Despite extensive research, the mechanisms behind cardiovascular effects of subchronic environmental tobacco smoke (ETS) remain unclear, but may be related to ETS-induced inflammation and oxidative stress. Additionally, the protective role of resveratrol (RES), a natural antioxidant available in red grapes, is controversial. We hypothesized that the polycyclic aromatic hydrocarbon (PAH) component of ETS is responsible for causing adverse cardiovascular effects. We also hypothesized that the administration of RES is protective against the adverse cardiovascular effects of ETS. In order to address these hypotheses, male juvenile pigs (4-weeks old) were exposed to ETS or ambient air for 28 consecutive days (1 hr/day) and effects compared to 7 days of i.v. injection of the PAH, benzo-a-pyrene (BAP; 5 mg/kg daily). In another experiment, pigs were sham-exposed or ETS-exposed, with or without oral RES treatment (5mg/kg daily). In all experiments, endothelial and left ventricular function were assessed by flow mediated dilation (FMD), and echocardiography, respectively, while blood pressure was evaluated by oscillometry. At the termination of each experiment, serum nitrotyrosine, total nitrate/nitrite (NOx) and C-reactive protein (CRP) were measured as well as hepatic and pulmonary ethoxyresorufin-o-deethylase (EROD) activity to indicate cytochrome P450 1A1 (CYP1A1) expression. Finally, the correlation between pulmonary inflammation and adverse cardiovascular effects was investigated by measuring total and differential white blood cell (WBC) count as well as leukocyte elastase activity in bronchoalveolar lavage fluid at the termination of each experiment. ETS exposure, but not BAP treatment, resulted in a significant impairment of FMD (P<0.0001) and increased left ventricular end diastolic volume (P=0.0032). Cotreatment with RES failed to restore the ETS induced impairment of FMD (P>0.05). However, a trend pointing to an increase in ejection fraction (EF) was noted (P=0.072). ETS, BAP and RES treatments failed to have any effect on blood pressure (P>0.05). BAP injection caused a significant increase in serum nitrotyrosine (P=0.0146) and CRP (P=0.012), but not serum NOx levels (P>0.05). In contrast, ETS exposure resulted in a significant increase in CRP serum levels (P=0.0092), a trend pointing to increased serum nitrotyrosine (P=0.105), and no change in serum NOx levels (P>0.05). The increased nitrotyrosine and CRP with ETS exposure was not reversed by RES administration (P>0.05). ETS exposure increased EROD activity in the lung (P=0.0093), but not the liver (P=0.12). In contrast, BAP treatment had the opposite effect (lung EROD: P=0.621, liver EROD: P=0.01), while RES administration had no effect (P>0.05). ETS exposure (P=0.0139), but not BAP treatment (P=0.723), resulted in increased WBC count in BAL fluid which was not affected by RES administration (P>0.05). These results show that ETS exposure causes lung inflammation, systemic inflammation, oxidative stress-mediated inactivation of nitric oxide and impaired endothelial function. In contrast, BAP failed to alter endothelial function, downstream of the lung, despite systemic inflammation and increased oxidative stress. Furthermore, RES failed to restore endothelial function, or decrease systemic inflammation and oxidative stress. Taken together, these results suggest either that pulmonary inflammatory responses or pulmonary increases in CYP1A1 activity may be more important links to endothelial dysfunction than systemic inflammation and nitric oxide bioactivity. The beneficial effects of RES by itself are manifested only at the cardiac level by improving the ejection fraction, but the work in this thesis failed to detect any ability of RES to ameliorate ETS cardiovascular effects.

A series of seven experiments were conducted to evaluate the risk of acute (sudden death syndrome; SDS) or chronic (congestive heart failure; CHF) heart failure associated with dietary over-supplementation of vitamin A, vitamin D3, vitamin E, vitamin C or cardiotoxic factors present in meat meal. The risk of heart failure associated with the above mentioned dietary factors was tested followed by gross, microscopic, ultrastructural and biochemical investigation for mechanisms associated with mentioned risk factors. Simultaneously, the molecular mechanisms underlying the deterioration of heart function in fast-growing commercial broilers were studied. Each compound was tested separately at a concentration higher than the recommended levels. The basic experimental unit comprised groups of 40 to 50 day old male broiler chickens at the start of experiment. Lowered thermal brooding temperature protocol, an approach resulting in clinical manifestation of heart failure in practically all broilers predisposed to heart disease, was used.<p> Broilers fed the vitamin D3 enriched diet were 2.5 fold more likely to succumb to acute heart failure (p<0.05). Simulated stress challenge with epinephrine revealed that broilers fed excess of vitamin D3 were more susceptible to ventricular arrhythmia. The risk of CHF was higher (P<0.05) in broilers fed the vitamin D3, vitamin A and methanol soluble extract from meat meal enriched diets as compared to groups fed the control diet. The incidence of CHF in broilers fed the diet fortified with vitamin E was not significantly different as compared to the control group, whereas supplementation of vitamin C in the diet tended (p=0.10) to reduce the incidence of CHF. The level of malondialdehyde equivalents, an indicator of lipid peroxidation, was significantly higher (p<0.05) in myocardium of broilers developing CHF irrespective of dietary factors. Antioxidant vitamins (E and C) did not prevent lipid peroxidation in broilers developing CHF. <p>In conclusion, the present findings indicate that over-supplementation of vitamin A and D3 increases the risk of heart failure in broilers. Meat meal contains some unknown cardiotoxic factors, capable of precipitating heart conditions in susceptible broilers. Oxidative stress is involved in the pathogenesis of heart failure in broilers, but supplementation of antioxidant vitamins did not prevent oxidative damage in broilers that developed CHF. The oversupplementation of vitamins (A and D3) should not be encouraged in broilers diet as they may increase the economic losses to broilers industry subsequent to heart related mortalities/morbidities.

Every year, several thousand individuals suffer spinal cord injury (SCI) in North America, while 1.5 million suffer traumatic brain injury in the U.S.A. alone. Primary mechanical trauma to the CNS is followed by a complex pathology, including vascular dysregulation, ischemia, edema and traumatic hemorrhage. Secondary damage is to a large extent caused by oxidative stress and inflammatory processes, resulting in necrosis and apoptosis of neural cells. If secondary tissue injury could be limited by interference with any of the pathomechanisms involved, preservation of structure and function would increase the potential for functional recovery. Experiments performed in other laboratories have shown that the polyphenolic flavonoid quercetin acts as an anti-oxidant and anti-inflammatory, reduces edema formation and apoptotic cell death. Quercetin is also an excellent iron chelator. This action profile suggested a high therapeutic potential for acute CNS trauma. Therefore, I used models of both spinal cord injury and head trauma in adult male rats to test the hypothesis that administration of quercetin is beneficial for the therapy of acute traumatic CNS injury. While the primary focus of my work was on therapy of acute traumatic spinal cord injury, quercetin was also evaluated in the settings of chronic SCI and acute head trauma. I found that, in a rat model of mid-thoracic spinal cord compression injury, 1) administration of quercetin, starting 1 hr after injury and continued every 12 hr, improved recovery of motor function in the hind limbs in more than half of the injured animals to a degree that allowed previously paraplegic animals to step or walk. The minimum quercetin dose that was efficacious was 5 µmol/kg. The minimum treatment duration for optimal outcome was determined to be 3 days. In control animals, some spontaneous recovery of motor function did occur, but never to an extent that allowed animals to step or walk. Quercetin administration was associated with more efficient iron clearance from the site of injury, decreased inflammatory response as reflected in decrease of myeloperoxidase activity and decreased apoptosis of neural cells at the site of injury. 2) Quercetin administered in the same injury model as late as 2 weeks after injury, given in a higher dose than that used for treatment in the acute phase, still resulted in significant recovery of motor function in 40% of the injured animals, although at a lower level of performance, when compared to early onset of treatment. 3) Quercetin administered after moderate fluid percussion brain injury resulted in decreased oxidative stress, as reflected in higher tissue glutathione levels at the site of injury. In animals receiving quercetin, the amplitude of compound action potentials was significantly better maintained at 24 hr and 72 hr after injury than in saline-treated control animals. My experiments have shown that the flavonoid quercetin is neuroprotective in a rat model of brain trauma and in a rat model of spinal cord injury. My data show that administration of quercetin after CNS trauma promotes iron clearance, decreases oxidative stress and inflammation. Quercetin also decreases apoptotic cell death following neurotrauma. These results suggest that quercetin may be a valuable adjunct in the therapy of acute CNS trauma. There is a possibility that administration of quercetin may be beneficial even in certain settings of chronic CNS trauma. These conclusions are based solely on the results from animal experiments. However, the fact that few adverse reactions have been noted to date in either animal experiments or human trials targeting other diseases is encouraging for the progression to human clinical trials for patients with spinal cord injury.

The pathology of multiple sclerosis (MS) is characterized by an inflammatory mononuclear infiltration in the white matter. There has been converging evidence of the oxidative stress playing a role in the onset and progression of MS. We postulated that the decreasing oxidative stress might help in the management of MS. We know that the induction of phase 2 enzymes decreases the oxidative stress. The experimental allergic encephalomyelitis (EAE) induced in the Lewis rats were used to test this hypothesis. The 24 animals were placed into two groups: 1) those on normal rat chow, 2) those on rat chow containing 7.5 g/kg of tetra-butylhydroxyanisole (BHA), a food preservative. All the animals were administered 100 µg of guinea pig myelin basic protein in their tails to induce EAE and examined daily in a double blinded fashion. On 29th day of the induction, the animals were sacrificed, blood collected for glutathione (GSH) measurements and tissues collected for histology. All the animals, regardless of their diet status, developed symptoms of EAE on different days ranging from tail weakness to hind limb paralysis and all of them reached remission of acute EAE before the 28th day of induction. The non-BHA fed animals developed hind limb weakness in 8 animals and hind limb paralysis in 4 cases, while that of BHA fed group developed tail paralysis in 2, hind limb weakness in 2 and hind limb paralysis in 8 cases. The histology of the non-BHA group correlated well with the clinical symptoms of perivascular mononuclear infiltration. However, the BHA group revealed complete pathological recovery. Animals with BHA in the diet had significantly raised GSH, indicating the induction of phase 2 enzymes. We conclude that dietary phase 2 enzyme inducers show potential therapeutic benefits in EAE and should be examined for this role in MS.

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.

Several lines of evidence implicate oxidative stress in the pathophysiology of bipolar disorder (BPD). The mood stabilizers lithium and valproate have been shown to protect against oxidative stress-induced cell death. This study examined whether an increase in cellular reductive potential due to glutathione (GSH) synthesis up-regulation underlies this neuroprotective effect. Using primary rat cortical neurons as a model, this study demonstrated that unlike lithium and valproate, carbamazepine and lamotrigine do not exert neuroprotective effects against H2O2-induced cell death. Moreover, the level of GSH and the GSH:GSSG ratio in neurons and in rat brain remained unchanged following chronic treatment with either lithium or valproate. Similarly, this study did not find a significant effect of treatment on the expression of genes encoding γ-glutamylcysteine ligase sub-units, Gclc and Gclm, in both neurons and the rat brain. These findings suggest that other molecular targets of lithium and valproate likely mediate the observed neuroprotective effects.

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.

Several lines of evidence implicate oxidative stress in the pathophysiology of bipolar disorder (BPD). The mood stabilizers lithium and valproate have been shown to protect against oxidative stress-induced cell death. This study examined whether an increase in cellular reductive potential due to glutathione (GSH) synthesis up-regulation underlies this neuroprotective effect. Using primary rat cortical neurons as a model, this study demonstrated that unlike lithium and valproate, carbamazepine and lamotrigine do not exert neuroprotective effects against H2O2-induced cell death. Moreover, the level of GSH and the GSH:GSSG ratio in neurons and in rat brain remained unchanged following chronic treatment with either lithium or valproate. Similarly, this study did not find a significant effect of treatment on the expression of genes encoding γ-glutamylcysteine ligase sub-units, Gclc and Gclm, in both neurons and the rat brain. These findings suggest that other molecular targets of lithium and valproate likely mediate the observed neuroprotective effects.