Browsing by Subject "Oxidative Stress"
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Item A Big Response to a ?Small? Problem: Identifying the Oxidative Potential of Nanomaterials and the Physicochemical Characteristics That Play a Role(2012-02-14) Berg, James MichaelNanotechnology as a science is emerging rapidly. As materials are synthesized and utilized at the nanometer size scale, concerns of potential health and safety effects are arising. In an effort to elucidate the physicochemical characteristics of nanoparticles influential in toxicological studies, surface properties of metal oxide and carbonaceous nanoparticles were measured. These properties include zeta potential, dissolution and surface-bound chemical components. Subsequently, the role of these properties in oxidative stress was examined in vitro. This work identifies the influence that pH has on the zeta potential of nanoparticles. The zeta potential has the ability to alter colloidal stability, as the largest nanoparticle agglomerate is seen at or near the isoelectric point for each of the particles tested. Furthermore, it was observed that metal oxide nanoparticles which exhibit a charged surface at physiological pH, lead to decreased in vitro cellular viability as compared to those that were neutral. Thus, nanoparticle zeta potential may be an important factor to consider when attempting to predict nanoparticle toxicity. Real world exposure to nanoparticles is a mixture of various particulates and organics. Therefore, to simulate this particle mixture, iron oxide (Fe2O3) and engineered carbon black (ECB) were utilized in combination to identify potential synergistic reactions. Following in vitro exposure, both nanoparticle types are internalized into endosomes, where liberated Fe3+ reacts with hydroquinone moieties on the ECB surface yielding Fe2+. This bioavailable iron may then generate oxidative stress through intracellular pathways including the Fenton reaction. As oxidative stress is common in particulate toxicology, a comparison between the antioxidant defenses of epithelial (A549) and mesothelial (MeT-5A) cell lines was made. The A549 cell line exhibits alterations in the NRF2-KEAP1 transcription factor system and therefore retains high basal levels of phase II antioxidants. Both cell types were exposed to 33 nm silica where intracellular oxidant generation coupled with markers of oxidative stress were observed. While the MeT-5A cells exhibited a decrease in cell viability, the A549 cell line did not. Therefore, proper characterization of both material and biological systems prior to toxicity testing will help to further define the risks associated with the use of nanotechnology.Item Differential Regulation of PHOSPHATIDYLINOSITOL PHOSPHATE 5 Kinase beta during Hypertonic and Oxidative stress(2009-01-08) Chen, Mark; Yin, HelenOf the total amount of phospholipids in the cell, phosphoinositides account for only a small fraction, yet they play an indispensable role in regulating cellular homeostasis. The phosphatidylinositol derivative, PI(4,5)P2 (PIP2)is an essential mediator of cellular processes such as signal transduction, membrane traffic, actin cytoskeleton, ion homeostasis, growth and apoptosis. Despite the importance of this signaling molecule in controlling a diverse array of functions, little is known about the regulation of the kinases that produce PIP2 as well as its precursor, phosphatidylinositol 4 phosphate (PI4P). Our laboratory found that hypertonic and oxidative stress increases and decreases PIP2 levels, respectively. In this thesis, I sought to understand how these two different types of stresses alter PIP2 by examining the effects on the phosphatidylinositol 5-phosphate 4 kinases that generate PIP2. Using RNAi and in vitro kinase activity assays, I found that PIP5Kbeta is regulated by both stimuli in opposite directions and is responsible for the net change in PIP2 levels. Hypertonic stress activates PIP5Kbeta through PP1 phosphatase dependent ser/thr dephosphorylation which leads to increased kinase activity and plasma membrane localization. The PIP2 produced by PIP5Kbeta during this event leads to an increase in actin polymerization and stress fiber formation. Conversely, oxidative stress leads to a decrease in PIP5Kbeta activity, membrane detachment and stress fiber dissolution. Oxidative stress induces simultaneous tyrosine phosphorylation and ser/thr dephosphorylation, establishing that tyrosine phosphorylation decreases PIP5Kbeta activity and is the dominant modification of PIP5Kbeta that overrides the activating effects of ser/thr dephosphorylation. I further identified the tyrosine kinase Syk as the kinase responsible for PIP5Kbeta phosphorylation during oxidative stress. The phosphorylation site was mapped to position Y105 which was shown to control both activity and localization of PIP5Kbeta. Oxidative stress also increased PI4P which is likely to be at least partly due to activation of a PI4K. Using RNAi and wortmannin to selectively inhibit the activity of different PI4K isoforms, I sought to identify the PI4K isoform that is responsible for this increase.Item Effect of Catalase/Superoxide Dismutase Mimetic EUK-134 on Damage, Inflammation, and Force Generation of the Diaphragm Muscle in mdx Mice(2010-10-12) Kim, Jong HeeDuchenne muscular dystrophy (DMD) is the most devastating form of muscular dystrophy caused by a mutation in the dystrophin gene. Defects in the dystrophin gene in DMD, are homologous to that found in mdx mice, and result in profound muscle damage, inflammation and weakness in diaphragm and limb muscles. Dystrophin, a scaffolding protein located in the sarcolemmal cytoskeleton, helps cells to maintain their structural integrity and associates with critical cell signaling molecules that regulate cell growth and repair (e.g., nNOS). While the contributing mechanisms leading to DMD-induced degenerative muscle function and damage are multi-factorial, elevated oxidative stress has been proposed as a central mechanism. In contrast, antioxidants can attenuate muscle damage as well as improve contractile function in dystrophin-deficient muscles. However, it is unknown if oxidative stress is a causal factor in dystrophin-deficient diaphragm muscle pathology and specifically targeted antioxidant (e.g., EUK-134) treated early in the course of the disease (3-4 weeks) can modulate oxidative stress, functional damage and weakness in mdx diaphragm. Therefore, the purpose of this study was to determine the effects of catalase/superoxide dismutase mimetic EUK-134 on damage, inflammation, and contractile function of the diaphragm muscle in mdx mice. We hypothesized that (a) EUK-134 would attenuate muscle damage and oxidative stress in mdx diaphragm, (b) EUK-134 would reduce inflammatory cells and an important transcription factor including nuclear factor-kappaB (NF-kB) in mdx diaphragm and (c) EUK-134 would restore proteins that attach to dystrophin such as nNOS and cytoskeletal proteins back to sarcolemmal region and improve muscle contractility in mdx diaphragm. C57BL/10ScSn wild type and mdx mice were given EUK-134 (30mg/kg, i.p., injection) beginning at 20 days of age for 8 days. The mice were euthanized and the diaphragm muscle was harvested at 4 weeks of age, the time of peak inflammation, and analyzed to measure myofiber inflammation, NF-kB activation, cytoskeletal proteins and oxidative stress markers using Western immunoblotting, ELISA, immunofluoresence, and immunohistochemistry. We found that EUK-134 ameliorated muscle damage and oxidative stress in mdx diaphragm. EUK-134 protected against inflammation by decreasing NF-kB activation in the nucleosome fraction of mdx diaphragm. Further, EUK-134 partially rescued nNOS and k-1 syntrophin back to sarcolemmal membranes and recovered force generation even in acute application in vitro in mdx diaphragm. These results are the first to demonstrate a causal relationship between oxidative stress and pathology caused by dystrophin-deficient diaphragm muscle. Moreover, the data indicate that EUK-134 has a protective effect against muscle damage, inflammation, and contractility in mdx diaphragm. We believe that the results from our investigation will provide clinical significance, as we expect to elucidate mechanisms by which oxidative stress contribute to tissue damage and weakness in dystrophic diaphragm.Item ROLE OF AGE-ASSOCIATED OXIDATIVE STRESS IN ALTERED RENAL D1 AND AT1 RECEPTOR FUNCTIONS AND HYPERTENSION(2012-04-19) Chugh, Gaurav; Lokhandwala, Mustafa F.; Asghar, Mohammad; Hussain, Tahir; Prince, Randall A.; Shek, EugeneBlood pressure (BP) and oxidative stress increase with aging. Renal dopamine D1 (D1R) and angiotensin AT1 (AT1R) receptors by maintaining sodium homeostasis regulate blood pressure. Impaired D1R and exaggerated AT1R functions in the kidneys contribute to hypertension in animal models, which also exhibit oxidative stress. However, the role of oxidative stress in age-related hypertension has not been studied. In this study, we hypothesized that age-associated increase in oxidative stress by altering renal D1R and AT1R functions cause high BP in aging. To test this hypothesis, we measured oxidative stress, BP, and D1 and AT1 receptor functions in adult (3-month) and old (21-month) Fischer 344 X Brown Norway F1 (FBN) rats supplemented without/with antioxidant tempol. We found age-related increases in oxidative stress and blood pressure; which were reduced with tempol treatment in old FBN rats. D1R and AT1R functions were determined by measuring diuretic and natriuretic responses to SKF-38393 (D1R agonist) and candesartan (AT1 receptor antagonist) respectively. Natriuresis in response to D1R activation was impaired in old rats, suggesting an age-associated decline in D1R function in old FBN rats. Increase in G protein coupled receptor kinase (GRK) expression/activity is associated with reduced D1R-G protein coupling and function in humans and animal models with hypertension. We found age-associated increase in GRK-4 levels accompanied with D1R-G protein uncoupling in the renal proximal tubules of old FBN rats. Tempol treatment reduced GRK-4 levels and restored D1R-G protein coupling in these old rats. Natriuretic and diuretic responses to candesartan; however, were exaggerated in old rats, suggesting an age-associated increase in renal AT1R function in old FBN rats. Age-related increases in angiotensin II-mediated G protein coupling leading to exaggerated Na,K-ATPase activity may have caused increased renal AT1R function observed in old FBN rats. Tempol treatment restored angiotensin II-mediated G protein coupling and Na,K-ATPase response and thus reduced candesartan-mediated natriuresis and diuresis in old FBN rats. Our results demonstrate that both diminished renal D1R and exaggerated AT1R functions are associated with high BP in old FBN rats. Furthermore, oxidative stress may cause altered renal D1R and AT1R functions and high BP in these old rats.Item The Role of Caspase-8 in Oligodendrocyte Development and Mechanisms of Oxidative Injury in Neurons and Glia(2012-12-01) Thompson, JeffreyApoptosis 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.