress, with a focus on protein carbonylation. Next, we determined the mechanisms by which carbonyl scavenging drugs (vitamin B6 included) protected against the intracellular targets of glyoxal-induced toxicity. Our results suggested that the agents used were cytoprotective by multiple mechanisms and glyoxal trapping was only observed when the agents were administered at concentrations equal to glyoxal. We also evaluated the protective capacity of vitamins B1 and B6 against iron-catalyzed cytotoxicity and found that hepatocytes could be rescued from protein and DNA damage when vitamins B1 or B6 were added up to one hour after treatment with iron. The vitamins also varied in their primary mechanisms of protection. Our improved understanding of Western diet-derived endogenous toxins enabled us to identify and prioritize the specific inhibitory mechanisms of vitamins B1 or B6. The ability to delay, inhibit or reverse toxicity using multi-functional B1 or B6 vitamins could prove useful as therapy to minimize oxidative stress in diet-induced chronic conditions.